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. 2020 Sep 1;21(17):6348. doi: 10.3390/ijms21176348

Figure 4.

Figure 4

Knock-down of B-Raf and treatment with UO126 (+UO) abrogates MAPK cascade activation and fetal remodeling. Isolated cardiomyocytes (n = 4) were allowed to recover for 1 day (Con 0 d) and then treated for 10 min (10 min) or 6 days (6 d) for western blot analysis. Cultures were treated with basic medium (Con), with serum (Serum), or with morphogens (Morpho) as indicated. MEK1/2 inhibitor UO126 (UO, 5 µM) was added 1 h before the experimental start as indicated. Quantitative evaluation and statistical analysis of western blots (WB) are shown. Single star and hash sign indicate p < 0.01 and p < 0.05, respectively. Black signs refer statistically to Con. Red signs compare Morpho (Morpho, Morpho siCon) statistically with and without UO/siB-Raf (+UO, siBRaf). (A) WB analysis of ERK1/2 (P-ERK1/2) activation and dedifferentiation (SM-actin) in the absence or presence of UO (+UO). (B) WB analysis of P-ERK1/2 of untreated cardiomyocytes (cultured in basic medium) at 0 d and after 6 d (n = 6). (C) After a three-day pretreatment period with siRNA, cardiomyocytes were cultured in basic medium or stimulated with Serum and Morpho for WB of α-actinin-1 (Actinin-1) and α-smooth muscle actin (SM-actin) for six days. In order to determine MEK1/2 and ERK1/2 phosphorylation (P-MEK1/2, P-ERK1/2) cultures were made quiescent after six days for one day in basic medium and then treated as indicated for 10 min.