fret-based atp biosensor  (ATeam Scientific)

Journal: Cell Death & Disease

Article Title: Misoprostol treatment prevents hypoxia-induced cardiac dysfunction through a 14-3-3 and PKA regulatory motif on Bnip3

doi: 10.1038/s41419-021-04402-3

Figure Lengend Snippet: A PVNC’s treated with 10 μM misoprostol (Miso) with or without exposure to 1% O 2 (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 (CoCl 2 , 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.

Article Snippet: The FRET-based ATP biosensor (ATeam1.03-nD/nA/pcDNA3) was a gift from Takeharu Nagai (Addgene plasmid #51958) [ ].

Techniques: Staining, Immunofluorescence, Fluorescence, Microscopy, Western Blot, Permeability, Transfection

Journal: Cell Death & Disease

Article Title: Misoprostol treatment prevents hypoxia-induced cardiac dysfunction through a 14-3-3 and PKA regulatory motif on Bnip3

doi: 10.1038/s41419-021-04402-3

Figure Lengend Snippet: A SIM scan of a mutated peptide where the PKA site at Threonine-181 is replaced with Alanine (left). On the right, phosphorylation of this mutate peptide is negligible at the predicted m/z that corresponds to the addition of a PO 3 (M = 80.00 Da). B H9c2 cells transfected with pcDNA3 (control) or Myc-T181A 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. C Quantification of cells in ( B ), 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. D H9c2 cells treated as in ( B ). 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 micros€y. E Quantification of cells in ( D ), where red fluorescent signal was normalized to cell area and quantified in 30 random fields, across 3 independent experiments. F Bnip3 −/− mouse embryonic fibroblasts (MEFs) treated as in ( B ) where either Myc-Bnip3(WT) or Myc-Bnip3(T181A) was transfected in Cells were stained with TMRM (red) and hoechst (blue) and imaged by standard fluorescence microscopy. G Quantification of cells in ( F ), where red fluorescent signal was normalized to cell area and quantified in 15 random fields, across 3 independent experiments. H H9c2 cells treated as in ( B ). ER-LAR-GECO (red) was included in all conditions to indicate ER calcium content. Cells were stained with hoechst (blue) and imaged by standard fluorescence microscopy. I Quantification of cells in (€as in ( E ) in 30 random fields, across 3 independent experiments. J H9c2 cells treated as in ( B ). Mito-CAR-GECO (red) was included in all conditions to indicate mitochondrial calcium content. Cells were stained with hoechst (blue) and imaged by standard fluorescence microscopy. K Quantification of cells i€J) as in ( E ) in 30 random fields, across 3 independent experiments. L Quantification of H9c2 cells treated as in ( B ) 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 (CoCl 2 , 5 μM) to assess permeability transition. Quantification was done by calculating the percentage of cells with mitochondrial puncta in 30 random fields, across 3 independent experiments. M Quantification of H9c2 cells treated as in (B). 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 Bnip3 treatment, determined by 1-way ANOVA or 2-way ANOVA where appropriate.

Article Snippet: The FRET-based ATP biosensor (ATeam1.03-nD/nA/pcDNA3) was a gift from Takeharu Nagai (Addgene plasmid #51958) [ ].

Techniques: Transfection, Staining, Fluorescence, Microscopy, Permeability

Journal: Cell Death & Disease

Article Title: Misoprostol treatment prevents hypoxia-induced cardiac dysfunction through a 14-3-3 and PKA regulatory motif on Bnip3

doi: 10.1038/s41419-021-04402-3

Figure Lengend Snippet: A H9c2 cells treated with 10 μM misoprostol (Miso) ± 1% O 2 (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% O 2 ) ± 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% O 2 (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 (CoCl 2 , 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 (CoCl 2 , 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% O 2 ) ± 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.

Article Snippet: The FRET-based ATP biosensor (ATeam1.03-nD/nA/pcDNA3) was a gift from Takeharu Nagai (Addgene plasmid #51958) [ ].

Techniques: Staining, Immunofluorescence, Confocal Microscopy, Transfection, Fractionation, Activity Assay, Fluorescence, Microscopy, Permeability, Immunoprecipitation, Expressing, Western Blot