Fig. 7. Bnip3 splice variants regulate nuclear calcium content.

a HCT-116 cells were transfected with Flag-BCL2 and HA-Bnip3ΔExon3, as indicated. Protein extracts were subjected to fractionation and were immunoblotted, as indicated. b HCT-116 cells were transfected with Mito-CAR-GECO, Bnip3-FL, Flag-BCL2, or MCL-1, as indicated. Fluorescence 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. NLS-R-GECO (red) was used to indicate nuclear calcium content. Cells were stained with Hoechst (blue) and imaged by standard fluorescence microscopy. d Quantification of (c), where red fluorescent signal was normalized to nuclear area and quantified in 10 random fields. e HCT-116 cells were transfected with NLS-R-GECO, Bnip3-FL, BNIP3ΔExon2, or an empty vector control. f Quantification of e. g HCT-116 cells were transfected with NLS-R-GECO, Bnip3ΔExon3 ± 2 μM 2-APB for 16 h. h Quantification of g. i HCT-116 cells transfected with NLS-R-GECO, Bnip3ΔExon3 ± 1 μM Thapsigargin (Thaps) for 4 h. j H9c2 cells transfected NLS-R-GECO, si-Bnip3ΔExon3 (indicated as si-ΔEx3) or scrambled control, and treated with 10 μM misoprostol or vehicle control for 20 h. Cells were stained with Hoechst (blue) and imaged by standard fluorescence microscopy. k Quantification of j. 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 or unpaired t-test