Fig. 3. HIF1α and P65 drive expression of pro-survival BNIP3 splice variants.

a HCT-116 cells were treated with 200 μM cobalt chloride ± 10 μM misoprostol or vehicle control for 20 h. RNA was isolated and RT-PCR was performed for BNIP3 isoforms. b HCT-116 cells were treated as in a. Protein extracts were immunoblotted, as indicated. c Immunoblot of protein extracts from HCT-116 cells treated with misoprostol and CoCl₂ in for 36 h. d HCT-116 cells were transfected with HIF1α ± P65. Protein extracts were immunoblotted, as indicated. e Quantification calcein-AM and ethidium homodimer-1 stained HCT-116 cells transfected with HIF1α and/or P65. f HCT-116 cells treated with 200 μM cobalt chloride ± 10 μM misoprostol or vehicle control for 20 h. Live cells were stained were stained with calcein-AM (green)and necrotic cells were stained with ethidium homodimer-1 (red), cells were imaged by standard fluorescence microscopy. g Fluorescent images were quantified by calculating the percent of necrotic cells (ethidium homodimer-1 positive) cells in 10 random fields. h Immunoblot of HCT-116 cells transfected with si-BNIP3-FL or scrambled control. Cells were treated with 200 μM cobalt chloride or vehicle control for 20 h. i HCT-116 cells treated as in h and cells were stained as in f, and quantified as indicated in g. j Quantification of HCT-116 cells transfected with Bnip3-FL, Bnip3ΔExon3 or empty vector control. Cells were stained, and quantified as indicated in f. k Quantification of HCT-116 cells transfected with Bnip3-FL, BNIP3ΔExon2 or an empty vector control. Cells were stained, and quantified as indicated in f. Data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with treatment, determined by 1-way ANOVA