Fig. 3: Misoprostol prevents Bnip3-induced mitochondrial perturbations and cell death in MEFs and H9c2 cells.

A PVNC’s treated with 10 μM misoprostol (Miso) with or without exposure to 1% O₂ (HPX) for 24 h. Cells were fixed, stained with hoechst (blue), and immunofluorescence was performed using a Bnip3 primary antibody (green). Cells were then imaged by standard fluorescence microscopy. B Quantification of cells in (A), where green fluorescent signal was normalized to cell area and quantified in 30 random fields, across 3 independent experiments. C Immunoblot for Bnip3 in protein extracts from WT and Bnip3^−/− MEFs treated as in (A). D Quantification of WT and Bnip3^−/− mouse embryonic fibroblasts (MEFs) treated as in (A). Cells were stained with TMRM (red) and hoechst (blue) and imaged by standard fluorescence microscopy. Red fluorescent signal was normalized to cell area and quantified in 15 random fields, across 3 independent experiments. E WT and Bnip3^−/− MEFs treated as in (A). Cells were stained with hoechst (blue) and dihydrorhod-2AM to stain mitochondrial calcium. F Quantification of (E) as in (D) in 15 random fields, across 3 independent experiments. G Quantification of WT and Bnip3^−/− MEFs treated as in (A). Cells were stained with hoechst (blue) and calcein-AM quenched by cobalt chloride (CoCl₂, 5 μM) to assess permeability transition. Green fluorescent signal was normalized to cell area and quantified in 15 random fields, across 3 independent experiments. H H9c2 cells transfected with pcDNA3 (control) or Myc-Bnip3 and treated with 10 μM misoprostol (Miso) or PBS control for 16 h. Mito-Emerald (green) was included in all conditions to show transfected cells and mitochondrial morphology. Cells were stained with hoechst (blue) and imaged by standard fluorescence microscopy. I Quantification of cells in (H), where the number of cells with elongated and fragmented mitochondria are expressed as a percentage of all transfected cells in 30 random fields, across 3 independent experiments. J Quantification of H9c2 cells transfected with pcDNA3 (control), Myc-Bnip3 and/or myc-Opa1. Mito-Emerald (green) was included in all conditions as in (H) and cells were stained with hoechst (blue) and imaged by standard fluorescence microscopy. Quantification as in (I). K H9c2 cells treated as in (H). CMV-GFP (outline) was included in all conditions to indicate transfected cells Cells were stained and imaged as in (D). L Quantification of cells in (K) as in (D). M Quantification of H9c2 cells treated as in (H). ER-LAR-GECO (red) was included in all conditions to indicate ER calcium content. Cells were stained and imaged as in (H). Quantification was performed as in (D) in 30 random fields, across 3 independent experiments. N Quantification of H9c2 cells treated as in (H). Mito-CAR-GECO (red) was included in all conditions to indicate mitochondrial calcium content. Cells were stained and imaged as in (D). Quantification was performed as in (D) in 30 random fields, across 3 independent experiments. O Quantification of H9c2 cells treated as in (H) and CMV-ds-RED was included in all conditions to indicate transfected cells. Cells were stained, imaged and quantified as in (G) across 30 random fields, across 3 independent experiments. P H9c2 cells treated as in (H). LC3-GFP (green) was included in all conditions to show transfected cells and autophagic puncta. Cells were stained with hoechst (blue) and MitoTracker red (red) and imaged by standard fluorescence microscopy. Q Quantification of cells in (P), where the number of cells with LC3-GFP and MitoTracker co-localization are expressed as a percentage of all transfected cells in 10 random fields. R Quantification of H9c2’s treated as in (H). ATeam was used to indicate cytosolic ATP content. Cells were imaged by FRET-based microscopy. FRET-YFP (ATP) signal was divided by the YFP (unbound biosensor) signal in 15 random fields across 3 independent experiments. S Quantification of H9c2 cells treated as in (H). Live cells were stained with calcein-AM (green), and necrotic cells were stained with ethidium homodimer-1 (red) and are expressed as percent (%) dead in 30 random fields, across 3 independent experiments. All data are represented as mean ± S.E.M. *P < 0.05 compared with control, while **P < 0.05 compared with hypoxia or Bnip3 treatment, determined by 1-way ANOVA or 2-way ANOVA where appropriate. Three * indicates P < 0.05 compared to both control and treatment conditions.