Portland Press

Figure 5.

$(A and B) Rat neonatal cardiomyocytes were exposed to the indicated concentrations of SB590885 for the times shown. Cytosolic and nuclear-enriched fractions were immunoblotted for phosphorylated (phospho-) or total ERK1/2. Representative blots are shown (A) with quantification for 0.1 µM SB590885 (B). Results are means ± SEM (n = 3). P values are relative to 0 min (one-way ANOVA with Holm–Sidak's post-test). Positions of relative molecular mass markers (kDa) are on the right of the immunoblots. (C) Rat neonatal cardiomyocytes were untreated or exposed to 0.1 µM SB590885 (1 h) with/without 2 µM PD184352. mRNA expression was measured by quantitative qPCR. Individual data points are shown with means ± SEM (n = 4). P values are for PD184352/SB590885 vs SB590885 (one-way ANOVA with Holm–Sidak's post-test). (D) Cardiomyocytes were incubated with 1 µM SB590885, 1 µM encorafenib or under control conditions (24 h) and immunostained for troponin T. Images (left panels) are representative of 6 (SB590885, controls) or 4 (encorafenib) independent myocyte preparations with analysis of the surface area of individual cardiomyocytes shown in the right panel. Images are from the same experiment. Individual data points are shown for the mean values for cardiomyocyte area for each experiment, normalised to the mean value of the controls. We also show the means ± SEM for all of the experiments. Statistical analysis used a two-tailed paired t-test. (E) Immunoblot analysis of phosphorylated (phospho-) and total ERK1/2 in extracts from neonatal rat cardiomyocytes exposed to the indicated concentrations of encorafenib (20 min). Representative blots are shown on the left with densitometric analysis on the right. Results are means ± SEM (n = 3 independent cell preparations). P value is relative to controls with no encorafenib (one-way ANOVA with Holm–Sidak's post-test). (F) Immunoblot analysis of phospho- and total MKK1/2 or ERK1/2 in extracts from neonatal rat cardiomyocytes exposed to the indicated concentrations of SB590885 or encorafenib (20 min) with addition of 50 nM A61603 for the final 5 min as indicated. Representative blots are shown in the upper panels with densitometric analysis for MKK1/2 in the lower panels. Results are means ± SEM (n = 3 independent cell preparations).$

SB590885 promotes immediate early gene expression and hypertrophy in cardiomyocytes.

(A and B) Rat neonatal cardiomyocytes were exposed to the indicated concentrations of SB590885 for the times shown. Cytosolic and nuclear-enriched fractions were immunoblotted for phosphorylated (phospho-) or total ERK1/2. Representative blots are shown (A) with quantification for 0.1 µM SB590885 (B). Results are means ± SEM (n = 3). P values are relative to 0 min (one-way ANOVA with Holm–Sidak's post-test). Positions of relative molecular mass markers (kDa) are on the right of the immunoblots. (C) Rat neonatal cardiomyocytes were untreated or exposed to 0.1 µM SB590885 (1 h) with/without 2 µM PD184352. mRNA expression was measured by quantitative qPCR. Individual data points are shown with means ± SEM (n = 4). P values are for PD184352/SB590885 vs SB590885 (one-way ANOVA with Holm–Sidak's post-test). (D) Cardiomyocytes were incubated with 1 µM SB590885, 1 µM encorafenib or under control conditions (24 h) and immunostained for troponin T. Images (left panels) are representative of 6 (SB590885, controls) or 4 (encorafenib) independent myocyte preparations with analysis of the surface area of individual cardiomyocytes shown in the right panel. Images are from the same experiment. Individual data points are shown for the mean values for cardiomyocyte area for each experiment, normalised to the mean value of the controls. We also show the means ± SEM for all of the experiments. Statistical analysis used a two-tailed paired t-test. (E) Immunoblot analysis of phosphorylated (phospho-) and total ERK1/2 in extracts from neonatal rat cardiomyocytes exposed to the indicated concentrations of encorafenib (20 min). Representative blots are shown on the left with densitometric analysis on the right. Results are means ± SEM (n = 3 independent cell preparations). P value is relative to controls with no encorafenib (one-way ANOVA with Holm–Sidak's post-test). (F) Immunoblot analysis of phospho- and total MKK1/2 or ERK1/2 in extracts from neonatal rat cardiomyocytes exposed to the indicated concentrations of SB590885 or encorafenib (20 min) with addition of 50 nM A61603 for the final 5 min as indicated. Representative blots are shown in the upper panels with densitometric analysis for MKK1/2 in the lower panels. Results are means ± SEM (n = 3 independent cell preparations).

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