EOS, mEar1, and IL4 activities in mouse cardiomyocyte apoptosis and cardiac fibroblast TGF-β signalling and fibrosis. (A) Cardiomyocyte death percentage (bar: 100 µm) after cells were pre-treated with or without mEar1 or different EOS lysates for 12 h, followed by ISO for 6 h. (B) Immunoblot analysis of Bcl-2 and cleaved caspase-3 from cardiomyocytes with the same treatments as in (A). (C) Immunoblot analysis of p-Smad2/3 after cardiac fibroblasts were pre-treated with or without mEar1, IL4, or different EOS lysates with or without IL4 for 12 h, followed by TGF-β for 30 min. (D) Immunoblot analysis of α-SMA, Collagen I and III after cardiac fibroblasts were pre-treated with or without mEar1 or different EOS lysate for 12 h, followed by TGF-β for 24 h. (E) RT-PCR determined the mRNA levels of α-SMA, Collagen I and III in cardiac fibroblasts after the same treatment as in (D). GAPDH was used as protein loading control for all immunoblots. Representative images for panels (A–D) are shown. All data are mean ± SEM from six experiments per group. P < 0.05 was considered statistically significant. One-way ANOVA followed by a post hoc Tukey’s test.