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. 2023 Mar 4;56(6):e13435. doi: 10.1111/cpr.13435

FIGURE 9.

FIGURE 9

Overexpression of p70 S6K induces activated myofibroblast differentiation and ECM production through downregulating apoptosis and autophagy while enhancing cellular senescence. Canine qVICs were transfected with human p70 S6K cDNA ORF plasmids, mouse p70 S6K cDNA ORF plasmids, pcDNA3.1 plasmids (vectors) and Lipofectamine 3000 (Mock) with or without 5 μM of baflomycin‐A1 (Baf‐A1). (A) Representative western blot of p70 S6K, phosphorylated p70 S6K (p‐p70 S6K), VIC phenotype and ECM protein expression and quantification of the relative protein expression (n = 6). (B) p70 S6K overexpressed qVICs were exposed to 5 μM Baflomycin‐A1. Representative western blot of ATG7 and LC3‐II protein expression and quantification of the relative protein expression (n = 6). (C) Representative confocal images of LC3‐II labelled autophagosomes (green) and quantitative analysis of the number of LC3‐II puncta, scale bar 20 μm (n = 98 cells/treatment). (D) Representative western blot of caspase‐3, cleaved caspase‐3, p16INK4A, p21CIP1 and p53 protein expression and quantification of the relative protein expression (n = 6). (E) Representative images of SA‐β‐gal (blue) staining and quantitative analysis of the percentage of SA‐β‐gal positive cells, scale bar 50 μm (n = 12 microscopic fields). (F) Quantitative RT‐PCR for senescence‐associated secretory phenotype (SASP) cytokine expression (left panel) in qVICs transfected with p70 S6K cDNA ORF plasmids (n = 6). Quantification of secreted TGF‐β1, IL‐6 and MMP‐9 (right panel) in collected supernatant from qVIC cultures overexpressing p70 S6K (n = 6). Results are presented as mean ± SEM. ANOVA followed by Tukey's range test. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control. ANOVA, analysis of variance; ECM, extracellular matrix; VIC, valve interstitial cell.