Journal: bioRxiv

Article Title: Misoprostol Attenuates Cardiomyocyte Proliferation in the Neonatal Heart through Bnip3 and Perinuclear Calcium Signaling

doi: 10.1101/681692

Figure Lengend Snippet: (A) H9c2 cells treated with 10 μM misoprostol (Miso) ± 10% O 2 (HPX) for 48 hours. Peredox-mCherry (where Red=NAD + , Green=NADH) was used to evaluate cytosolic reduced and oxidized NAD content in all conditions. Cells were imaged by standard fluorescence microscopy. (B) Quantification of cells in (A), where the ratio of green fluorescence (NADH) to red fluorescent signal (NAD + ) in 15 random fields across 3 independent experiments. (C) H9c2 Extracellular Acidification Rate (ECAR) in cells treated as in (A) determined by Seahorse Extracellular Flux Analysis at baseline, following 1 μM oligomycin injection (a), and 50 mM 2DG injection (b) run across 3 independent experiments. (D) Quantification of H9c2 glycolytic capacity, calculated from (C). (E) Quantification of H9c2 glycolytic reserve, calculated from (C). (F) Ratiometric images of H9c2’s treated as in (A). Laconic was used to indicate cytosolic lactate content. Cells were imaged by FRET-based microscopy. (G) Quantification of cells in (F), where the FRET-YFP (lactate) signal was divided by the YFP (unbound biosensor) signal in 15 random fields across 3 independent experiments. (I) Ratiometric images of H9c2’s treated as in (A). Pyronic was used to indicate cytosolic pyruvate content. Cells were imaged by FRET-based microscopy. (J) Quantification of cells in (I), where the FRET-YFP (Pyruvate) signal was divided by the YFP (unbound biosensor) signal in 15 random fields across 3 independent experiments. (K) H9c2’s treated as in (A) stained with hoechst (blue) and 200 μM 2-NBDG (green) and imaged by standard fluorescence microscopy. (L) Quantification of cells in (K), where green fluorescent signal was normalized to cell area and quantified in 15 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 treatment, determined by 1-way ANOVA or 2-way ANOVA where appropriate.

Article Snippet: GW1-Peredox-mCherry (Peredox-mCherry) was a gift from Gary Yellen (Addgene plasmid #32380) [ ].

Techniques: Fluorescence, Microscopy, Injection, Staining

Journal: bioRxiv

Article Title: Misoprostol Attenuates Cardiomyocyte Proliferation in the Neonatal Heart through Bnip3 and Perinuclear Calcium Signaling

doi: 10.1101/681692

Figure Lengend Snippet: (A) H9c2 cells treated with 10 μM misoprostol (Miso) ± 10% O 2 (HPX) for 48 hours. NLS-R-GECO (red) was used to indicate nuclear calcium content in all conditions. Cells were stained with hoechst (blue) and imaged by standard fluorescence microscopy. (B) Quantification of cells in (A), where red fluorescent signal was normalized to cell area and quantified in 10 random fields. (C) H9c2 cells treated as in (A) and Myc-NFATc3 was transfected into each condition. Cells were fixed, stained with hoechst (blue), and immunofluorescence was performed using a Myc primary antibody (green). Cells were then imaged by standard fluorescence microscopy. (D) Quantification of cells in (C), where the number of cells with nuclear Myc-NFATc3, is presented as a percentage of the number of cells/field in 5 random fields. (E) H9c2 cells treated as in (A), Protein extracts were subjected to nuclear/cytosolic fractionation and were immunoblotted, as indicated. (F) H9c2’s treated as in (A). 2 μM CsA was added to half of the conditions for 48 hours in order to inhibit calcineurin-NFAT signalling. Peredox-mCherry (where Red=NAD + , Green=NADH) was used to indicate cytosolic reduced or oxidized NAD content in all conditions. Cells were imaged by standard fluorescence microscopy. (G) Quantification of cells in (F), where the ratio of green fluorescence (NADH) to red fluorescent signal (NAD + ) in 15 random fields across 3 independent experiments. (H) Quantification of H9c2 cells treated with 10% O 2 (HPX) for 48 hours and transfected pcDNA3 (control) or myc-NFATc3. Peredox-mCherry was used to indicate cytosolic NAD content. The ratio of green fluorescence (NADH) to red fluorescent signal (NAD + ) was measured in >15 random fields across 6 independent experiments. (I) H9c2’s treated as in (H), cells were stained with Tag-it Violet (blue) and calcein-AM (green) and imaged by standard fluorescence microscopy. (J) Quantification of Tag-it Violet positive cells in (I), where the number of blue cells is represented as a percentage (%) of the total number of (green) cells in 10 random fields. (K) Immunoblot for MEF2C and MEF2A in protein extracts from PVNCs treated as in (A). (L) PVNCs treated as in (A). RNA was isolated and qRT-PCR was performed for BMP-10 expression. All data are represented as mean ± S.E.M. *P<0.05 compared with control, while **P <0.05 compared with hypoxia treatment, determined by 1-way ANOVA or 2-way ANOVA where appropriate.

Article Snippet: GW1-Peredox-mCherry (Peredox-mCherry) was a gift from Gary Yellen (Addgene plasmid #32380) [ ].

Techniques: Staining, Fluorescence, Microscopy, Transfection, Immunofluorescence, Fractionation, Western Blot, Isolation, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Misoprostol Attenuates Cardiomyocyte Proliferation in the Neonatal Heart through Bnip3 and Perinuclear Calcium Signaling

doi: 10.1101/681692

Figure Lengend Snippet: (A) Immunoblot for sNip expression in H9c2 protein extracts treated with 10 μM misoprostol (Miso) ± 10% O 2 (HPX) for 48 hours. (B) PVNCs treated with pLenti-HA-sNip ± 10% O 2 (HPX) for 48 hours and stained with Tag-it Violet (blue) and calcein-AM (green) and imaged by standard fluorescence microscopy. (C) Quantification of Tag-it Violet positive cells in (B), where the number of blue cells is represented as a percentage (%) of the total number of (green) cells in 10 random fields. (D) Quantification of cells in (B), where cell surface area was calculated for >80 cells/condition in 10 random fields. (E) H9c2 cells treated as in (A), transfected with scrambled control si-RNA or si-sNip, and stained with Tag-it Violet (blue) and calcein-AM (green) and imaged by standard fluorescence microscopy. (F) Quantification of Tag-it Violet positive cells in (E), where the number of blue cells is represented as a percentage (%) of the total number of (green) cells in 10 random fields. (G) Quantification of H9c2 cells treated with 10% O 2 (HPX) for 48 hours and transfected pcDNA3 (control) or HA-sNip. Peredox-mCherry was used to indicate cytosolic NAD content in all conditions. The ratio of green fluorescence (NADH) to red fluorescent signal (NAD + ) was measured in >15 random fields across 3 independent experiments. (H) Ratiometric images of H9c2’s treated as in (G). Laconic was used to indicate cytosolic lactate content. Cells were imaged by FRET-based microscopy. (I) Quantification of cells in (H), where the FRET-YFP (lactate) signal was divided by the YFP (unbound biosensor) signal in 15 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 treatment, determined by 1-way ANOVA or 2-way ANOVA where appropriate.

Article Snippet: GW1-Peredox-mCherry (Peredox-mCherry) was a gift from Gary Yellen (Addgene plasmid #32380) [ ].

Techniques: Western Blot, Expressing, Staining, Fluorescence, Microscopy, Transfection