Research Paper Volume 13, Issue 17 pp 21547—21570

HSP90 acts as a senomorphic target in senescent retinal pigmental epithelial cells

Induction of senescent ARPE-19 cells by H2O2in vitro. (A) Proliferation of ARPE-19 during recovery after 2 hours of H2O2 treatment using the CCK-8 assay (WST-8, 2-methoxy-4-nitrophenyl)-3- (4-nitrophenyl)-5- (2, 4-disulfophenyl)-2H-tetrazolium). (B) Staining for SA-β-Gal activity in ARPE-19 cells treated with 200 μM H2O2 for 2 hours followed by recovery for 1, 2, 3, 4 and 5 days. (C–H) Quantitative PCR to detect mRNA expression of IL-1β, IL-6, IL-8, MCP-1, TGF-β1 and P21 in ARPE-19 cells treated with H2O2 in the same way as in B. (I) Immunoblot of P53 and p21 protein expression in ARPE-19 cells treated with H2O2 in the same way as in B. (J) Detection of SA-β-Gal activity in passage 3 primary monkey RPE cells treated with H2O2 for two hours followed by recovery in normal media for 0 and 5 days. The two-tailed unpaired t-test was used for statistical analysis. The data were collected from three independent experiments (n = 3).

Figure 1. Induction of senescent ARPE-19 cells by H2O2in vitro. (A) Proliferation of ARPE-19 during recovery after 2 hours of H2O2 treatment using the CCK-8 assay (WST-8, 2-methoxy-4-nitrophenyl)-3- (4-nitrophenyl)-5- (2, 4-disulfophenyl)-2H-tetrazolium). (B) Staining for SA-β-Gal activity in ARPE-19 cells treated with 200 μM H2O2 for 2 hours followed by recovery for 1, 2, 3, 4 and 5 days. (CH) Quantitative PCR to detect mRNA expression of IL-1β, IL-6, IL-8, MCP-1, TGF-β1 and P21 in ARPE-19 cells treated with H2O2 in the same way as in B. (I) Immunoblot of P53 and p21 protein expression in ARPE-19 cells treated with H2O2 in the same way as in B. (J) Detection of SA-β-Gal activity in passage 3 primary monkey RPE cells treated with H2O2 for two hours followed by recovery in normal media for 0 and 5 days. The two-tailed unpaired t-test was used for statistical analysis. The data were collected from three independent experiments (n = 3).