Figure 6.

Both catalase (CAT) and superoxide dismutase 2 (SOD2) attenuate PQ‐induced senescent phenotype in Forkhead box O3 (FoxO3)‐silenced H9c2 cells. (a) shFoxO3‐H9c2 cells were transfected with pcDNA3.1‐SOD or pcDNA3.1‐CAT plasmid for 48 hr, respectively. The overexpression of target genes was analyzed by qRT–PCR analysis. The pcDNA3.1 empty plasmid was used as control. Values are presented as mean ± SEM (n = 4), ***p < 0.001. (b–d) The shFoxO3‐H9c2 cells were transfected with or without overexpression plasmids for 24 hr prior to treatment with PQ (400 μM) for another 4 hr, followed by proliferation analysis by 5‐ethynyl‐2′‐deoxyuridine (EdU) labeling. Representative images (b), quantification of EdU⁺ cells (c), and total cells (d) revealed that both SOD and CAT attenuate PQ‐induced inhibition of cell proliferation. Values are presented as mean ± SEM (n = 5), *p < 0.05, **p < 0.01. (e) The shFoxO3‐H9c2 cells were transfected with or without overexpression plasmids for 24 hr prior to treatment with PQ (400 μM) for another 24 hr, followed by cell growth activity analysis. Values are presented as mean ± SEM (n = 5), **p < 0.01, ***p < 0.001. (f and g) The shFoxO3‐H9c2 cells were treated with as mentioned above, followed by TUNEL labeling. Representative images (f) and quantification of TUNEL⁺ cells revealed that SOD, but not CAT, inhibits PQ‐induced apoptosis. Values are presented as mean ± SEM (n = 5), ***p < 0.01. (h–k) The shFoxO3‐H9c2 cells were transfected with or without overexpression plasmids for 24 hr prior to treatment with PQ (400 μM) for 3 days (4 hr/day), followed by senescence‐associated β‐galactosidase (SA‐β‐Gal) staining and nucleus staining, respectively. The representative images of SA‐β‐Gal⁺ cells (h) and total nuclei (j), and the quantification of SA‐β‐Gal⁺ cells (i) and nuclei size (k) are shown. Values are presented as mean ± SEM (n = 5), *p < 0.05, **p < 0.01, ***p < 0.001