Fig. 6: Bnip3 phosphorylation at Thr-181 retains Bnip3 in the cytosol through inhibitory interactions with 14-3-3β in H9c2 cells.

A H9c2 cells treated with 10 μM misoprostol (Miso) ± 1% O₂ (HPX) for 24 h. Myc-Bnip3 and Mito-Emerald (green) were included in each condition to visualize localization. Cells were fixed, stained with hoechst (blue), and immunofluorescence was performed using a Myc-tag primary antibody (Red). Cells were then imaged by standard confocal microscopy. B Quantification of cells in (A), where colocalization coefficient was calculated for 30 cells per condition across 10 random fields. C Quantification of immunofluorescence in PND10 hearts exposed to hypoxia (10% O₂) ± 10 μg/kg misoprostol from PND3-10. Hearts were probed for Bnip3 (Red), Opa1 (Green), and stained with DAPI (Blue). Hearts were imaged by standard confocal microscopy and the colocalization coefficient was calculated in 20 fields per condition (n = 4 animals/conditions). D Quantification of H9c2 cells treated as in (A). ER-Emerald (green) was transfected in all conditions. Cells were fixed, stained with hoechst (blue), and immunofluorescence was performed using a Myc-tag primary antibody (Red). Cells were then imaged by standard confocal microscopy. Colocalization coefficient was calculated for 30 cells per condition across 10 random fields. E Fractionation of control treated H9c2. Protein extracts were fractionated and immunoblotted, as indicated. F Quantification of primary ventricular neonatal cardiomyocytes (PVNCs) treated with 10 μM misoprostol (Miso) ± 1% O₂ (HPX) for 24 h. 5 μM BvO2 was included in half of the conditions to inhibit 14-3-3 protein activity. 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 20 random fields, across 2 independent experiments. G Quantification of PVNCs treated as in (E). Cells were stained with hoechst (blue) and calcein-AM quenched by cobalt chloride (CoCl₂, 5 μM) to assess permeability transition, and imaged by standard fluorescence microscopy. The percentage of cells with mitochondrial puncta was calculated in 20 random fields, across 2 independent experiments. H Quantification of H9c2 cells transfected with pcDNA3 (control) or Myc-Bnip3 with and without HA-14-3-3β. CMV-GFP (outline) was included in all conditions to indicate transfected cells 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 30 random fields, across 3 independent experiments. I Quantification of H9c2s treated as in (G), with and without 14-3-3ε. Cells were stained and imaged as in (H) and quantified as an (I) across 30 random fields, in 3 independent experiments. J Quantification of H9c2’s 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). Red fluorescent signal was normalized to cell are in 30 random fields, across 3 independent experiments. (K) Quantification of H9c2’s treated as in (H). Mito-CAR-GECO (red) was included in all conditions to indicate mitochondrial calcium content. Quantification performed as in (J) in 30 random fields, across 3 independent experiments. L Quantification of H9c2’s treated as in (H), and CMV-ds.RED was included in all conditions to indicate transfected cells. Cells were stained with hoechst (blue) and calcein-AM quenched by cobalt chloride (CoCl₂, 5 μM) to assess permeability transition. Where the percentage of cells with mitochondrial puncta was calculated in 30 random fields, across 3 independent experiments. M Quantification of immunofluorescence in PND10 hearts exposed to hypoxia (10% O₂) ± 10 μg/kg misoprostol from PND3-10. Hearts were probed for Bnip3 (Red), 14-3-3β (Green), and stained with DAPI (Blue). Hearts were imaged by standard confocal microscopy and the colocalization coefficient was calculated in 20 fields per condition (n = 4 animals/conditions). N H9c2 cells transfected with Myc-Bnip3 ± 10 μM misoprostol (Miso) 18 h. Cells were fixed, stained with hoechst (blue), and immunofluorescence was performed using a Myc-tag (Red), and 14-3-3β (Green). Cells were then imaged by standard confocal microscopy. O Quantification of cells in (N), where colocalization coefficient was calculated for 30 cells per condition across 10 random fields. P Co-immunoprecipitation of HCT-116 cells expressing HA-14-3-3 and Myc-Bnip3. Proteins were pulled down with Myc and probed for HA-tag. Immunoblot was probed as indicated. 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.