Figure 4. Remifentanil preconditioning protects against hypoxia-induced senescence in HCM cells. (A) Senescence-associated β-galactosidase (SA-β-gal) activity. Representative microphotographs are shown. Scale bars 50 μm, objective 20×. The levels of SA-β-gal positive (blue) and negative (no blue) cells were calculated [%]. (B) Western blot analysis of the levels of cell cycle inhibitor p21. Data were normalized to β-actin. (C) Western blot analysis of the levels of pro-inflammatory cytokine IL-8. IL-8 levels in supernatants (Myocyte Growth Medium, MGM) were calculated per 10000 cells. Bars indicate SD, n = 3, ***p < 0.001 compared to normoxic control (CTR), ###p < 0.001, ##p < 0.01 compared to hypoxic control (CTR) (ANOVA and Dunnett's a posteriori test). CTR, control; R, remifentanil preconditioning.