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

FIGURE 5.

FIGURE 5

Pharmacological inhibition of PI3K signalling reverses VIC phenotype and reduces ECM protein expression in canine aVICs. aVICs were exposed to DMSO (Control), LY294002 (60 μM), copanlisib (5 μM) and alpelisib (50 μM) treatment for 3 days. (A, B) Representative confocal images of α‐SMA (red) staining in canine aVICs and quantitative analysis of the percentage of α‐SMA positive cells, scale bar 20 μm (n = 12 microscopic fields/treatment). (C) Quantitative RT‐PCR for α‐SMA, SM22 and Smemb mRNA expression in aVICs (n = 6) at Day 3. (D, E) Representative western blot of ECM and VIC phenotype protein expression and quantification of the relative protein expression (n = 6). (F, G) Representative western blot of PI3K 110α, AKT, phosphorylated AKT (p‐AKT), mTOR, phosphorylated mTOR (p‐mTOR), p70 S6K, phosphorylated p70 S6K (p‐p70 S6K) protein expression and quantification of the relative protein expression (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.