Requirement of the BmANTI1 for cell proliferation of BmN4-SID1 cells.

<p>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 <i>t</i>-test. (*<i>P</i> < 0.05; **<i>P</i> < 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. (**<i>P</i> < 0.01).