Resistance of cancer cells to Bnip3FL isoform and hypoxia-induced cell death. (A) Schematic depicting the targeting strategy of shRNA for knockdown of endogenous Bnip3FL. shRNA was designed to specifically target sequences within exon 3 of Bnip3FL to knock down Bnip3FL without interfering with Bnip3Δex3, as we have previously reported (Gang et al., 2011). (B and C) Real-time qPCR analysis of Bnip3FL (B) and Bnip3Δex3 (C) mRNA levels in cancer cells. Cells were subjected to hypoxia condition for 18 h in the absence or presence of shRNA-Bnip3FL. Data are expressed as mean ± SD (error bars). *, statistically different from control (CTRL); **, statistically different from HPX; †, not statistically different from HPX. Data were obtained from at least n = 3–4 independent cell cultures for each condition tested. (D) Cell viability of cancer cells after Bnip3FL knockdown with shRNA-Bnip3FL under hypoxic conditions. Shown are representative epifluorescence images of cells stained with vital dyes calcein-AM and ethidium homodimer-1 to visualize live (green) and dead (red) cells, respectively. Bars, 40 µm. (E) Histogram for quantitative data shown in E. Data were obtained from at least n = 3–4 independent cell cultures counting ≥500 cells from n = 3 glass coverslips for each condition tested.