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

FIGURE 8.

FIGURE 8

Pharmacological antagonism of PI3K signalling attenuates cellular senescence in canine aVICs. aVICs were exposed to DMSO (Control), 60 μM LY294002, 5 μM copanlisib and 50 μM alpelisib treatment for 24 h. (A, B) 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/treatment). (C, D) Representative confocal images of γ‐H2AX (green) immunostaining in nuclei and quantitative analysis of the number of γ‐H2AX puncta, scale bar 20 μm (n = 50 cells/treatment). (E) Representative western blot of p16INK4A, p21CIP1, p53 and β‐actin protein expression and (F–H) quantification of the relative protein expression (n = 6). (I) Quantitative RT‐PCR for SASP cytokine expression in aVICs exposed to DMSO and PI3K inhibitors (n = 6). (J–L) Quantification of secreted TGF‐β1, IL‐6 and MMP‐9 in collected supernatant from aVIC cultures (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; aVIC, activated myofibroblast phenotype; SASP, senescent‐associated secretory phenotype.