Fig. 6: Bnip3 splice variants differentially regulate subcellular calcium micro-domains.

a HCT-116 cells were transfected with Bnip3-FL, Bnip3ΔExon3, or an empty vector control. ER-LAR-GECO (red) was used to indicate endoplasmic reticulum calcium content in all conditions. Cells were stained with Hoechst (blue) and imaged by standard fluorescence microscopy. b Quantification of a, where red fluorescent signal was normalized to cell area and quantified in 10 random fields. c HCT-116 cells were transfected with Bnip3-FL, Bnip3ΔExon3, or an empty vector control. Mito-CAR-GECO (red) was used to indicate mitochondrial calcium content in all conditions. Cells were stained and imaged as in a. d Quantification of c. e Quantification of HCT-116 cells transfected with Mito-CAR-GECO, Bnip3-FL, BNIP3ΔExon2, or an empty vector control. f Quantification of HCT-116 cells transfected with Mito-CAR-GECO and treated with 200 μM cobalt chloride ± 10 μM misoprostol or vehicle control for 20 h. g Quantification of HCT-116 cells transfected with Mito-CAR-GECO, si-BNIP3-FL, or scramble control. Cells were treated with 200 μM cobalt chloride or vehicle control for 16 h. h Quantification of H9c2 cells treated with 200 μM cobalt chloride ± 10 μM misoprostol or vehicle control for 20 h, and transfected with Mito-CAR-GECO and si-Bnip3ΔExon3 or a scrambled control. i Quantification of HCT-116 cells transfected with Mito-CAR-GECO, Bnip3-FL ± 2 μM 2-APB for 16 h. j Quantification of HCT-116 cells treated with 10 μM DIDs and/or 10 μM Ru360 for 16 h (where both indicates DIDs + Ru360) and transfected Mito-CAR-GECO, Bnip3-FL, Bnip3ΔExon3 and/or empty vector control. k Quantification of HCT-116 cells treated with 1 μM Thapsigargin (Thaps) for 4 h and transfected with Mito-CAR-GECO, Bnip3ΔExon3, or empty vector control. 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