Research Paper Volume 13, Issue 17 pp 21547—21570

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

NF-kB pathway is involved in HSP90-regulated expression of senescence-associated cytokines in senescent RPE cells in vitro. (A) Immunoblot of IKKα AKT, p65, p21, HSP90, HSP70, HSC70 and β-actin in day-4 senescent ARPE-19 cells treated with PBS (sham) (lane 1) or IPI-504 at concentrations 0.25, 0.5, 1 and 5 μM (lanes 2–5). (B) Densitometry quantitation of IKKα in A. The results shown are mean ± SD. The two-tailed unpaired t-test was used for statistical analysis (n = 4). (C) Immunoblot of IKKα and GAPDH in day-4 senescent ARPE-19 cells that were treated with media containing PBS (sham, lane 1), 1 μM IPI-504 for 2, 4, 8, 12, 24 hours (lanes 2–6). (D–I) mRNA expression of IL-1β, IL-6, IL-8, MCP-1, TGF-b and VEGFA in day-4 senescent ARPE-19 cells treated with or without IKKα/IKKβ inhibitor TPCA-1. The data were collected from 5 independent experiments, the two-tailed unpaired t-test was used for statistical analysis, *p ***p

Figure 4. NF-kB pathway is involved in HSP90-regulated expression of senescence-associated cytokines in senescent RPE cells in vitro. (A) Immunoblot of IKKα AKT, p65, p21, HSP90, HSP70, HSC70 and β-actin in day-4 senescent ARPE-19 cells treated with PBS (sham) (lane 1) or IPI-504 at concentrations 0.25, 0.5, 1 and 5 μM (lanes 2–5). (B) Densitometry quantitation of IKKα in A. The results shown are mean ± SD. The two-tailed unpaired t-test was used for statistical analysis (n = 4). (C) Immunoblot of IKKα and GAPDH in day-4 senescent ARPE-19 cells that were treated with media containing PBS (sham, lane 1), 1 μM IPI-504 for 2, 4, 8, 12, 24 hours (lanes 2–6). (DI) mRNA expression of IL-1β, IL-6, IL-8, MCP-1, TGF-b and VEGFA in day-4 senescent ARPE-19 cells treated with or without IKKα/IKKβ inhibitor TPCA-1. The data were collected from 5 independent experiments, the two-tailed unpaired t-test was used for statistical analysis, *p < 0.05, ***p < 0.001.