Figure 2

Effect of miR-210 and miR-196b overexpression on the protective effect of hypoxia on the etoposide-induced apoptosis. HepG2 cells were transfected with pre-miR negative control (pre-miR CTL-), pre-miR-210 or pre-miR-196b (50 nM) during 24 h or 48 h and then cells were incubated under normoxia (N) or hypoxia (H) with or without etoposide (E) (50 μM) during 16 h. (A, C) Cleaved caspase 3 and cleaved PARP were detected in total cell extracts by western blotting analysis, using specific antibodies. Immunodetection of α-tubulin was used to assess the total amount of proteins loaded on the gel. Numbers correspond to the quantification of the abundance of protein of interest normalized to the abundance of α-tubulin. (B, D) 48 h post-transfection, the caspase 3/7 activity was assayed by measuring free AFC released from the cleavage of the caspase 3/7 substrate Ac-DEVD-AFC. Results are expressed in fluorescence intensity, as means ± 1 SD (n = 3). *, **, ***: (p < 0.05, p < 0.01, p < 0.001), °, °°, °°°: significantly different from untransfected cells (Cells) (p < 0.05, p < 0.01, p < 0.001), $$$: significantly different from pre-miR negative control transfected cells (p < 0.001) respectively for each group (N, H, NE, HE).