ins-7 Gene Expression Is Partially Regulated by the DAF-16/IIS Signaling Pathway in Caenorhabditis elegans under Celecoxib Intervention

DAF-16 target genes are employed as reporters of the insulin/IGF-1 like signal pathway (IIS), and this is notably true when Caenorhabditis elegans (C. elegans) is used to study the action of anti-aging compounds on IIS activity. However, some of these genes may not be specific to DAF-16, even if their expression levels are altered when DAF-16 is activated. Celecoxib was reported to extend the lifespan of C. elegans through activation of DAF-16. Our results confirmed the function of celecoxib on aging; however, we found that the expression of ins-7, a DAF-16 target gene, was abnormally regulated by celecoxib. ins-7 plays an important role in regulating aging, and its expression is suppressed in C. elegans when DAF-16 is activated. However, we found that celecoxib upregulated the expression of ins-7 in contrast to its role in DAF-16 activation. Our subsequent analysis indicated that the expression level of ins-7 in C. elegans was negatively regulated by DAF-16 activity. Additionally, its expression was also positively regulated by DAF-16-independent mechanisms, at least following external pharmacological intervention. Our study suggests that ins-7 is not a specific target gene of DAF-16, and should not be chosen as a reporter for IIS activity. This conclusion is important in the study of INSs on aging in C. elegans, especially under the circumstance of drug intervention.


Introduction
From invertebrates to vertebrates, the insulin/IGF-1-like signaling pathway (IIS) is evolutionary conservation and plays an important role in regulating animal development and pathology [1][2][3][4]. In vertebrates, phosphatidylinositol 3-kinase (PI3K) is activated to generate phosphatidylinositol 3, 4, 5-triphosphate (PIP3) when the cell membrane-localized IIS receptor is stimulated by insulin or IGF-1. Subsequently, protein kinase B (PKB) is localized to the cell membrane by PIP3 and activated by PDK-1/2 to phosphorylate FOXO transcription factors [5]. In C. elegans, insulin-like proteins activate the PI3K homolog AGE-1 through the IIS receptor DAF-2, ultimately directing the AKT-1/2 and SGK-1 kinases to phosphorylate the FOXO protein DAF-16 with phosphorylated DAF-16 accumulating in the nuclei [6]. The IIS signaling pathway has been shown to regulate dauer formation, improve heat and oxidative stress resistance, extend lifespan, and delay the onset of many age-related diseases in C. elegans [7][8][9][10][11]. Decreased IIS signal transduction releases the FOXO protein DAF-16 into the nucleus to regulate the expression levels of many genes.
Many DAF-16 target genes have been reported to control aging in C. elegans [6]. ins-7, one of about 40 insulin-like genes that have been identified in the C. elegans genome [12,13], is downregulated in daf-2 loss-of-function mutants and upregulated in daf-16 null mutants [6]. Many INS proteins are present in the nervous system of C. elegans, and killing these sensory neurons or inhibiting their functions triggers DAF-16 nuclear localization [14][15][16]. However, to the best of our knowledge, the functions of the 40 INS proteins in C. elegans have not been well characterized. Wild-type (N2) worms treated with ins-7 RNAi have an extended lifespan, and ins-7 RNAi also results in increased nuclear accumulation of DAF-16::GFP in intestine cells [16]. Murphy et al. reported that DAF-2 and DAF-16 regulate ins-7 expression in the intestine and that downregulation of ins-7 expression in the intestine lowers INS-7 levels in other tissues, which in turn triggers DAF-16 activity in the muscles and hypodermis [16]. It has also been reported that ins-7 expression in URX neurons can regulate aversive olfactory learning by antagonizing DAF-2, the receptor of the IIS signaling pathway [17]. Together, these studies reveal that the expression of ins-7 is regulated by the DAF-16/IIS signal pathway and that ins-7 can also influence the function of IIS by means of its feedback regulation of DAF-2 [16].
C. elegans has been widely used as a model to screen for antiaging compounds and to study the mechanisms of aging. Many compounds have been reported to have effects on the aging of C. elegans [18]. A well-characterized compound with an influence on aging would be a valuable tool/drug to study the mechanisms of aging and investigate how endogenous systems change with aging.
We used celecoxib as our experimental compound due to its established effect on the aging of C. elegans. Celecoxib extends the lifespan of C. elegans by activating DAF-16 and subsequently alters the expression of DAF-16 target genes [19]. According to our study, celecoxib extended the lifespan of C. elegans via DAF-16 activity and regulated the expression levels of DAF-16 target genes: scl-20, K09F6.6, sod-3, and ins-7. Interestingly, the expression of ins-7, which is typically downregulated when DAF-16 is activated, was significantly upregulated in celecoxib-treated N2 worms. Our results also showed that ins-7 expression was upregulated even in daf-16 and daf-2;daf-16 double mutants following treatment with celecoxib. Consequently, our work indicated that the expression of ins-7 was negatively regulated by DAF-16 activity, but that it could also be positively regulated by other DAF-16-independent mechanisms, at least under external pharmacological intervention. We confirmed that ins-7 was not a specific target gene of DAF-16 and also expanded our understanding of the regulation of ins-7 expression, further supporting its importance in the study of aging and the function of insulin-like genes.

Lifespan Tests
Lifespan assays were performed on nematode growth media (NGM) plates. Synchronized L4 or young adult worms were transferred to 5 NGM plates (6 cm diameter). A total of 20-30 worms were transferred on to each NGM plate containing 40 mM FUDR. All lifespan assays were performed at 20uC. The worms were scored as live, dead, or lost and transferred to new NGM plates every other day. Worms that failed to display touchprovoked movement were scored as dead. For celecoxib treatment, 500 mL of 10 mM celecoxib (Sigma-Aldrich, St. Louis, MO, USA) was added on to the surface of NGM media 8 hours before transferring worms onto the plates. Celecoxib was dissolved in DMSO for storage and diluted in water to work concentration before use. After adding the compound to the NGM plates, the final DMSO concentration was 0.1%. Control plates contained 0.1% DMSO too. All lifespan tests were repeated three times. Survival curves and statistical analyses were carried out using SPSS software (SPSS, Chicago, IL, USA). P values were calculated using the log-rank test.

Quantitative Real-time PCR Experiments
Young adult N2 worms were transferred on to control plates or those containing 10 mM celecoxib, cultured at 20uC, and harvested after 24 hours, 7 days, or 14 days. Total RNA was isolated from about 2000 worms using an RNA isolation kit (RNAiso Plus; TaKaRa Bio, Shiga, Japan). A total of 5 mg RNA was used to prepare cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The real-time PCR reactions were performed using Power SYBR Green PCR Master Mix and an ABI 7500 system (Applied Biosystems, Foster City, CA, USA). The relative expression levels of genes were determined using the 2 2DDCT method and normalized to cdc-42 and act-1 [21][22][23]. Primer sequences used are summarized in supplement information (Table S6).

RNAi Knockdown of Gene Expression
An RNAi bacterial strain (HT115) expressing a double-stranded daf-16 RNA (vector, L4440) was cultured and used to inactivate daf-16 function. The identity of the clones was confirmed by sequencing. Eggs from HT1702 or UL1735 worms were transferred to fresh NGM plates containing daf-16 RNAi bacteria and allowed to grow at 15uC for 3 days. Larva stage 4 HT1702 or UL1735 worms were then transferred to the same RNAi NGM plates in the presence or absence of 10 mM celecoxib and cultured at 20uC for 24 hours. The RNAi NGM plates contained 1 mM isopropyl-B-D-thiogalactopyranoside (IPTG) for the induction of double stranded RNA [19,24].

GFP Fluorescent Analysis
After the HT1702 worms were cultured on RNAi NGM plates, about 20 control or celecoxib-treated worms were mounted on 1% agarose slides (10-20 per slide). The induction of INS-7::GFP in the body of worms was assayed using a Ti fluorescent microscope (Nikon, Tokyo, Japan). The average GFP intensity was calculated using the Metamorph software package (Molecular Devices, Sunnyvale, CA, USA).
GFP nuclear localization was analyzed as described previously [19]. Young adult worms (TJ356, UL1735 or UL1735;daf-16 RNAi) were seeded on to either control or celecoxib plates and cultured at 20uC for 24 hours. GFP expression was then analyzed using a Ti fluorescent microscope (Nikon, Tokyo, Japan) at 106or 406magnification. Worms were scored for the presence or absence of GFP accumulation. Animals were scored as having nuclear GFP if more than one intestinal nuclei contained GFP.

Celecoxib Extends the Lifespan of C. elegans via DAF-16
We used celecoxib to study the pharmacological intervention of aging on C. elegans. According to previous studies, celecoxib extends the lifespan of C. elegans by decreasing IIS signal transduction, which serves to activate DAF-16 [19]. We treated young adult N2 worms with 10 mM celecoxib and found that the mean lifespan of N2 worms was extended by up to 18% at 20uC (mean increase of three independent experiments) ( Figure 1A, Table 1). We also treated daf-16 null-mutant worms with 10 mM celecoxib. Our results indicated that celecoxib had no effect on the lifespan of daf-16 (mu86) I worms ( Figure 1B, Table 1). These results were consistent with previous studies indicating that celecoxib extended the lifespan of C. elegans by way of DAF-16 activity.
The Expression of ins-7 in N2 Worms is Positively Regulated by Celecoxib sod-3 has been reported to be a specific target gene of DAF-16, and its expression level is upregulated when DAF-16 is activated [25][26][27][28]. ins-7 has been reported to be downregulated in daf-2 mutants or when DAF-16 is activated [6]. We used daf-2 (e1370) III to test the expression of sod-3 and ins-7. According to our results, the expression profiles of sod-3 and ins-7 were consistent with these previous studies ( Figure 2A, Table S1). In order to confirm that celecoxib extends the lifespan of C. elegans by activating DAF-16, we tested the expression of DAF-16 target genes in celecoxib-treated worms. About 2000 young adult N2 ins-7 Partially Regulated by DAF-16 worms were cultured on NGM plates containing 10 mM celecoxib or control plates for 24 hours, 7 days, or 14 days. Total RNA was then isolated from these worms. The relative expression of sod-3 and ins-7 were calculated using real-time PCR. Our results showed that sod-3 expression was significantly upregulated in celecoxibtreated N2 worms ( Figure 2B, Table S2), this result was consistent with previous reports [19]. Celecoxib extends the lifespan of C. elegans by decreasing IIS signal transduction to activate DAF-16, so  (2) worms (B) were cultured on NGM plates in the presence or absence of 10 mM celecoxib. Mean lifespan of N2 worms treated with 10 mM celecoxib was significantly longer than that of control worms. Celecoxib had no effect on aging of daf-16 (2) mutants. One representative result from three independent experiments is presented. Survival curves were generated using SPSS software (SPSS, Chicago, IL, USA). P values and mean lifespans were calculated using a log-rank test ( celecoxib-treated N2 worms should have lower the expression levels of ins-7 according to previous reports [6,16]. However, our results indicated that ins-7 was significantly upregulated following celecoxib treatment ( Figure 2B, Table S2). Furthermore, we also observed that the mean lifespan of ins-7 mutant worms treated with celecoxib was increased by up to 21.92% (mean increase of three independent experiments; Table 1), a greater increase when compared to celecoxib-treatment alone. This discrepancy led us to speculate that there were other mechanisms regulating ins-7 expression in C. elegans following celecoxib intervention. IIS regulates two classes of genes through DAF-16. Class I genes are upregulated when the IIS signal is reduced or DAF-16 is translocated to the nuclei. Class II genes display the opposite profile [6]. There are two elements in the promoters of DAF-16 targets: a DAF-16 binding element (DBE) and a DAF-16 associated element (DAE). According to the previous studies, both DAE and DBE are present in the promoters of both gene classes and DAF-16 directly regulates the expression of class I genes through DBE [29,30]. The class II genes may be regulated by other co-factors in addition to DAF-16. sod-3 is a class I gene and only contains a DBE. ins-7 is a class II gene and only contains a DAE. Our results suggested that ins-7 was positively regulated by celecoxib, so we also tested the effects of celecoxib on the expression of scl-20 (class I, DAE only) and K09F6.6 (class II, DBE only). We found that expression of scl-20 was upregulated and that of K09F6.6 was downregulated following treatment with celecoxib in N2 worms ( Figure 2B, Table S2). These expression profiles were consistent with the altered activity of DAF-16 following celecoxib treatment. Together, these results led us to speculate that ins-7 was not directly regulated by DAF-16. Figure 2. Celecoxib regulates DAF-16 target gene expression. scl-20, K09F6.6, ins-7, and sod-3 expression profiles in daf-2 (e1370) III mutants compared to N2 worms (A). ins-7 was significantly upregulated in N2 worms treated with 10 mM celecoxib (B). Celecoxib was previously reported to regulate DAF-16 target genes by decreasing IIS signal transduction activity. ins-7 was reported to be a target gene of DAF-16 and downregulated when IIS signal activity was decreased. These results indicated ins-7 was uncharacteristically upregulated in celecoxib-treated N2 worms. The results of at least three independent experiments are presented. Data are averages of real-time PCR results 6 Standard Deviation (SD). Error bars represent SD. P values were calculated by using a T-test, ***P,0.001. Also see Table S2. doi:10.1371/journal.pone.0100320.g002 ins-7 Partially Regulated by DAF-16 ins-7 Expression Partially Depends on DAF-16/IIS Activity INS-7, an insulin-like protein, is reported to be a possible DAF-2 agonist and negatively regulated by DAF-16 activity [6]. It has also been reported that ins-7 might regulate DAF-16 activity by way of feedback regulation of DAF-2 [16]. We tested ins-7 expression levels in daf-2(e1368) III, daf-2(e1370) III, and daf-16 (mu86) I mutants. The results were consistent with previous reports and indicated that ins-7 expression is downregulated in daf-2 mutants and upregulated in daf-16 mutants (Figure 2A and 3A, Table S1).
According to our results, ins-7 was upregulated approximately 4-fold following celecoxib treatment of N2 worms ( Figure 2B, Table S2), which was in contrast to its expression pattern when DAF-16 was activated. If ins-7 expression is specifically related to DAF-16 activity, its expression in celecoxib-treated daf-16 (2) mutant worms should demonstrate no significant change compared to controls. However, we found that the expression of ins-7 was significantly upregulated in daf-16 (2) worms when treated with celecoxib ( Figure 3B, Table S3). This result suggested that ins-7 expression was not specific to DAF-16. When we treated daf-2 mutant worms with celecoxib, we found ins-7 expression was downregulated about 50% compared to controls ( Figure 3C and D, Table S3). This result was consistent with the idea that celecoxib negatively regulates IIS signal transduction by decreasing PDK-1 activity, as the expression of ins-7 was downregulated when IIS signal transduction was decreased [16,19]. The above results led us to speculate that ins-7 gene expression in N2 worms could be regulated by some other mechanisms in addition to being regulated by IIS and DAF-16. In order to test this hypothesis, we analyzed ins-7 expression in daf-2; daf-16 double mutants treated with celecoxib. The resulting expression level of ins-7 was upregulated by up to 70% ( Figure 3E, Table S3). This result was consistent with the idea that ins-7 expression could be positively regulated by other pathways independent or partially independent of IIS activity.

ins-7 is Partially Regulated by PQM-1 Activity
It has been reported that the transcription factor PQM-1 is strongly dependent on IIS activity and directly controls class II gene expression via binding to DAE motifs [6,30]. We tested the effect of celecoxib on pqm-1 expression. Our results showed that celecoxib had no effect on the expression level of pqm-1 in N2 worms. IIS regulation of PQM-1 is modulated primarily through posttranslational regulation of its subcellular localization [30]. We found that 10 mM celecoxib could enhance DAF-16 nuclear translocation ( Figure 4A and B, Table S4). This result is consist with a previous report [19], but we found that celecoxib had no obvious function on PQM-1::GFP nuclear translocation ( Figure 4A and C, Table S4). The nuclear localization of PQM-1::GFP is opposite of that of DAF-16::GFP [30], and when we treated PQM-1::GFP worms with daf-16 RNAi we found that the PQM-1::GFP nuclear location was further enhanced by celecoxib ( Figure 4A and D, Table S4). According to this result, we speculated that ins-7 was partially regulated by PQM-1 in celecoxib-treated N2 worms. Subsequently, we found that the expression level of ins-7 was significantly upregulated following celecoxib treatment in pqm-1(2) worms ( Figure 4E, Table S2), but the degree of upregulation was lower than that of N2 worms. And, pqm-1 expression level in daf-16 (2) worms was not significantly changed by10 mM celecoxib (Table S2). These results suggested that celecoxib positively regulated ins-7 and that this was partially dependent on PQM-1.

ins-7 Expression in the Intestine is Upregulated by Celecoxib in daf-16 (RNAi) Worms
To further confirm our speculation, we performed RNAi knockdown of daf-16 in an ins-7::gfp strain to test whether ins-7 expression was regulated in daf-16 null worms following celecoxib treatment. Eggs isolated from synchronous HT1702 worms were cultured on fresh RNAi plates containing bacterial strain HT115 expressing a double-stranded daf-16 RNA and allowed to grow at 15uC; 3 days later, L4 stage nematodes were transferred to new plates seeded with the same bacteria in the presence or absence of 10 mM celecoxib and switched to 20uC for 24 hours. We used a fluorescent microscope to analysis ins-7::gfp expression. Our results were consistent with the previous reports indicating that ins-7 could be expressed both in neurons and intestine cells ( Figure 5A, Table S5) and indicated that the relative intensity of INS-7::GFP in the intestines of celecoxib-treated daf-16 (RNAi) worms was significantly higher than in the controls ( Figure 5A and B, Table  S5). This test confirmed our speculation that ins-7 expression was positively regulated by other mechanisms independent of the DAF-16/IIS signal pathway.

Discussion
A number of compounds and plant extracts of have been reported to influence aging in C. elegans [18], and pharmacological approaches to investigate aging are ongoing. A well-studied compound can be used to improve health and could also be very helpful for investigating the mechanisms of aging. Some compounds have been reported to extend the lifespan of C. elegans by regulating DAF-16 in the IIS signaling pathway. Celecoxib is a well-studied molecule that extends the lifespan of C. elegans by decreasing IIS signal transduction required to activate DAF-16 [19]. We noticed that this compound could not extend the lifespan of daf-2 mutant worms, so we speculated that celecoxib could also influence DAF-2 ligands in C. elegans. In addition, when we tested the expression of DAF-16 target genes in celecoxib-treated worms and found that ins-7 was abnormally upregulated by celecoxib, which conflicted with the idea that ins-7 was downregulated when DAF-16 was activated or IIS signal transduction was decreased. This discrepancy led us to investigate the expression of ins-7 in C. elegans.
In C. elegans, most insulin-like genes are expressed in the nervous system [12]. There are reports that ins-7 is specifically expressed in nervous cells, but recently Murphy et al. [16] and Chen et al. [17] have reported that ins-7::gfp is also expressed in the intestine cells of C. elegans. Our results confirmed that ins-7 is expressed in the intestine using the transgenic strain HT1702. ins-7 expression was reported to be regulated by DAF-16 and DAF-2 in the intestine and to be downregulated in daf-2(2) worms [6]. We found that ins-7 expression levels were upregulated in daf-16 (2) worms when treated with celecoxib and that the ins-7::gfp expression in the intestines of celecoxib-treated daf-16 (RNAi) worms was higher than in controls. According to these results, we speculated that ins-7 expression was likely upregulated by other DAF-16-independent mechanisms. Additionally, we found that ins-7 expression levels were upregulated to a lower but significant level in daf-2;daf-16 double mutants when compared to daf-16 mutants when both strains were treated with celecoxib. We speculated that the celecoxib upregulated ins-7 expression levels were partially dependent on DAF-2 or that ins-7 could regulate IIS activity via its feedback regulation of DAF-2 [16]. Our results also indicated that the ins-7 gene expression in celecoxib-treated daf-2 (2) worms was significantly downregulated compared to daf-2 (2) control worms. This result was consistent with previous reports indicating ins-7 Partially Regulated by DAF-16 that celecoxib negatively regulates IIS activity by decreasing PDK-1 activity [19]. Because PDK-1 is a downstream kinase in the IIS signaling pathway [31], ins-7 expression levels could be downregulated by DAF-16 in daf-2 (2) worms when treated with celecoxib. However, according to our results, ins-7 expression was signifi-cantly upregulated in celecoxib-treated N2 worms and downregulated in celecoxib-treated daf-2 (2) worms. Thus, the relationship between ins-7 gene expression and DAF-2 function appeared to be interdependent (at least under celecoxib intervention) or, alternatively, the effect of celecoxib on ins-7 expression could be altered Figure 3. ins-7 expression patterns in daf-2 and daf-16 mutant worms with or without celecoxib treatment. ins-7 was downregulated in daf-2 (e1370) III mutants and upregulated in daf-16 (mu86) I mutants and daf-2 (e1370) III; daf-16 (mu86) I double mutants (A). ins-7 was significantly upregulated in daf-16 (mu86) I worms treated with 10 mM celecoxib (B). ins-7 was significantly downregulated in daf-2 (e1370) III and daf-2 (e1368) III worms treated with 10 mM celecoxib (C, D). ins-7 expression was still significantly upregulated in daf-2(e1370) III; daf-16 (mu86) I double mutants treated with 10 mM celecoxib (E). The results of at least three independent experiments are presented. Data are averages of real-time PCR results 6 SD. Error bars represent SD. P values were calculated using a T-test, *P,0.05; **P,0.01; ***P,0.001. Also see Table S1 and S3. doi:10.1371/journal.pone.0100320.g003 ins-7 Partially Regulated by DAF-16 by DAF-16 function in daf-2 (2) worms. However, these possibilities have yet to be confirmed. As such, our analysis showed that the expression level of ins-7 in the intestine of C. elegans was negatively regulated by DAF-16 activity and could also be concurrently regulated in a positive manner by other mechanisms, at least following external pharmacological intervention.
DAF-16 directly regulates class I genes, but it may require some co-factors to regulate class II target genes. Tepper et al. reported that PQM-1 activates class II target gene transcription via DAE motifs at the posttranslational level [30]. Our results showed that celecoxib could also upregulate ins-7 expression in pqm-1 mutants, and we found that celecoxib could not enhance the PQM-1::GFP nuclear translocation in N2 worms. When we treated UL1735; daf-16 (RNAi) worms with celecoxib, we found that PQM-1::GFP nuclear localization was further increased. We speculated that there might be two possible reasons for this: first, the effect of celecoxib on PQM-1::GFP nuclear localization might be negated by a role for DAF-16 in the localization of PQM-1. Alternatively, the effect of celecoxib on PQM-1::GFP nuclear localization in otherwise wild-type worms might simply be below the threshold of our detection methods. However, pqm-1 mutants did not completely repress the effect of celecoxib on ins-7 expression, and this was also consisted with our speculation that ins-7 was partially regulated by IIS and DAF-16.
In intestine cells, INS-7 can function as an agonist of DAF-2 and is negatively regulated by DAF-16 activity [6,16]. ins-7 (RNAi) worms have a longer lifespan than wild-type worms, and ins-7 expression in intestine cells of N2 worms is regulated by DAF-16 and the IIS signaling pathway [16]. We found that the expression level of ins-7 in intestine cells of celecoxib-treated daf-16 (RNAi) worms was higher than in controls, indicating that ins-7 expression could also be regulated by other mechanisms. Additionally, we  ). However, PQM-1::GFP nuclear localization was not significantly changed following celecoxib treatment in N2 worms (A, D). ins-7 was upregulated following celecoxib treatment in pqm-1 mutants (E). Error bars represent SD. P values were calculated using a T-test, *P,0.05; **P,0.01; ***P,0.001. Also see Table S2  found that the mean lifespan of ins-7 mutant worms treated with celecoxib could be further increased compared to celecoxib alone. As such, we speculated that the lifespan-decreasing function of high ins-7 expression levels in celecoxib-treated N2 worms might be suppressed or neutralized by the interaction of multiple mechanisms and the function of the IIS signaling pathway. As to whether or not celecoxib also regulates other anti-aging mechanisms in addition to the IIS signaling pathway has not been confirmed. However, the function of ins-7 in nerve cells conflicts with that proposed for intestine cells. Chen et al. [17] reported that INS-7 in nerve cells can function as an antagonist of DAF-2 to activate DAF-16 activity. Recently, Ritter et al. [32] used 40 C. elegans insulin genes to study the balance between specificity and redundancy. They reported that no single insulin mutation or deletion completely recapitulated the phenotypes associated with perturbation of daf-2 and that no single insulin gene was the sole agonist or antagonist for coordinating dauer formation via the DAF-2 receptor. As such, we feel that a more detailed investigation into the expression and function of insulin-like peptides in C. elegans is still required.
There are about 40 INS peptides that are reported to be ligands of DAF-2 in C. elegans, but the functions of many of these are not clear. Some studies have reported that ins-1, ins-4, ins-5, ins-17, ins-18, ins-28, and ins-30 might have a function in the aging of C. elegans [33][34][35][36]. As such, we also tested if the expression of these genes could be influenced by celecoxib. We found there were no significant changes in ins-1, ins-4, ins-5, ins-17, ins-18, ins-28, or ins-30 expression levels in celecoxib-treated N2 worms (data not show). There is, however, speculation that 2 or more INS peptides might combine to form a complex and that different INS peptides might combine to regulate aging, disease, and development of C. elegans. These different sets of INS peptides could have different functions in the recognition and response to many environmental stimuli [32]. Future studies will be needed to illuminate the expression patterns and functions of INS peptides in worms when they are under environmental or pharmacological stimuli or at different normal developmental stages. Our study confirmed that celecoxib extended the lifespan of C. elegans by decreasing IIS signaling and indicated that ins-7 expression in N2 worms treated with celecoxib was abnormally upregulated. Together, our results suggested that the expression level of ins-7, a DAF-16 target gene, was negatively regulated by decreased IIS signal transduction and was positively regulated by other mechanisms when IIS signal transduction was decreased by pharmacological intervention. Accordingly, we suggest that ins-7 is not an ideal reporter gene for DAF-16 activity and that the functions and regulated expression of INS peptides still require detailed study. Table S1 The expression profiles of DAF-16 target genes: scl-20, K09F6.6, sod-3, and ins-7 in daf-2 and daf-16 mutants. Young adult day 1 worms were used in these tests. The relative expression levels of the genes were determined using the 2 2DDCT method and normalized to cdc-42 and act-1.

Supporting Information
(DOCX) Table S2 The expression profiles of DAF-16 target genes when N2, daf-16 (2) or pqm-1(2) worms were treated with 10 mM celecoxib. The relative expression levels of the genes were determined using the 2 2DDCT method and normalized to cdc-42 and act-1.

(DOCX)
Table S3 ins-7 expression levels in daf-2 and daf-16 mutants when treated with celecoxib. Young adult day 1 worms were transferred on to celecoxib-contained plates, and cultured for 24 hours at 20uC. The relative expression levels of the genes were determined using the 2 2DDCT method and normalized to cdc-42 and act-1.

(DOCX)
Table S4 Nuclear translocation of DAF-16::GFP and PQM-1::GFP. %: the percentage of worms demonstrating GFP nuclear localization. N: the number of worms that were analyzed in one experiment. n: the number of worms that demonstrated GFP nuclear localization. (DOCX) Table S5 Average INS-7::GFP intensity in N2 worms treated with celecoxib is higher than that of controls. The induction of the INS-7::GFP in the body of worms was assayed based on fluorescence using a Ti microscope (Nikon, Tokyo, Japan). The average GFP intensity was calculated by using  Figure B shows the mean average flourencent intensity of about 20 worms. Data are presented as mean 6 SD. Error bars represent SD. P values were calculated using a T-test. ***P,0.001. Also see Table S5.