Table 1.
A list of arthropod species examined.
Fig 1.
Amino acid sequences of insect ANT.
Sequence alignment of the ANT proteins. The Genetyx software and CLUSTAL-W program were used for arrangement. Three homologous repeated domains are shown with arrows. The loops of the matrix side and intermembrane space side of mitochondria are indicated as mat-loop and int-loop on dashed line, respectively. Transmembrane segments located on the inner membrane of mitochondria are highlighted in light gray. Conserved residues marked by arrowheads represent the PX(D/E)XX(K/R) sequence. The RRRMMM signature is observed in all the ANT proteins aligned. HsANTI1 (NP_001142), HsANTI2 (NP_001143), HsANTI3 (NP_001627), HsANTI4 (NP_112581), DpANTI1 (EHJ74594), DpANTI2 (EHJ77067), DmANT1 (NP_727448), DmANT2 (NP_511110), TcANTI1 (XP_968561), TcANTI2 (XP_973257), and TcANTI3 (EFA07039) were retrieved from the NCBI/GenBank database. Abbreviations: Bm, Bombyx mori; Dp, Danaus plexippus; Px, Plutella xylostella; Ah, Adoxophyes honmai; Dm, Drosophila melanogaster; Tc, Tribolium castaneum; Nl, Nilaparvata lugens; Bt, Bemisia tabaci; Sr, Stenotus rubrovittatus; Sg, Schistocerca gregaria; Hs, Homo sapience; Tu, Tetranychus urticae.
Fig 2.
Phylogenetic tree of ANT proteins.
Phylogenetic analysis of the ANT proteins. The neighbor-joining tree was generated in Genetyx software with the multiple sequence alignment. The numbers at the nodes denote bootstrap values (%). Bootstrap values <50% are not indicated. Sequences of mouse and anole lizard ANT genes, MsANT1 (NP_031476), MsANT2 (NP_031477), MsANT4 (NP_848473), AcANT1 (ENSACAP00000002268), AcANT2 (ENSACAP00000012600), AcANT3 (ENSACAP00000005895), and AcANT4 (ENSACAP00000011672) were retrieved from the NCBI/GenBank and Ensembl databases. Abbreviations: Mm, Mus musculus; Ac, Anolis carolinensis.
Fig 3.
Expression profiles of BmANTI1 and BmANTI2 genes.
The developmental and tissue-specific expression of BmANT genes. (A) Developmental expression profiles of BmANTI1 and BmANTI2 genes. Whole body RNA samples were extracted from embryo to adult, and subjected to semi-quantitative reverse transcription (semi-qRT)-PCR analysis. Total RNA from BmN4-SID1 cells was also included. Amplifications of GAPDH cDNA were used as an internal control. Individuals from 4th instar larvae to adult were divided into female and male. (B) Tissue expression profiles of BmANTI1 and BmANTI2 genes. The fat body (FB), gut (GU), testis (TE), Malpighian tubules (MT), and silk gland (SG) were retrieved from individuals at day 5 of 5th instar male larvae, and their total RNAs were subjected to semi-qRT-PCR analysis. (C) Testis expression profiles of BmANTI1 and BmANTI2 genes at the 4th and 5th instar larvae. The expression levels of BmANTs in the testis were measured by real-time PCR. Silkworms started to spin silk between day 7 and 8 of the 5th instar larvae. Relative expression levels against the Bmrp49 gene in the testis are shown. Error bars represent the SD values of the means of triplicates.
Fig 4.
Tissue expression profiles of SgANTI1 and SgANTI2 genes.
Brain (BR), testis (TE), ovary (OV), thoracic integument (IN), fat body (FB), and muscle (MS) were retrieved from desert locusts at day 1 of 3th instar nymphs, and their total RNAs were subjected to semi-qRT-PCR analysis. Amplifications of GAPDH cDNA were used as an internal control.
Fig 5.
Requirement of the BmANTI1 for cell proliferation of BmN4-SID1 cells.
BmANTI1 is required for cell proliferation in BmN4-SID1 cells. (A) Double-stranded (ds)RNA mediated gene silencing of BmANTI1 mRNA. To confirm the knockdown efficiency of dsRNAs on BmANTI1, semi-qRT-PCR analysis was performed. Lines on a schematic diagram of BmANTI1 represent three relative positions of dsRNA-targeted regions. The length of dsRNAs is shown in parentheses. PCR amplifications were carried out on cDNAs obtained from BmN4-SID1 cells soaked in VENUS (green fluorescent protein variant), BmANTI1-a, BmANTI1-b, and BmANTI1-UTR dsRNAs for 3 days. VENUS was used as a negative control that is unrelated sequence to silkworm genome. Transcript levels of the BmANTI1 gene were quantitated by IMAGEJ software. Amplifications of GAPDH cDNA were used as an internal control. (B) BmANTI1 depletion inhibits cell proliferation of BmN4-SID1 cells. Cell proliferations of BmANTI1 knockdown cells were assessed after 4, 7, 9 and 14 days culture. The data represent the percent growth as compared with dsRNA-untreated cells. Data are from one of four independent experiments with similar results. Error bars represent the SD values of the means of triplicate wells. (C) Mitochondrial localization of GFP-fused BmANTI1 and BmANTI2 in BmN4-SID1 cells. GFP alone or each GFP-fused BmANTI1 and BmANTI2 (green) was transiently expressed in BmN4-SID1 cells, and the subcellular localizations of these constructs were observed using confocal microscope. Mitochondria in cells were labeled with MitoTracher (red). (D) BmANTI1 and BmANTI2 stably expressed in BmN4-SID1 cells were efficiently transported to mitochondria. FLAG-tagged BmANTI1 or BmANTI2 was stably expressed in BmN4-SID1 cells, and the cells were fractionated into cytosolic (Cyto) and mitochondrial (Mito) compartments. Whole-cell lysates (WCL) were included to confirm protein expression. Each fraction was immunoblotted with anti-FLAG M2 and anti-α-tubulin antibodies. (E) Decreased cell proliferation of BmN4-SID1 cells depleted of endogenous BmANTI1 can be restored by expression of FLAG-tagged BmANTI1. Endogenous BmANTI1 was silenced by dsRNAs in BmN4-SID1 cells stably expressing FLAG-tagged BmANTI1. After 7 days incubation, the cells were subjected to cell proliferation experiment as described in (B). Data are from one of four independent experiments with similar results. Error bars represent the SD values of the means of triplicate wells. Differences in cell proliferation rate between BmANTI1-knockdown cells and cells soaked in VENUS dsRNA were evaluated with a two-tailed Student’s t-test. (*P < 0.05; **P < 0.01) (F) Expression of FLAG-tagged BmANTI2 fails to restore decreased cell proliferation of BmN4-SID1 cells depleted of endogenous BmANTI1. Using BmN4-SID1 cells stably expressing FLAG-tagged BmANTI2, cell proliferation assay was performed as described in (E). Data are from one of four independent experiments with similar results. Error bars represent the SD values of the means of triplicate wells. (**P < 0.01).
Fig 6.
Cell proliferation of BmN4-SID1 cells expressing insect ANTs under knockdown of the endogenous BmANTI1.
Decreased cell proliferation of BmN4-SID1 cells silenced the endogenous BmANTI1 gene can be restored by expression of PxANTI1. Using BmN4-SID1 cells stably expressing FLAG-tagged PxANTI1, PxANTI2, PxANTI3, SgANTI1, SgANTI2, NlANTI1, NlANTI2, DmANT1, or TuANT, cell proliferation assay was performed as described in Fig. 5E. Data are from one of three independent experiments with similar results. Error bars represent the SD values of the means of triplicate wells. Differences in cell proliferation rate between BmANTI1-knockdown cells and cells soaked in VENUS dsRNA were evaluated with a two-tailed Student’s t-test. (*P < 0.05; **P < 0.01).
Table 2.
Phenotypes of Tribolium larvae depleted for TcANTI1 and TcANTI2 genes.