Figure 7
Hypoxia-induced apoptosis is CHOP dependent. Min6 cells stably expressing a negative control shRNA (C) or shRNAs targeting CHOP (CHOP#1, CHOP#2) were generated and analyzed. (a) Protein levels of CHOP in stable cell lines. HIF-1α and CHOP protein levels were assessed by immunoblot analysis using anti-HIF-1α, anti-CHOP. (b) Cleavage of caspase-3 induced by exposure to 1% O2 in Min6 cells is mediated by CHOP. Whole-cells extracts were prepared and proteins separated by immunoblotting using anti-procaspase-3 (procasp-3), anti-cleaved caspase-3 (cleaved Casp-3) and anti-β-actin antibodies. (c) CHOP mediates hypoxia-induced apoptosis in Min6 cells. CHOP-knockdown cell lines were cultured under normoxia or hypoxia for 48 h. Apoptosis was assessed by quantification of BrdU-FITC-labeled DNA breaks (BrdU-FITC) using a flow cytometry-based TUNEL assay. (d) CHOP contributes to the reduced cell viability observed at hypoxia. Min6 stable cell lines were cultured under normoxia or hypoxia (1% O2) for the indicated time points. Cell viability was measured (n=3; values are mean±S.D.; **P<0.05 relative to control by two-tailed Student's t test)
