https://orcid.org/0000-0002-3925-9154
Figures
Abstract
Oxidative stress response is a fundamental biological process mediated by conserved mechanisms. The identities and functions of some key regulators remain unknown. Here, we report a novel role of C. elegans casein kinase 1 gamma CSNK-1 (also known as CK1γ or CSNK1G) in regulating oxidative stress response and ROS levels. csnk-1 interacted with the bli-3/tsp-15/doxa-1 NADPH dual oxidase genes via genetic nonallelic noncomplementation to affect C. elegans survival in oxidative stress. The genetic interaction was supported by specific biochemical interactions between DOXA-1 and CSNK-1 and potentially between their human orthologs DUOXA2 and CSNK1G2. Consistently, CSNK-1 was required for normal ROS levels in C. elegans. CSNK1G2 and DUOXA2 each can promote ROS levels in human cells, effects that were suppressed by a small molecule casein kinase 1 inhibitor. We also detected genetic interactions between csnk-1 and skn-1 Nrf2 in oxidative stress response. Together, we propose that CSNK-1 CSNK1G defines a novel conserved regulatory mechanism for ROS homeostasis.
Author summary
Oxidative stress response is a fundamental life process that regulates the generation and elimination of reactive oxygen species (ROS) and manages ROS-elicited damages. Some key regulatory mechanisms remain to be elucidated. While analyzing mutations promoting the survival of the nematode Caenorhabditis elegans in excess iodide, an oxidative stressor, we identified the casein kinase 1 gamma gene csnk-1. Casein kinase 1 gamma proteins are highly conserved and proposed to have important biomedical functions. However, they are understudied and their effects on oxidative stress response are unknown. We combined genetic, biochemical and cell biology approaches to show that casein kinase 1 gamma members in C. elegans and humans are novel regulators of oxidative stress response and promoters of ROS levels. These activities involve interactions with the NADPH dual oxidase complex, which are likely mediated by physical interactions between casein kinase 1 gamma and the dual oxidase maturation factor. Our findings provide new mechanistic insights into the regulation of ROS homeostasis and uncover an important function of the “dark” casein kinase 1 gamma.
Citation: Hu Y, Xu Z, Pan Q, Ma L (2023) Casein kinase 1 gamma regulates oxidative stress response via interacting with the NADPH dual oxidase complex. PLoS Genet 19(4): e1010740. https://doi.org/10.1371/journal.pgen.1010740
Editor: Danielle A. Garsin, The University of Texas Health Science Center at Houston, UNITED STATES
Received: September 19, 2022; Accepted: April 10, 2023; Published: April 26, 2023
Copyright: © 2023 Hu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting information files.
Funding: The study is supported by National Natural Science Foundation of China grants (No. 31972877) and Natural Science Foundation of Hunan Province (2020JJ4109) to LM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Reactive oxygen species (ROS) are universal life molecules. ROS at physiological levels are essential signaling molecules that regulate metabolism, cell growth, cell survival and cell proliferation [1]. Endogenous ROS are primarily generated by the NADPH oxidases and mitochondria [1,2]. Abnormal cellular processes, xenobiotics, some metals and pathogens can induce excessive ROS generation [3–5], which may damage proteins, lipids and nucleic acids and cause oxidative stress [2]. Animals use conserved molecular mechanisms to regulate ROS levels and mitigate oxidative stress [1,2]. For example, the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway plays a major role in the defense response to oxidative stress triggered by endogenous or exogenous stressors [1,2,6].
Studies in the nematode Caenorhabditis elegans have provided important mechanistic understandings of oxidative stress response [6,7]. C. elegans genome encodes a Nrf2 homolog SKN-1 [8] that mediates oxidative stress response [9] and is also broadly involved in aging, immunity, lipid metabolism and other stress responses [6]. SKN-1 activity is regulated by p38 MAPK, GSK-3 and other signals [6,10,11]. Similar to the mammalian Keap1-mediated ubiquitination and proteasome degradation of Nrf2 [12], SKN-1 levels can be negatively regulated by the WD40 repeat protein WDR-23 via the ubiquitin-proteosome pathway [13].
C. elegans dual oxidase BLI-3 is the only functional NADPH oxidase encoded by its genome [14]. BLI-3, the tetraspanin protein TSP-15 and the dual oxidase maturation factor DOXA-1 form a NADPH oxidase complex to regulate C. elegans cuticle formation and response to oxidative stress [7,14–20]. BLI-3 affects innate immunity by interacting with p38 MAPK, SKN-1, proline catabolism and DAF-16 pathways [21–27]. BLI-3 also plays roles in longevity [15,28], vulva development [29] and the response to manganese toxicity [30]. The mammalian orthologs of BLI-3, DUOX1 and DUOX2, are involved in immunity and required for thyroid hormone synthesis, respectively [31].
Casein kinase 1 (CSNK1) is a family of conserved serine/threonine kinases that regulates diverse signaling processes by phosphorylating a large number of protein substrates [32]. Mammals have six common casein kinase 1 members, CSNK1A (CK1α), CSNK1D (CK1δ), CSNK1E (CK1ε) and CSNK1G1/2/3 (CK1γ1/2/3) [33], among which CSNK1Gs require palmitoylation-mediated membrane localization for their activities [34–36]. Studies in Drosophila and mammals found that CSNK1Gs can regulate Wnt, Hedgehog, JNK and RIPK3 signals [34,36–39], and can also phosphorylate the ceramide transport protein [40] and Lyn tyrosine kinase [41]. Human CSNK1G1 mutations are associated with developmental delay and autism spectrum disorder [42].
In C. elegans, kin-19, kin-20 and csnk-1 encode orthologs of CSNK1A, CSNK1D and CSNK1G, respectively [43] (wormbase.org). csnk-1 is an essential gene for C. elegans embryonic asymmetric spindle positioning [44–48] and oocyte meiosis [49]. The function of CSNK1G in oxidative stress response is unclear.
Iodine is an essential mineral nutrient for the synthesis of thyroid hormones and is routinely added to salt and foods. Surprisingly, some studies found that excess intake of iodide salt was associated with several thyroid diseases with unclear molecular mechanisms [50]. We became interested in this phenomenon and used C. elegans as a model to examine the biological effects of excess iodide intake. We found that C. elegans treated with excess iodide had a significant increase of ROS levels and wildtype animals exhibited larval arrest potentially due to oxidative stress [19,20]. This is likely a conserved effect of iodide because several earlier studies showed that excess iodide can induce ROS generation in mammalian thyroid cells [51–54]. To investigate whether any genes might be involved in the larva-arresting effect of excess iodide, we screened for mutants that can survive into adults in excess iodide and isolated multiple loss-of-function (lf) mutations in bli-3, tsp-15, doxa-1, wdr-23 and gain-of-function (gf) mutations in skn-1 [19,20]. These findings suggest that new genes involved in oxidative stress response might be identified by examining whether their mutations can promote C. elegans survival in excess iodide [19,20].
In this study, we found that mac397, a previously isolated mutation that promoted C. elegans survival in excess iodide [20], caused a missense mutation in csnk-1. Further analyses found that csnk-1 genetically interacted with the bli-3/tsp-15/doxa-1 genes by nonallelic noncomplementation to affect animal survival in excess iodide. Consistent with this special genetic interaction, we detected specific biochemical interactions between CSNK-1 and DOXA-1 and potentially between their human orthologs. We also found that CSNK-1 was required for maintaining ROS levels in C. elegans and human CSNK1G2 can promote ROS levels in cultured cells. We propose that CSNK-1 CSNK1G is a conserved novel regulator of ROS levels and oxidative stress response by interacting with the NADPH dual oxidase complex.
Results
csnk-1 loss of function promotes C. elegans survival in excess iodide
We previously performed an ethyl methanesulfonate (EMS) screen for F1 mutants that can grow into adults in 5 mM NaI (the survived-in-sodium-iodide phenotype, abbreviated as Sisi hereafter) and isolated six unidentified mutants [20]. We mapped one of the mutations, mac397, to Chr. I. Within the mapped region of mac397, genomic sequencing detected a point mutation in csnk-1 (exon4:c.G482A:p.R161H, named mac397. Fig 1A and S1 Table), a point mutation in tsp-15 (exon3:c.G331A:p.G111R, named mac499. S1 Table), and nine other coding variants (S1 Table). Deficiencies in csnk-1 or tsp-15 caused by RNAi or genetic mutations led to the Sisi phenotype (S1, S2 and S3 Tables). Transgene rescue and genocopy experiments suggest that tsp-15(mac499) caused a loss of function (S3 Table). The analyses of other isolates are ongoing.
(A) csnk-1 gene structure (based on wormbase.org). The positions of mac397, mac494 and mac495 mutations are indicated. The position of the sgRNA used for CRISPR/Cas9-based mutagenesis is shown as a red bar. (B) Percentage of L1 larva that grew into adults on plates with 5 mM NaI. Results were based on two biological replicates. 100 L1 larva were analyzed in each replicate. Statistics: two-tailed unpaired Student’s t-test. *: p < 0.05. (C-H) Expression pattern of a csnk-1p::GFP transgene in adults. (C) Fluorescent picture of a transgenic adult. The head, tail and vulva are indicated. (D-H) Higher-resolution pictures of transgenic adults. Cells with obvious GFP expression are indicated.
csnk-1 encodes a casein kinase 1 gamma [43] orthologous to the mammalian CSNK1G1, CSNK1G2 and CSNK1G3 [33]. The kinase domains and the C-terminal palmitoylation signals of CSNK1Gs are highly conserved (S1 Fig). The R161H (arginine to histidine) change caused by mac397 is in the kinase domain of CSNK-1 (S1 Fig).
To verify the effect of csnk-1 on the Sisi phenotype, we generated two frameshift mutations, mac494 and mac495 (Fig 1A and S2 Table), using the CRISPR/Cas9 method. Homozygous progeny of csnk-1(mac494) or csnk-1(mac495) heterozygotes exhibited the Sisi phenotype, while the heterozygous progeny did not (Fig 1B and S2 Table).
To investigate how csnk-1 transcript levels change in these mutants, we performed RT-qPCR experiments using primer pairs that are supposed to recognize all transcripts (wildtype and mutants) or wildtype-only transcripts (S2A Fig). We found that both mutants exhibited significantly reduced levels of total csnk-1 transcripts at the L4 larval stage (S2B Fig), implying a positive feedback effect of CSNK-1 on the expression of its own transcripts or that the mutant transcripts were degraded via nonsense-mediated decay (NMD). Furthermore, wildtype transcripts were essentially depleted in both mutants at the L4 larval stage (S2C and S2D Fig).
We observed other phenotypes of these mutants. For example, homozygotes can lay plenty of eggs (S3A Fig), but few would hatch (S3B Fig). csnk-1 mutants exhibited significant molting defects (S3C–S3E Fig). However, if the molting was normal, the mutants exhibited grossly regular cuticle stripe patterns (S3F and S3G Fig). Like wild type, csnk-1(mac494) mutants were rarely stained by the nuclear dye Hoechst 33258 (S3H Fig), implying relatively normal cuticle integrity.
The egg-hatching, egg-laying and molting defects of csnk-1(mac495) mutants were obviously more severe than those of csnk-1(mac494) mutants. Since mac494 and mac495 both caused frameshifts close to the start codon and are predicted to encode severely truncated proteins (Figs 1 and S1), it is intriguing that the severity of their phenotypes was quite different. We have yet to determine whether this difference is caused by a potential hypomorphic nature of mac494 or unknown modifier mutations in them.
To further understand csnk-1 function, we expressed a csnk-1 cDNA transgene driven by an endogenous csnk-1 promoter (3.1 kb upstream of the start codon) in csnk-1(mac494) mutants. The transgene significantly rescued the Sisi phenotype of the mutants (Table 1). Because the majority of csnk-1(mac494) mutants were able to survive into adults probably due to apparently weaker molting defects (S3E Fig), which makes our analyses of the Sisi phenotype significantly easier, we used this mutation as our reference loss-of-function (lf) allele in the following studies, if not specified.
We next examined how mac397 R161H affects CSNK-1 activity. Driven by the endogenous csnk-1 promoter, a csnk-1(R161H) cDNA transgene failed to rescue or only weakly rescued the Sisi phenotype of csnk-1(lf) mutants (Table 1). This transgene also failed to cause Sisi in wildtype animals, suggesting that mac397 was not dominant negative (Table 1). We propose that mac397 caused a loss of function.
CSNK-1 primarily functions in the epidermis to affect the Sisi phenotype and its function is conserved between C. elegans and human
We next examined the expression pattern of csnk-1. A GFP transgene driven by the csnk-1 endogenous promoter was broadly expressed in the wildtype background (Fig 1C). The expression was obvious in the pharynx, head neurons, epidermis (hypodermis), intestine, ventral cord, dorsal cord, vulval muscles and body-wall muscles (Fig 1D–1H).
To investigate in which tissue csnk-1 affects the Sisi phenotype, we performed tissue-specific transgene rescue experiments. Driven by an epidermis-specific promoter (dpy-7p), csnk-1 cDNA transgenes strongly rescued the Sisi phenotype of csnk-1(lf) mutants (Table 1). However, csnk-1 transgenes driven by an intestine-specific promoter (nhx-2p) failed to rescue (Table 1). Therefore, csnk-1 likely functions in the epidermis to affect the Sisi phenotype. We previously reported similar results for tsp-15 [20].
To examine whether the function of CSNK1G was conserved, we expressed human CSNK1G1, CSNK1G2 or CSNK1G3 cDNA transgenes in the epidermis of csnk-1(lf) mutants. We found that these transgenes significantly rescued the Sisi phenotype of the mutants and CSNK1G2 transgenes appeared to be more effective (Table 1). Hence, the function of csnk-1 is likely conserved and human CSNK1G1/2/3 exhibited similar activities.
CSNK1Gs contain a conserved C-terminal palmitoylation signal (TKCCCFFKR) required for membrane localization and the activities of the kinases [34,55]. A conserved palmitoylation sequence was also found in CSNK-1 (VKCCCFRRR, aa 386–394, S1 Fig). We generated two mutant csnk-1 transgenes disrupting this sequence (p.C388-K407del or p.C388-390S, S1 Fig). These transgenes failed to or barely rescued the Sisi phenotype of csnk-1(lf) mutants (Table 1).
Nonallelic noncomplementation genetic interaction between csnk-1 and bli-3/tsp-15/doxa-1
Since csnk-1(mac397) and tsp-15(mac499) mutations were both detected in the descendants of the original mac397 isolate (S1 Table), which was picked as a Sisi F1 progeny of EMS-mutagenized wildtype P0 animals, we speculated that this F1 progeny probably carried a tsp-15(mac499) csnk-1(mac397)/+ + genotype and the rare co-presence of csnk-1(mac397)/+ and tsp-15(mac499)/+ might have caused the Sisi phenotype in this animal [20]. [tsp-15(mac499) is recessive (S3 Table) so its heterozygous presence would not cause Sisi in our F1 screen.] It is worth noting that we previously observed similar genetic interactions between bli-3(lf)/+ and doxa-1(lf)/+ mutations [20]. Such interactions, called nonallelic noncomplementation [56,57], are often exhibited by two genes encoding proteins physically interacting with each other or functioning in the same pathway [56–58]. To examine whether there were broader nonallelic noncomplementation interactions, we generated more double mutants between csnk-1 and bli-3/tsp-15/doxa-1.
We found that tsp-15(lf) +/+ csnk-1(lf) males grew like wild type on regular NGM plates (Table 2). On plates with 5 mM NaI, some of these males were weakly Sisi (Table 2). Similarly, bli-3(lf) csnk-1(lf)/+ + hermaphrodites exhibited a wildtype-like phenotype on regular NGM plates (Table 2 and S4 and S5 Figs) and some were weakly Sisi in 5 mM NaI (Table 2).
Interestingly, csnk-1(lf)/+; doxa-1(mac55lf)/+ hermaphrodites were obviously blistered and dumpy on regular NGM plates (Table 2 and S4 and S5 Figs). These animals showed a strong Sisi phenotype (Table 2). A different doxa-1(lf) allele (mac67) that we previously isolated [20] exhibited similar interactions with csnk-1(lf) (Table 2).
Meanwhile, we examined the morphology of double homozygous mutants between csnk-1(lf) and bli-3(lf) or doxa-1(lf). Compared to single mutants, bli-3(lf) csnk-1(lf) double mutants were strongly scrawny and blistered (S4 and S6 Figs). Differently, csnk-1(lf); doxa-1(lf) double homozygotes were apparently larger (S6 Fig) and appeared like csnk-1(lf)/+; doxa-1(lf)/+ double heterozygotes (S5 Fig).
Conserved interaction between CSNK-1 and DOXA-1
The genetic findings imply that CSNK-1 and BLI-3/TSP-15/DOXA-1 might form a protein complex or function in the same pathway. Furthermore, the more special interaction between csnk-1 and doxa-1 prompted us to speculate whether DOXA-1 might physically interact with CSNK-1. To investigate the possibility, we co-expressed a DOXA-1::GFP fusion protein and a CSNK-1::mCherry fusion protein in C. elegans epidermis. Here we detected colocalization of the fluorescent signals in epidermal subcellular structures (Fig 2A–2C). The colocalization is likely caused by specific interaction between DOXA-1 and CSNK-1 because GFP alone did not colocalize with CSNK-1::mCherry, nor did mCherry alone colocalize with DOXA-1::GFP (S7 Fig).
(A, B, C) Confocal pictures of a transgenic L3 larva co-expressing dpy-7p::doxa-1::GFP and dpy-7p::csnk-1::mCherry transgenes. Enclosed areas are enlarged in lower panels. Arrows indicate typical subcellular structures co-labeled by GFP and mCherry. (D, E) Western blotting showing the specific interaction between DOXA-1::HA and FLAG::CSNK-1 expressed in HEK293T Cells. Immunoprecipitation was performed using an anti-HA antibody (D) or an anti-FLAG antibody (E). (F, G) Western blotting showing that His::TF::DOXA-1 or His::TF::CSNK-1 purified from BL21 bacteria can specifically pull-down FLAG::CSNK-1 (F) or DOXA-1::HA (G) expressed in HEK293T Cells, respectively. Coomassie blue (CB) staining of purified proteins is shown in lower panels. (H, I) Relative fluorescent intensities of L4 larva stained with DCFDA or Amplex Red. Results were based on three biological replicates, with 2–5 samples per replicate. Each datapoint represents one sample. Colors represent different replicates. Statistics: two-tailed unpaired Student’s t-test. ***: p < 0.001. (J) Representative fluorescent pictures of the jrIs1 reporter in wildtype or csnk-1(lf) L4 larva. Picture exposure time was 432.6 ms. (K) Relative oxidized/reduced HyPer reporter signals. Results were based on three biological replicates, with 2–3 samples per replicate. Each data point represents one sample. Colors represent different replicates. Statistics: two-tailed unpaired Student’s t-test. **: p < 0.01. Error bars: standard deviation.
We next transiently expressed a FLAG::CSNK-1 fusion protein with HA tag alone or with a DOXA-1::HA fusion protein in HEK293T cells. From the cell extracts, FLAG::CSNK-1 could be coimmunoprecipitated with DOXA-1::HA using an anti-HA antibody, while FLAG::CSNK-1 co-expressed with HA tag alone could not be coimmunoprecipitated using this antibody (Fig 2D). We further performed reverse immunoprecipitation using an anti-FLAG antibody. Similarly, DOXA-1::HA could be coimmunoprecipitated with FLAG::CSNK-1 but not with the FLAG tag alone (Fig 2E).
To examine the interaction between CSNK-1 and DOXA-1 by a different approach, we purified a bacterially expressed His::TF::DOXA-1 fusion protein and co-incubated the purified protein with the lysates of HEK293T cells expressing FLAG::CSNK-1. Here FLAG::CSNK-1 could be specifically pulled down by His::TF::DOXA-1 (Fig 2F). In reverse, a His::TF::CSNK-1 fusion protein purified from bacterial expression could specifically pull down a DOXA-1::HA fusion protein expressed in HEK293T cells (Fig 2G). Together, these results suggest a specific interaction between CSNK-1 and DOXA-1. Nevertheless, caution should be exercised in interpreting the pulldown results as proteins expressed in E. coli may be incorrectly folded, which might cause non-specific interactions.
DOXA-1 is orthologous to the human DUOX maturation factor 1 and 2 (DUOXA1 and DUOXA2) (S8 Fig), which are required for dual oxidase activities [17,59]. To investigate whether human CSNK1Gs and DUOXAs exhibit similar interactions like that between CSNK-1 and DOXA-1, we co-expressed FLAG::CSNK1G2 and DUOXA2::HA fusion proteins in HEK293T cells. In these cells we detected co-localization of the two proteins on the plasma membrane and subcellular structures (Fig 3A). Similar colocalization was also observed in a transfected HeLa cell (S3I Fig). [CSNK1G2 was chosen because its transgenes showed stronger rescuing effects (Table 1).] We also detected a potentially specific interaction between these proteins using immunoprecipitation (Fig 3B).
(A) Immunofluorescent staining showing membrane and subcellular colocalization of DUOXA2::HA (green) and FLAG::CSNK1G2 (red) co-expressed in HEK293T cells. Enlarged pictures of two cells are shown on right. We observed that CSNK1G2 overexpression often changed the fibroblast morphology of the cells to a round shape. (B) Western blotting showing the interaction between DUOXA2::HA and FLAG::CSNK1G2 co-expressed in HEK293T Cells. Immunoprecipitation was performed using an anti-HA antibody. (C, D) Dose-dependent inhibitory effect of D4476 on DCFDA or Amplex Red signals in HEK293T cells. Three biological replicates were performed with two wells per replicate. Colors represent different replicates. Statistics: Bonferroni’s multiple comparison test with one-way ANOVA. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. ns: not significant. (E) Effects of D4476 on DCFDA signals in HEK293T cells overexpressing CSNK1G2 or DUOXA2. Controls were transfected with pCMV-Tag2B backbone vector. Three biological replicates were performed with two wells per replicate. Colors represent different replicates. See raw data for all results. Statistics: two-tailed unpaired Student’s t-test. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
CSNK-1 promotes ROS levels
The interaction between CSNK-1 and DOXA-1 suggests that CSNK-1 might affect ROS levels. Using 2’,7’-dichlorofluorescin diacetate (DCFDA) and Amplex Red staining as readouts of ROS levels, we found that csnk-1(lf) mutants exhibited significantly reduced ROS levels (Figs 2H, 2I, S3J and S3K). We further crossed the jrIs1 reporter [60], an YFP-based hydrogen peroxide sensor that detects endogenous ROS levels, into csnk-1(lf) mutants and observed obviously reduced levels of oxidized sensor proteins in the mutants (Fig 2J and 2K).
To investigate whether human CSNK1 might affect ROS levels, we treated HEK293T cells with D4476, a selective small molecule inhibitor of CSNK1 [61]. Using DCFDA and Amplex Red staining, we detected a dose-dependent inhibition of ROS levels by D4476 (Fig 3C and 3D). Furthermore, overexpression of CSNK1G2 led to increased ROS levels in the cells as measured by DCFDA fluorescence, which were inhibited by D4476 (Fig 3E). The increased ROS levels caused by DUOXA2 overexpression were also inhibited by D4476 (Fig 3E).
The interaction between CSNK-1 and DOXA-1 prompted us to explore whether DOXA-1 activity might be related to any potential phosphorylation sites. We inspected DOXA-1 sequence using Scansite 4 (www.scansite4.mit.edu) and the top three predicted sites were shown in S8 Fig (red boxes and red arrowheads). These sites have the characters of the consensus CSNK-1 phosphorylation sites, D/E/(p)S/T(X)1-3S/T [55], in which the phospho-acceptor residue is underlined. We mutated each individual residue to alanine (A) in doxa-1 transgenes. Interestingly, only the T343A mutation significantly weakened the rescuing effects of the transgene (Table 3). These results suggest that T343 is important for DOXA-1 activity. However, it remains unclear whether the importance of this site depends on its phosphorylation.
csnk-1 and skn-1 genetically interact to affect oxidative stress response
skn-1(gf) mutants are Sisi while skn-1(lf) mutants are not [20]. To understand the interaction between csnk-1 and skn-1, we examined the Sisi phenotype of csnk-1; skn-1 double mutants.
Different from skn-1(lf) mutants, which are not Sisi, csnk-1(lf); skn-1(lf) double mutants became Sisi (Table 4). This implies an epistatic effect of csnk-1 on the Sisi phenotype. We next examined the Sisi phenotype of csnk-1(lf); skn-1(gf) double mutants in 50 mM NaI, a test that we previously used to examine whether bli-3/tsp-15/doxa-1 loss-of-function mutations and skn-1 gain-of-function mutations had synergistic effects [20]. Though single mutant failed to show the Sisi phenotype in such a high concentration of NaI, csnk-1(lf); skn-1(gf) double mutants became Sisi (Table 5).
Finally, to investigate whether CSNK-1 might affect the expression of genes regulated by SKN-1, we crossed a SKN-1 target gene reporter, dvIs19 (gst-4p::GFP) [62] into csnk-1(lf) mutants. The GFP signals were obviously increased in both csnk-1(lf) mutants (S9A Fig) and the increase was still visible when the animals were treated with skn-1(RNAi) (S9B Fig).
Discussion
In this study, we identified a novel role of CSNK-1 CSNK1G in oxidative stress response. We uncovered nonallelic noncomplementation interactions between C. elegans csnk-1 and bli-3/tsp-15/doxa-1 NADPH dual oxidase genes, which led to the findings of a potentially conserved biochemical interaction between CSNK-1 and DOXA-1 and a conserved function of CSNK-1 in promoting ROS levels. We also detected genetic interactions between csnk-1 and skn-1 in oxidative stress response.
CSNK-1 is an embryonically essential kinase with novel function in postembryonic oxidative stress response
csnk-1 is an essential gene that affects the embryonic asymmetric spindle positioning and oocyte meiosis in C. elegans [44–49]. Consistently, we found that eggs laid by csnk-1(lf) mutants failed to hatch. The effect of csnk-1 on the Sisi phenotype is specific, as RNAi knockdown of kin-19, kin-20 and three other kinase genes did not obviously affect the phenotype (S2 Table). The importance of the conserved C-terminal palmitoylation for CSNK-1 activity is supported by the finding that mutations of the site caused a loss of the function.
Our transgene rescue results suggest functional similarity among the three human CSNK1Gs in affecting oxidative stress response. This is consistent with a recent finding that human CSNK1Gs exhibited functional redundancy in regulating Wnt signaling [63]. Together with our findings that CSNK-1 and human CSNK1G2 can promote ROS levels, we suggest that CSNK1Gs are important regulators of ROS homeostasis across species.
CSNK1G interacts with the NADPH dual oxidase complex
Mammals have seven membrane-localized NADPH oxidases as major generators of endogenous ROS [2,31], among which the dual oxidase DUOX2 is essential for thyroid hormone synthesis, while DUOX1 is more involved in immune cell functions [31]. It is not well understood how DUOX activities are regulated. DUOXA1 and DUOXA2 are conserved facilitators for the ER-Golgi-plasma membrane translocation of DUOX1 and DUOX2 [59]. In C. elegans, BLI-3 DUOX, DOXA-1 DUOXA and TSP-15 tetraspanin form a dual oxidase complex [16,17]. Recently, the highly conserved MEMO-1 protein was identified as a negative regulator of BLI-3 activity via binding the RHO-1/RhoA/GTPase [15], providing a new regulatory mechanism for dual oxidase activities.
We suggest that CSNK-1 interacts with the dual oxidase complex and might regulate dual oxidase activity. First, the nonallelic noncomplementation interactions between csnk-1 and bli-3/tsp-15/doxa-1 imply physical and/or functional interactions between CSNK-1 and BLI-3/TSP-15/DOXA-1. Second, CSNK-1 was colocalized with DOXA-1 on C. elegans epithelial subcellular structures. So did human CSNK1G2 and DUOXA2 in HEK293T cells. Third, that CSNK-1 and DOXA-1 were detected in a same protein complex by immunoprecipitation and pull-down experiments suggests a possible direct physical interaction between CSNK-1 and DOXA-1, and human CSNK1G2 and DUOXA2 were detected in a same protein complex by immunoprecipitation. Fourth, CSNK-1 is required for normal ROS levels in C. elegans and CSNK1G2 and DUOXA2 each can promote ROS levels in HEK293T cells.
We found that D4476, a commonly used CSNK1 inhibitor, can decrease ROS levels in HEK293T cells in a dose-dependent manner and suppress the promotion of ROS levels by overexpressed CSNK1G2 and DUOXA2, supporting the involvement of CSNK1 in regulating ROS levels. However, our evidence is insufficient for connecting CSNK1s directly with ROS level regulation before extensive analyses of the expression and function of each CSNK1 in HEK293T are performed.
Mutagenesis analyses suggest that T343, the phospho-acceptor of a potential CSNK1 site in DOXA-1 C-terminal domain (S8 Fig), was required for DOXA-1 activity, implying phosphorylation as a mechanism for regulating DOXA-1 activity. However, the lack of conservation in the aligned DUOXA2 sequence raises the question about the importance of this site in other species. Considering that four potential CSNK1 phosphorylation sites are detected in the C-terminal domain of DUOXA2 (S8 Fig), two of which appear to be conserved in DOXA-1, we suspect that the interaction between CSNK-1 and DOXA-1 and the phosphorylation of DOXA-1, if it exists, might be located at different sites. To answer this question, it is necessary to identify the interacting domains between these proteins, the potential phosphorylation sites on DOXA-1, and the functional importance of these sites in future.
csnk-1 genetically interacts with skn-1
SKN-1 is the C. elegans ortholog of Nrf2 [8,9] and a key regulator of oxidative stress response and other cellular processes [6]. We previously found that skn-1 can genetically interact with bli-3/tsp-15/doxa-1 to affect the Sisi phenotype [20]. Similarly in this study we found that csnk-1 and skn-1 can genetically interact to affect the Sisi phenotype. Specifically, the lack of Sisi phenotype in skn-1(lf) mutants in 5 mM NaI was suppressed by csnk-1(lf), while csnk-1(lf) and skn-1(gf) exhibited synergy in promoting the Sisi phenotype in 50 mM NaI. These interactions imply an opposite effect of csnk-1 on oxidative stress response that is in parallel with skn-1. However, the molecular genetic mechanism underlying such interactions remains to be understood considering that the genetic analyses were performed on mutants with maternal effects and that reducing ROS levels (like in bli-3/tsp-15/doxa-1 loss-of-function mutants) [19,20], increasing antioxidant expression (like in skn-1(gf) mutants) [20] or adding antioxidants such as vitamin C or NAC to NGM plate [20] all can cause the Sisi phenotype.
We found that the expression of the SKN-1 reporter transgene dvIs19 (gst-4p::GFP) was increased in csnk-1(lf) mutants and the increase existed even with skn-1(RNAi) treatment. However, considering that RNAi knockdown of skn-1 could be partial, we have yet to interpret such an effect of csnk-1 as skn-1-independent. To gain a deeper understanding of the interaction between CSNK-1 and SKN-1, it will be important to determine how ROS levels are affected in csnk-1; skn-1 double mutants, whether endogenous gst-4 expression is similarly affected by csnk-1(lf), or whether any other gene expression is affected by csnk-1. We hope to address these questions in future using multidisciplinary approaches such as transcriptome and phosphoproteomic analyses.
Conclusions
Among casein kinase 1 members, CSNK1Gs are understudied and are classified by the NIH program “Illuminating the Druggable Genome” as “dark kinases” calling for extra exploration (https://commonfund.nih.gov/IDG). We uncovered an important role of CSNK-1 CSNK1G in regulating oxidative stress response and ROS levels, which involves potentially conserved interactions between CSNK1G and the NADPH dual oxidase complex. Our findings provide novel insights into redox biology and may facilitate the understanding of diseases caused by mutations in these genes and more broadly by defective oxidative stress response.
Materials and methods
Mapping and cloning of csnk-1
mac397 was previously isolated as a Sisi F1 progeny of EMS-mutagenized wildtype P0 animals grown on plates with 5 mM NaI [20]. To map mac397, males of the Hawaiian strain CB4856 were crossed with mac397 hermaphrodites. F1 male progeny were crossed with CB4856 hermaphrodites on plates with 5 mM NaI to generate Sisi F2 male progeny. Similar crosses were performed three more rounds to generate Sisi F5 hermaphrodites, which were picked to individual plates with 5 mM NaI and allowed to propagate. Animals on these plates were examined for single nucleotide polymorphisms (SNPs) [64,65]. We established a linkage on Chr. I to the right of SNP WBVar00240395 (SNP W03D8: 34384 D1 = T, genetic location: -5.68; GenBank accession no. FO081764) and to the left of SNP WBVar00245221 (SNP F58D5: 17029–17032 D4 = ACTA, genetic location: 13.02; GenBank accession no. AL137227).
To determine the genomic sequences of mac397 mutants, progeny of unbackcrossed original isolate were washed and starved for ~4 hrs in H2O. Genomic DNAs were extracted by proteinase K digestion, followed by RNase A treatment and two rounds of phenol–chloroform extraction. Three genomic DNA libraries (350 bp inserts) were constructed by Annoroad Gene Technology Corporation (Beijing) using Illumina’s paired-end protocol. Paired-end sequencing (100-bp reads) was performed on the Illumina HiSeq X Ten. 4G clean bases were mapped to the N2 genome (Wormbase release 220) after removal of duplicated reads.
Genomic sequencing identified 7 homozygous and 4 heterozygous missense mutations between the mapped SNPs on Chr. I, among which were a homozygous mutation in tsp-15 and a heterozygous mutation in csnk-1 (S1 Table). The homozygosity of the tsp-15 mutation might be caused by prolonged propagation of the original isolate in 5 mM NaI before the strain was sequenced (see below).
We initially suspected that the tsp-15 mutation (exon3:c.G331A:p.G111R, named mac499) (S1 and S3 Tables) might be a dominant negative allele, thus causing the Sisi phenotype of the mac397 isolate as a heterozygote. To test this, we generated two knock-in mutations genocopying mac499 (S3 Table, mac500 and mac501). The heterozygous knock-in mutants were not Sisi, while the homozygous mutants were (S3 Table). In addition, a tsp-15 transgene can rescue the Sisi phenotype of tsp-15(mac499) homozygous mutants (S3 Table). Therefore, tsp-15(mac499) is recessive, which led us to suspect that a linked mutation, either alone as heterozygote or together with tsp-15(mac499)/+, caused the Sisi phenotype of the original isolate.
To identify this gene, we treated wildtype animals with feeding RNAi targeting the genes listed in S1 Table. Besides tsp-15(RNAi), we found that csnk-1(RNAi) also made animals Sisi (S1 and S2 Tables). The corresponding mutation in csnk-1 was name mac397.
After we found that csnk-1(RNAi) can cause Sisi, we re-examined our mac397 frozen stocks but were only able to detect tsp-15(mac499) mutation. mac397, either as heterozygote or homozygote, was not found in different batches of thawed animals.
Considering that mac397 was a loss-of-function mutation (Table 1) and csnk-1(lf) homozygous mutations cause lethality, we postulate that a prolonged propagation of the mutants before freezing probably caused this phenomenon. In the original isolate, csnk-1(mac397) was likely heterozygous (S1 Table, genomic sequencing results in the early propagation phase). A prolonged propagation of the strain in 5 mM NaI, with tsp-15(mac499) csnk-1(mac397)/+ + as the presumptive starting genotype, would generate crossover between tsp-15 and csnk-1 and allow tsp-15(mac499) (viable as heterozygotes or homozygotes) to outcompete csnk-1(mac397) (only viable as heterozygotes) in the population. At the time of freezing, most animals, if not all, might have carried only a tsp-15(mac499) homozygous mutation and lost the csnk-1(mac397) mutation.
We previously found that bli-3(lf)/+; doxa(lf)/+ double heterozygous mutants were Sisi in 5 mM NaI, while bli-3(lf)/+ or doxa-1(lf)/+ single mutants were not [19,20], suggesting a nonallelic noncomplementation interaction between the two genes. Similarly, we postulate that csnk-1(mac397) and tsp-15(mac499) probably interacted by nonallelic noncomplementation to cause the Sisi phenotype in the original mac397 isolate [20].
C. elegans survival assay in excess iodide (Sisi phenotype assay)
The survival assay was performed as described [19] with modification.
In general, bleached eggs were grown on OP50-seeded NGM plates with different concentrations of NaI (5 mM or 50 mM). The number of L1 larva was counted and the number of adults was counted 3 days post L1.
For heterozygous males in Table 2, males and hermaphrodites of the desired genotypes were crossed on plates with 5 mM NaI. The cross progeny were allowed to grow in the same plate for 4–6 days and the appearance of young adult males was recorded.
Generation of csnk-1 or tsp-15 mutations using CRISPR/Cas9
We followed the method [66] with modifications. The DNA mixture for injection contained 50 ng/μl Peft-3::Cas9-SV40_NLS, 25 ng/μl PU6::sgRNA (specific for csnk-1 or tsp-15), and 25 ng/μl PU6::sgRNA (specific for dpy-10) as co-injection marker. To generate tsp-15 knockin mutations, we included a 130 nt synthesized oligo (500 nM) containing the desired tsp-15 mutation as the repair template and a repair template for dpy-10 as described [67]. F1 animals with the Dpy/Rol phenotype were picked to individual plates and their progeny were analyzed for the desired mutations by Sanger sequencing. Target sequences of sgRNAs and the sequence of tsp-15 repair template are shown in S6 Table.
mac494 and mac495 homozygous mutants derived from heterozygous parents can grow into adults and were able to lay multiple eggs. However, these eggs would not or rarely hatch. We maintained csnk-1 mutations using the hT2 [bli-4(e937) let-?(q782) qIs48] (I; III) balancer.
Transgene experiments
Germline transgene experiments were performed as described [68].
For csnk-1 rescue experiments, the transgene mixture containing 0.5 ng/μl or 5 ng/μl of the transgenes of interest with 20 ng/μl pPD95_86 (myo-3p::GFP) (a gift from Andrew Fire) as co-injection marker was injected to wildtype animals to generate at least two stable lines. The transgenes were crossed into csnk-1(mac494lf)/hT2[qIs48] animals and csnk-1(mac494lf) homozygous progeny carrying the transgenes were examined. For the Sisi phenotyping, transgenic adults were bleached, and eggs were placed on regular or 5 mM NaI NGM plates for 12 hrs to hatch. ~100 transgenic homozygous csnk-1(mac494lf) L1 larva were picked to a new 5 mM NaI NGM plates. The numbers of adults were counted after 3 days.
Hoechst 33258 staining
Hoechst 33258 staining was performed as described [16,19] with modifications. Young adult animals (24 hrs post mid-L4) were washed off plates and incubated for 15 min in M9 containing 1 μg/ml Hoechst 33258 (Sigma, 861405) at 20°C with gentle shaking. After staining, animals were washed three times with M9 and observed under a Leica DM5000B fluorescence microscope.
RNA interference
Bleached eggs were transferred to plates seeded with HT115 (DE3) bacteria expressing RNAi plasmids on NGM plates with 1 mM IPTG and 0.1 mg/ml Ampicillin. The progeny were examined under dissecting microscope for the Sisi phenotype. RNAi feeding bacterial strains for lrp-2, rbpl-1, uggt-2, K07A1.1, sys-1 and gsk-3 were picked from a whole-genome RNAi library [69], and the inserts were confirmed by sequencing. RNAi plasmids for other genes were generated in this study (see Plasmids).
Confocal microscopy
Fluorescent images of immunostained cells or transgenic animals expressing GFP and/or mCherry reporters were captured using a Leica TCS SP5 II or Zeiss LSM 880 laser confocal microscope.
Cell culture and transfection
HeLa and HEK293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco, 12484028) and 1% penicillin/streptomycin (ThermoFisher Scientific,15140163). Cells were maintained at 37°C with 5% CO2. Plasmid transfection was performed at 80% cell confluency using Lipofectamine 3000 Reagent (Invitrogen, L3000015) according to the manufacturer’s instructions. Cells were analyzed after 48 hrs.
Immunostaining
For immunostaining, HEK293T or HeLa cells grown on coverslips were fixed with 4% PFA in PBS at room temperature for 15 min and permeabilized with 0.25% PBST (PBS containing 0.25% Triton X–100) at room temperature for 15 min. Cells were blocked in 5% normal goat serum for 1 hr at room temperature. The samples were incubated overnight at 4°C with primary antibodies: rabbit anti-HA-tag (1:500; Cell Signaling Technology, #3724) and mouse anti-FLAG-tag (1:500; Sigma, F9291). Cells were washed in PBST and Alexa Fluor 488-anti-rabbit (1:200; Jackson Immunoresearch, 111545144) and Cy3-anti-mouse (1:200; Jackson Immunoresearch, 115165003) were used as the secondary antibodies. The samples were then stained with DAPI (1 μg/mL, Sigma, D9542) for 5 min at room temperature, washed, and mounted with Fluoromount aqueous mounting medium (Sigma, F4680).
Immunoprecipitation, pull-down and western blotting
For immunoprecipitation, HEK293T cells were lysed in ice-cold IP buffer (Beyotime, P0013) with 1% protease inhibitor cocktail and phosphatase inhibitor cocktail (Bimake, B14001). Lysed cells were incubated at 4°C for 4 hrs and centrifuged at 12000 rpm for 20 min at 4°C. The clear supernatants were transferred to a new tube and incubated with an anti-HA antibody (1 μl per sample, Cell Signaling Technology, #3724) or anti-FLAG antibody (1 μl per sample, Sigma, F9291) and protein A/G-beads (Bimake, B23201, 10 μl) at 4°C overnight. The beads were centrifuged and washed 5 times in TBST. Proteins associated with the beads were mixed with 32 μl 2x SDS lysis buffer and 8 μl 5x SDS loading buffer and boiled for 5 min at 95°C.
For pull-down, BL21 bacteria transformed with pCold TF:: doxa-1_cDNA or pCold TF:: csnk-1_cDNA plasmid were cultured in Luria broth (LB) at 37°C to OD 0.6, and 1 mM IPTG was added to induce protein expression at 16°C for 24 hrs. Bacteria were collected and lysed in PBST (1% Triton X-100) with 1 mM lysozyme (Beyotime, ST206) on ice for 30 min, followed by centrifugation at 12000 rpm, 4°C for 20 min. The clear supernatant was transferred to a new tube and incubated with His-tag resin (Beyotime, P2229S-1) at 4°C for 2 hrs. The resin was collected after centrifugation at 1200 rpm for 20 sec and used to incubate at 4°C overnight with the lysate supernatant of HEK293T cells transfected with pCMV-Tag2B(FLAG)::csnk-1_cDNA or pcDNA3.1-doxa-1_cDNA::HA plasmid. On the next day the mixture was washed three times in TBST. Proteins associated with the resin were mixed with 20 μl 2x SDS lysis buffer and 5 μl 5x SDS loading buffer and boiled for 5 min at 95°C.
Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon-P, IPVH00010). The membranes were incubated with primary antibodies (rabbit anti-HA-tag, 1:5000; Cell Signaling Technology, #3724 or mouse anti-FLAG-tag, 1:5000; Sigma, F9291) at 4°C overnight. On the next day, the membranes were washed three times in TBST at room temperature, incubated with secondary antibodies (1:10000; Jackson Immunoresearch, anti-mouse 115035146 or anti-rabbit 111035144) at room temperature for 1 hr. Protein bands were visualized using an ECL detection system (YEASEN, 36222ES60).
ROS measurement using DCFDA
Whole-animal ROS levels were measured using 2,7-dichlorofluorescein diacetate (DCFDA) (Sigma, D6883) following previous methods [70,71] with modifications. Synchronized L4 larval animals were washed with H2O for three times. The population density was adjusted with H2O to 50 to 200 animals per 100 μl. Two to three 100 μl aliquots were transferred to individual wells of a black-walled 96-well plate. DCFDA dissolved in DMSO was added to a final concentration of 50 μM. Animals were incubated at 20°C for 30 min and measured for DCFDA fluorescence intensity using a fluorimeter (BioTek, Synergy 2) at the excitation wavelength of 485 nm and the emission wavelength of 528 nm at room temperature. After the measurement, animals in each well were emptied to an agar plate and counted.
For DCFDA signals of individual animals, synchronized L4 animals were washed three times in H2O and resuspended in M9 to a final volume of 95 μl. 5 μl 1 mM DCFDA solution was added to a final concentration of 50 μM. Animals were incubated at 20°C for 30 min, washed in M9 three times, transferred to a glass slide, and covered with a cover glass. Fluorescent pictures of multiple animals were taken immediately with the same exposure intervals. Whole body fluorescence intensities of individuals were measured with ImageJ.
To measure dose-dependent effects of D4476 (Sigma, D4476) on ROS levels, HEK293T cells at 70% confluency in 24-well plates (coated with 0.01 mg/ml poly-D-lysine (Sigma, P6407)) were treated with different concentrations of D4476 in duplicate wells. After 24 hrs, the culture media was removed, and cells were washed once with DMEM. A 500 μl volume of 50 μM DCFDA in DMEM was added to each well, followed by incubation at 37°C for 20 min. The cells were washed three times with PBS and DCFDA fluorescence intensities were immediately measured using a fluorimeter (BioTek, Synergy 2) at the excitation wavelength of 485 nm and the emission wavelength of 528 nm at room temperature.
For CSNK1G2 or DUOXA2 overexpression, cells at 70% confluency were transfected with 200 ng plasmids per well in a 24-well plate. After 24 hrs, D4476 was added to the media at a final concentration of 30 μM and DCFDA staining was performed after 24 hrs.
ROS measurement using Amplex Red
Whole-animal ROS levels were measured using Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen, A22182) in a 96-well plate following manufacturer’s instructions and previous methods with modifications [72,73]. Amplex Red was added to a final concentration of 50 μM. Synchronized L4 animals were incubated at 20°C for 30 min and measured for fluorescence intensity using a fluorimeter (BioTek, Synergy 2) at the excitation wavelength of 530 nm and the emission wavelength of 590 nm at room temperature. After the measurement, animals in each well were emptied to an agar plate and counted.
To measure dose-dependent effects of D4476 (Sigma, D4476) on ROS levels, HEK293T cells at 70% confluency in 24-well plates coated with PDL were treated with different concentrations of D4476 in duplicate wells. After 24 hrs, the culture media was removed, and cells were washed once with DMEM. A 300 μl volume of 50 μM Amplex Red was added to each well followed by incubation at 37°C for 20 min and measuring for fluorescence intensity using a fluorimeter (BioTek, Synergy 2) at the excitation wavelength 530 nm and the emission wavelength of 590 nm at room temperature.
ROS measurement using transgenic HyPer reporter jrIs1
Synchronized L4 animals were picked to triplicate wells of a 96-well plate in 100 μl H2O, with 20 animals per well. Fluorescent signals were measured using a fluorimeter (BioTek, Synergy 2) with excitation wavelength of either 490 nm (for oxidized HyPer) or 405 nm (for reduced HyPer) and emission filter of 535 nm. The quantification of the oxidized/reduced HyPer signals was based on the method described by Back et al [60].
Supporting information
S1 Fig. CSNK1G protein sequence alignment.
C.e.: C. elegans CSNK-1; D.m.: Drosophila Gilgamesh; M.m.: mouse CSNK1G2; H.s.: human CSNK1G1, CSNK1G2, CSNK1G3. Green bar: frameshift regions caused by mac494 and mac495 mutations. Red box: kinase domain. Red arrowhead: mac397 mutation. Blue box: conserved palmitoylation signal.
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S2 Fig. Depletion of wildtype csnk-1 transcripts in L4 csnk-1(lf) mutants.
(A) Positions and sequences of PCR primers for detecting all or wildtype-only csnk-1 transcripts. Partial wildtype and mutant csnk-1 sequences are aligned to show the specificity of the primers for wildtype-only transcripts. (B) Relative total csnk-1 transcript levels. (C, D) Relative wildtype-only csnk-1 transcript levels in wildtype, csnk-1(mac494lf) or csnk-1(mac495lf) animals. tba-1 was the loading control. Statistics: two-tailed unpaired Student’s t-test. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
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S3 Fig. Phenotypic analyses of csnk-1(lf) mutants.
(A) Number of all eggs laid per adult. Results were based three biological replicates, with three animals per replicate. Colors represent different replicates. Statistics: two-tailed unpaired Student’s t-test. *: p < 0.05. ns: not significant. (B) Hatching rate of eggs laid by csnk-1(lf) homozygous mutants. All eggs laid by a single adult on regular NGM agar plates were examined. Results were based on three biological replicates, with three animals per replicate. Statistics: two-tailed unpaired Student’s t-test. ****: p < 0.0001. (C, D) Representative molting defects of csnk-1(lf) mutants. Arrows indicate attached cuticles. (E) Quantification of young adults (24 hrs after mid-L4 larval stage) with molting defects. Results were based on three biological replicates, with 100 animals analyzed in each replicate. Statistics: two-tailed unpaired Student’s t-test. **: p < 0.01; ***: p < 0.001. (F, G) Typical cuticle stripe patterns of csnk-1(lf)/+ and csnk-1(lf) mutants labeled by a DPY-7::sfGFP reporter. (H) Percentage of young adults positively stained by the nuclear dye Hoechst 33258. Results were based on three biological replicates, with 59–141 animals in each replicate. Statistics: two-tailed unpaired Student’s t-test. ****: p < 0.0001. ns: not significant. (I) Colocalization of overexpressed DUOXA2::HA and FLAG::CSNK1G2 in a HeLa cell. (J) Representative fluorescent pictures of wildtype and csnk-1(lf) L4 animals stained with DCFDA. Pictures were taken with the same exposure time of 600 ms and fluorescent intensity of each animal was measured using ImageJ. (K) Quantification of DCFDA fluorescent signals of individual L4 animals. Results were based on three biological replicates, with 21–100 animals in each replicate. Statistics: two-tailed unpaired Student’s t-test. ****: p < 0.0001.
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S4 Fig. Representative morphologies of csnk-1(lf), bli-3(lf) and doxa-1(lf) single mutants.
All images are of the same scale.
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S5 Fig. Representative morphologies of double heterozygous mutants between csnk-1(lf) and bli-3(lf) or doxa-1(lf).
bli-3(lf) csnk-1(lf)/hT2 (treated as bli-3(lf) csnk-1(lf)/+ +) mutants had wildtype-like morphology, while csnk-1(lf)/hT2; doxa-1(lf)/hT2 (treated as csnk-1(lf)/+; doxa-1(lf)/+) mutants exhibited obviously blistered and dumpy phenotype. Arrows point to blisters. All images are of the same scale.
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S6 Fig. Representative morphologies of double homozygous mutants between csnk-1(lf) and bli-3(lf) or doxa-1(lf).
bli-3(lf) csnk-1(lf) double homozygous mutants were derived from bli-3(lf) csnk-1(lf)/hT2 heterozygous mutants. csnk-1(lf); doxa-1(lf) double homozygous mutants were derived from csnk-1(lf)/hT2; doxa-1(lf)/hT2 heterozygous mutants. Arrows point to typical blisters. All images are of the same scale.
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S7 Fig. CSNK-1::mCherry does not colocalize with GFP and DOXA-1::GFP does not colocalize with mCherry.
(A, B, C) A transgenic L3 larvae co-expressing GFP and CSNK-1::mCherry in epithelial cells. (D, E, F) A transgenic 3-fold embryo co-expressing DOXA-1::GFP and mCherry in epithelial cells. For unclear reason, DOXA-1::GFP was strongly expressed in embryos but was not visible at larval stages in these transgenic lines. We therefore observed whether DOXA-1::GFP colocalizes with mCherry in embryos.
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S8 Fig. Sequence alignment of DOXA-1 and its orthologs.
Top three predicted (scansite4.mit.edu) CSNK1 phosphorylation sites (similar to the conserved D/E/(p)S/T(X)1-3S/T sequence) in DOXA-1 C-terminal region (start indicated by blue arrow above, predicted by uniprot.org) are enclosed in red boxes with the phospho-acceptor pointed out by red arrowheads. Four potential CSNK1 phosphorylation sites in DUOXA2 C-terminal region (start indicated by blue arrow below, predicted by uniprot.org) are enclosed in green boxes with the phospho-acceptor pointed out by green arrowheads. Two of these sites appear to be conserved in DOXA-1 (purple box and purple arrowheads). C.e.: C. elegans DOXA-1; D.m.: Drosophila mol-PF; M.m.: mouse DUOXA1; H.s.: human DUOXA1 and DUOXA2.
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S9 Fig. Effects of csnk-1(lf) on the expression of gst-4p::GFP transgene dvIs19.
(A) Synchronized L1 animals of the indicated genotypes were observed after 4 hrs on food. For csnk-1(lf) animals, a mixed population of csnk-1(lf)/hT2; dvIs19 and csnk-1(lf); dvIs19 were shown, in which the csnk-1(lf)/hT2 genotype can be identified by pharyngeal GFP signals (arrowheads) expressed from an integrated GFP reporter in hT2 balancer. Animals without pharyngeal GFPs were identified as csnk-1(lf) homozygous (arrows). Pictures were taken with the same exposure time of 100 ms. Total GFP intensity of individual animal was measured using ImageJ and compared with those of dvIs19 controls. Results were based on over 130 individuals for each genotype. Statistics: two-tailed unpaired Student’s t-test. ***: p < 0.001. (B) Effects of csnk-1(lf) on dvIs19 expression with or without skn-1(RNAi). Animals were treated with feeding RNAi for 72 hrs after hatching. For each experiment, seven animals were aligned and measured for total GFP intensities using ImageJ. The average GFP intensity per animal was adjusted to that of dvIs19; ctrl RNAi group. Pictures were taken with the same exposure time of 100 ms. Results were based on three biological replicates. Statistics: two-tailed unpaired Student’s t-test. *: p < 0.05; **: p < 0.01.
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S1 Table. Deleterious genetic variations detected by whole-genome sequencing in the mapped region of the mac397 isolate.
Ratios of mutated sequences are shown in parentheses. A ratio of 1.00 suggests homozygous, and a ratio less than 1.00 suggests heterozygous. A: adults. ND: not determined.
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S2 Table. Effects of csnk-1 mutations or variable feeding RNAis on the Sisi phenotype.
Mammalian orthologs or homologs are indicated in the parentheses.
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S3 Table. Characterization of tsp-15(mac499lf) mutation.
tsp-15(mac500lf) and tsp-15(mac501lf) are knockin genocopies of tsp-15(mac499lf) generated using the CRISPR/Cas9 method.
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S4 Table. PCR primers for generating the listed DNA fragments.
Restriction sites are shown in uppercase.
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S5 Table. PCR primers for generating RNAi plasmids targeting the listed genes.
Restriction sites are shown in uppercase.
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S6 Table. Genomic target sequences for generating tsp-15 knockin and csnk-1 knockout strains using the CRISPR/Cas9 method.
ND: not determined. For tsp-15 repair template, the letter in red was for introducing the missense mutation and letters in blue were for introducing silent mutations.
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S1 Raw Data. Raw data for Figures and Tables.
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Acknowledgments
We thank Dr Xiaochen Wang for providing the XW18042 qxIs722 reporter strain. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).
References
- 1. Schieber M, Chandel NS. ROS function in redox signaling and oxidative stress. Curr Biol. 2014;24: R453–462. pmid:24845678
- 2. Sies H, Berndt C, Jones DP. Oxidative Stress. Annu Rev Biochem. 2017;86: 715–748. pmid:28441057
- 3. Fang FC. Antimicrobial Actions of Reactive Oxygen Species. mBio. 2011;2: e00141–11. pmid:21896680
- 4. Forrester SJ, Kikuchi DS, Hernandes MS, Xu Q, Griendling KK. Reactive Oxygen Species in Metabolic and Inflammatory Signaling. Circulation Research. 2018;122: 877–902. pmid:29700084
- 5. Henkler F, Brinkmann J, Luch A. The Role of Oxidative Stress in Carcinogenesis Induced by Metals and Xenobiotics. Cancers (Basel). 2010;2: 376–396. pmid:24281075
- 6. Blackwell TK, Steinbaugh MJ, Hourihan JM, Ewald CY, Isik M. SKN-1/Nrf, stress responses, and aging in Caenorhabditis elegans. Free Radic Biol Med. 2015;88: 290–301. pmid:26232625
- 7. Ewald CY. Redox Signaling of NADPH Oxidases Regulates Oxidative Stress Responses, Immunity and Aging. Antioxidants (Basel). 2018;7: E130. pmid:30274229
- 8. Walker AK, See R, Batchelder C, Kophengnavong T, Gronniger JT, Shi Y, et al. A conserved transcription motif suggesting functional parallels between Caenorhabditis elegans SKN-1 and Cap’n’Collar-related basic leucine zipper proteins. J Biol Chem. 2000;275: 22166–22171. pmid:10764775
- 9. An JH, Blackwell TK. SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response. Genes Dev. 2003;17: 1882–1893. pmid:12869585
- 10. An JH, Vranas K, Lucke M, Inoue H, Hisamoto N, Matsumoto K, et al. Regulation of the Caenorhabditis elegans oxidative stress defense protein SKN-1 by glycogen synthase kinase-3. Proc Natl Acad Sci U S A. 2005;102: 16275–16280. pmid:16251270
- 11. Inoue H, Hisamoto N, An JH, Oliveira RP, Nishida E, Blackwell TK, et al. The C. elegans p38 MAPK pathway regulates nuclear localization of the transcription factor SKN-1 in oxidative stress response. Genes Dev. 2005;19: 2278–2283. pmid:16166371
- 12. Baird L, Yamamoto M. The Molecular Mechanisms Regulating the KEAP1-NRF2 Pathway. Mol Cell Biol. 2020;40: e00099–20. pmid:32284348
- 13. Choe KP, Przybysz AJ, Strange K. The WD40 repeat protein WDR-23 functions with the CUL4/DDB1 ubiquitin ligase to regulate nuclear abundance and activity of SKN-1 in Caenorhabditis elegans. Mol Cell Biol. 2009;29: 2704–2715. pmid:19273594
- 14. Edens WA, Sharling L, Cheng G, Shapira R, Kinkade JM, Lee T, et al. Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox. J Cell Biol. 2001;154: 879–891. pmid:11514595
- 15. Ewald CY, Hourihan JM, Bland MS, Obieglo C, Katic I, Moronetti Mazzeo LE, et al. NADPH oxidase-mediated redox signaling promotes oxidative stress resistance and longevity through memo-1 in C. elegans. Elife. 2017;6: e19493. pmid:28085666
- 16. Moribe H, Yochem J, Yamada H, Tabuse Y, Fujimoto T, Mekada E. Tetraspanin protein (TSP-15) is required for epidermal integrity in Caenorhabditis elegans. J Cell Sci. 2004;117: 5209–5220. pmid:15454573
- 17. Moribe H, Konakawa R, Koga D, Ushiki T, Nakamura K, Mekada E. Tetraspanin is required for generation of reactive oxygen species by the dual oxidase system in Caenorhabditis elegans. PLoS Genet. 2012;8: e1002957. pmid:23028364
- 18. Thein MC, Winter AD, Stepek G, McCormack G, Stapleton G, Johnstone IL, et al. Combined extracellular matrix cross-linking activity of the peroxidase MLT-7 and the dual oxidase BLI-3 is critical for post-embryonic viability in Caenorhabditis elegans. J Biol Chem. 2009;284: 17549–17563. pmid:19406744
- 19. Xu Z, Luo J, Li Y, Ma L. The BLI-3/TSP-15/DOXA-1 dual oxidase complex is required for iodide toxicity in Caenorhabditis elegans. G3 (Bethesda). 2014;5: 195–203. pmid:25480962
- 20. Xu Z, Hu Y, Deng Y, Chen Y, Hua H, Huang S, et al. WDR-23 and SKN-1/Nrf2 Coordinate with the BLI-3 Dual Oxidase in Response to Iodide-Triggered Oxidative Stress. G3 (Bethesda). 2018;8: 3515–3527. pmid:30166349
- 21. Chávez V, Mohri-Shiomi A, Garsin DA. Ce-Duox1/BLI-3 generates reactive oxygen species as a protective innate immune mechanism in Caenorhabditis elegans. Infect Immun. 2009;77: 4983–4989. pmid:19687201
- 22. Hoeven R van der, McCallum KC, Cruz MR, Garsin DA. Ce-Duox1/BLI-3 generated reactive oxygen species trigger protective SKN-1 activity via p38 MAPK signaling during infection in C. elegans. PLoS Pathog. 2011;7: e1002453. pmid:22216003
- 23. van der Hoeven R, Cruz MR, Chávez V, Garsin DA. Localization of the Dual Oxidase BLI-3 and Characterization of Its NADPH Oxidase Domain during Infection of Caenorhabditis elegans. PLoS One. 2015;10: e0124091. pmid:25909649
- 24. Kim DH, Feinbaum R, Alloing G, Emerson FE, Garsin DA, Inoue H, et al. A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity. Science. 2002;297: 623–626. pmid:12142542
- 25. McCallum KC, Garsin DA. The Role of Reactive Oxygen Species in Modulating the Caenorhabditis elegans Immune Response. PLoS Pathog. 2016;12: e1005923. pmid:27832190
- 26. Tang H, Pang S. Proline Catabolism Modulates Innate Immunity in Caenorhabditis elegans. Cell Rep. 2016;17: 2837–2844. pmid:27974198
- 27. Zou C-G, Tu Q, Niu J, Ji X-L, Zhang K-Q. The DAF-16/FOXO transcription factor functions as a regulator of epidermal innate immunity. PLoS Pathog. 2013;9: e1003660. pmid:24146615
- 28. Sasakura H, Moribe H, Nakano M, Ikemoto K, Takeuchi K, Mori I. Lifespan extension by peroxidase and dual oxidase-mediated ROS signaling through pyrroloquinoline quinone in C. elegans. J Cell Sci. 2017;130: 2631–2643. pmid:28676501
- 29. Kramer-Drauberg M, Liu J-L, Desjardins D, Wang Y, Branicky R, Hekimi S. ROS regulation of RAS and vulva development in Caenorhabditis elegans. PLoS Genet. 2020;16: e1008838. pmid:32544191
- 30. Benedetto A, Au C, Avila DS, Milatovic D, Aschner M. Extracellular dopamine potentiates mn-induced oxidative stress, lifespan reduction, and dopaminergic neurodegeneration in a BLI-3-dependent manner in Caenorhabditis elegans. PLoS Genet. 2010;6: e1001084. pmid:20865164
- 31. Taylor JP, Tse HM. The role of NADPH oxidases in infectious and inflammatory diseases. Redox Biol. 2021;48: 102159. pmid:34627721
- 32. Cheong JK, Virshup DM. Casein kinase 1: Complexity in the family. Int J Biochem Cell Biol. 2011;43: 465–469. pmid:21145983
- 33. Knippschild U, Krüger M, Richter J, Xu P, García-Reyes B, Peifer C, et al. The CK1 Family: Contribution to Cellular Stress Response and Its Role in Carcinogenesis. Front Oncol. 2014;4: 96. pmid:24904820
- 34. Davidson G, Wu W, Shen J, Bilic J, Fenger U, Stannek P, et al. Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction. Nature. 2005;438: 867–872. pmid:16341016
- 35. Gault WJ, Olguin P, Weber U, Mlodzik M. Drosophila CK1-γ, gilgamesh, controls PCP-mediated morphogenesis through regulation of vesicle trafficking. J Cell Biol. 2012;196: 605–621. pmid:22391037
- 36. Li S, Li S, Han Y, Tong C, Wang B, Chen Y, et al. Regulation of Smoothened Phosphorylation and High-Level Hedgehog Signaling Activity by a Plasma Membrane Associated Kinase. PLoS Biol. 2016;14: e1002481. pmid:27280464
- 37. Li S, Tian A, Li S, Han Y, Wang B, Jiang J. Gilgamesh (Gish)/CK1γ regulates tissue homeostasis and aging in adult Drosophila midgut. J Cell Biol. 2020;219: e.201909103. pmid:32328627
- 38. Li D, Ai Y, Guo J, Dong B, Li L, Cai G, et al. Casein kinase 1G2 suppresses necroptosis-promoted testis aging by inhibiting receptor-interacting kinase 3. Elife. 2020;9: e61564. pmid:33206046
- 39. Lee S-Y, Kim H, Li CM, Kang J, Najafov A, Jung M, et al. Casein kinase-1γ1 and 3 stimulate tumor necrosis factor-induced necroptosis through RIPK3. Cell Death Dis. 2019;10: 923. pmid:31801942
- 40. Goto A, Sakai S, Mizuike A, Yamaji T, Hanada K. Compartmentalization of casein kinase 1 γ CSNK1G controls the intracellular trafficking of ceramide. iScience. 2022;25: 104624. pmid:35800758
- 41. Kinoshita-Kikuta E, Utsumi T, Miyazaki A, Tokumoto C, Doi K, Harada H, et al. Protein-N-myristoylation-dependent phosphorylation of serine 13 of tyrosine kinase Lyn by casein kinase 1γ at the Golgi during intracellular protein traffic. Sci Rep. 2020;10: 16273. pmid:33004926
- 42. Gold NB, Li D, Chassevent A, Kaiser FJ, Parenti I, Strom TM, et al. Heterozygous de novo variants in CSNK1G1 are associated with syndromic developmental delay and autism spectrum disorder. Clin Genet. 2020;98: 571–576. pmid:33009664
- 43.
Manning G. Genomic overview of protein kinases. WormBook. 2005; 1–19.
- 44. Panbianco C, Weinkove D, Zanin E, Jones D, Divecha N, Gotta M, et al. A casein kinase 1 and PAR proteins regulate asymmetry of a PIP(2) synthesis enzyme for asymmetric spindle positioning. Dev Cell. 2008;15: 198–208. pmid:18694560
- 45. Redemann S, Pecreaux J, Goehring NW, Khairy K, Stelzer EHK, Hyman AA, et al. Membrane invaginations reveal cortical sites that pull on mitotic spindles in one-cell C. elegans embryos. PLoS One. 2010;5: e12301. pmid:20808841
- 46. Fievet BT, Rodriguez J, Naganathan S, Lee C, Zeiser E, Ishidate T, et al. Systematic genetic interaction screens uncover cell polarity regulators and functional redundancy. Nat Cell Biol. 2013;15: 103–112. pmid:23242217
- 47. Singh D, Pohl C. Coupling of rotational cortical flow, asymmetric midbody positioning, and spindle rotation mediates dorsoventral axis formation in C. elegans. Dev Cell. 2014;28: 253–267. pmid:24525186
- 48. Rodriguez-Garcia R, Chesneau L, Pastezeur S, Roul J, Tramier M, Pécréaux J. The polarity-induced force imbalance in Caenorhabditis elegans embryos is caused by asymmetric binding rates of dynein to the cortex. Mol Biol Cell. 2018;29: 3093–3104. pmid:30332325
- 49. Flynn JR, McNally FJ. A casein kinase 1 prevents expulsion of the oocyte meiotic spindle into a polar body by regulating cortical contractility. Mol Biol Cell. 2017;28: 2410–2419. pmid:28701347
- 50. Bürgi H. Iodine excess. Best Pract Res Clin Endocrinol Metab. 2010;24: 107–115. pmid:20172475
- 51. Corvilain B, Collyn L, van Sande J, Dumont JE. Stimulation by iodide of H2O2 generation in thyroid slices from several species. American Journal of Physiology-Endocrinology and Metabolism. 2000;278: E692–E699. pmid:10751204
- 52. Golstein J, Dumont JE. Cytotoxic effects of iodide on thyroid cells: difference between rat thyroid FRTL-5 cell and primary dog thyrocyte responsiveness. J Endocrinol Invest. 1996;19: 119–126. pmid:8778164
- 53. Many MC, Mestdagh C, van den Hove MF, Denef JF. In vitro study of acute toxic effects of high iodide doses in human thyroid follicles. Endocrinology. 1992;131: 621–630. pmid:1639011
- 54. Vitale M, Di Matola T, D’Ascoli F, Salzano S, Bogazzi F, Fenzi G, et al. Iodide excess induces apoptosis in thyroid cells through a p53-independent mechanism involving oxidative stress. Endocrinology. 2000;141: 598–605. pmid:10650940
- 55. Jiang J. CK1 in Developmental Signaling: Hedgehog and Wnt. Curr Top Dev Biol. 2017;123: 303–329. pmid:28236970
- 56.
Yook K. Complementation. WormBook. 2005 [cited 30 Jul 2022].
- 57. Yook KJ, Proulx SR, Jorgensen EM. Rules of nonallelic noncomplementation at the synapse in Caenorhabditis elegans. Genetics. 2001;158: 209–220. pmid:11333231
- 58. Kusch M, Edgar RS. Genetic studies of unusual loci that affect body shape of the nematode Caenorhabditis elegans and may code for cuticle structural proteins. Genetics. 1986;113: 621–639. pmid:3732788
- 59. Grasberger H, Refetoff S. Identification of the maturation factor for dual oxidase. Evolution of an eukaryotic operon equivalent. J Biol Chem. 2006;281: 18269–18272. pmid:16651268
- 60. Back P, De Vos WH, Depuydt GG, Matthijssens F, Vanfleteren JR, Braeckman BP. Exploring real-time in vivo redox biology of developing and aging Caenorhabditis elegans. Free Radic Biol Med. 2012;52: 850–859. pmid:22226831
- 61. Rena G, Bain J, Elliott M, Cohen P. D4476, a cell-permeant inhibitor of CK1, suppresses the site-specific phosphorylation and nuclear exclusion of FOXO1a. EMBO Rep. 2004;5: 60–65. pmid:14710188
- 62. Link CD, Johnson CJ. Reporter transgenes for study of oxidant stress in Caenorhabditis elegans. Methods Enzymol. 2002;353: 497–505. pmid:12078522
- 63. Agajanian MJ, Potjewyd FM, Bowman BM, Solomon S, LaPak KM, Bhatt DP, et al. Protein proximity networks and functional evaluation of the casein kinase 1 gamma family reveal unique roles for CK1γ3 in WNT signaling. J Biol Chem. 2022;298: 101986. pmid:35487243
- 64. Wicks SR, Yeh RT, Gish WR, Waterston RH, Plasterk RH. Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map. Nature genetics. 2001;28: 160–4. pmid:11381264
- 65. Davis MW, Hammarlund M, Harrach T, Hullett P, Olsen S, Jorgensen EM. Rapid single nucleotide polymorphism mapping in C. elegans. BMC genomics. 2005;6: 118. pmid:16156901
- 66. Farboud B, Meyer BJ. Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design. Genetics. 2015;199: 959–971. pmid:25695951
- 67. Arribere JA, Bell RT, Fu BXH, Artiles KL, Hartman PS, Fire AZ. Efficient marker-free recovery of custom genetic modifications with CRISPR/Cas9 in Caenorhabditis elegans. Genetics. 2014;198: 837–846. pmid:25161212
- 68. Mello CC, Kramer JM, Stinchcomb D, Ambros V. Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 1991;10: 3959–3970. pmid:1935914
- 69. Kamath RS, Fraser AG, Dong Y, Poulin G, Durbin R, Gotta M, et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature. 2003;421: 231–237. pmid:12529635
- 70. Gruber J, Ng LF, Fong S, Wong YT, Koh SA, Chen C-B, et al. Mitochondrial changes in ageing Caenorhabditis elegans—what do we learn from superoxide dismutase knockouts? PLoS One. 2011;6: e19444. pmid:21611128
- 71. Schulz TJ, Zarse K, Voigt A, Urban N, Birringer M, Ristow M. Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress. Cell Metab. 2007;6: 280–293. pmid:17908557
- 72. Chávez V, Mohri-Shiomi A, Maadani A, Vega LA, Garsin DA. Oxidative Stress Enzymes Are Required for DAF-16-Mediated Immunity Due to Generation of Reactive Oxygen Species by Caenorhabditis elegans. Genetics. 2007;176: 1567–1577. pmid:17483415
- 73. Karakuzu O, Cruz MR, Liu Y, Garsin DA. Amplex Red Assay for Measuring Hydrogen Peroxide Production from Caenorhabditis elegans. Bio Protoc. 2019;9: e3409. pmid:32699812