Fig 8.
Overexpression of D25SIRT3 deregulates stress-induced gene expressions. (A) D25SIRT3 overexpression inhibits NaAsO2-induced gene expression. The graphs depict qRT-PCR analysis of stress-induced gene expression in control versus D25SIRT3 (D25)-expressing U2OS cells (top and bottom panels) and the catalytically inactive D25SIRT3 (D25HY)-expressing U2OS cells (middle panels). U2OS cells were infected with a retrovirus expressing D25, D25HY, or a control vector (mock). The cells were then treated with 50 μM NaAsO2 (As) for the times (hours) indicated. The mRNA levels of the indicated genes were analyzed by real-time qPCR. The values shown represent the induction ratios (log2) relative to mRNA levels at zero time in each case. One asterisk indicates a P value of <0.05, and the pound sign indicates a P value of <0.1 compared to results from mock cells. (B) Expression levels of D25SIRT3 in NaAsO2-treated U2OS cells. Mock and D25SIRT3 (D25)-expressing U2OS cells as described for panel A were treated with 50 μM NaAsO2 (As) for 2 h and lysed with RIPA buffer. Endogenous SIRT3 and D25 proteins were immunoprecipitated with anti-SIRT3 from equivalent amounts of lysates and blotted with anti-SIRT3. (C) SIRT3 binding correlates with deacetylation of H4K16 at promoter regions of PTK2, GAL3ST1, and OTOG genes. The bar graphs depict ChIP-qPCR of H4K16 acetylation at SIRT3 binding regions in untreated U2OS cells and mock-infected (As/mock), D25SIRT3-expressing (As/D25), and D25HY-expressing (As/D25HY) NaAsO2-treated U2OS cells. The results are normalized to unmodified H3-ChIP data.