Research Paper Volume 13, Issue 17 pp 21547—21570

Figure 6. IPI-504 inhibits VEGFA expression by down-regulating HIF1α protein expression in senescent RPE cell in vitro. (A) Quantitative PCR determined VEGFA expression in control ARPE-19 cells (Con) or ARPE-19 cells treated with 2 h H₂O₂ followed by recovery in normal media for 0, 1, 2, 3, 4 and 5 days. (B) Immunoblot of VEGFA protein in the cells used in A. (C) Immunoblot of VEGFA in the supernatant of proliferative ARPE-19 cells (Con) or senescent ARPE-19 cells that were cultured for 3, 4 and 5 days. (D) Quantitative PCR determine VEGFA mRNA in day 4 senescent ARPE-19 cells treated with IPI-504 at 0.2, 0.5, 1 and 5 μM. (E) ELISA determines the VEGFA protein in the supernatants of day 4 senescent ARPE-19 cells treated with 1 μM IPI-504 for 24 hours. (F) Quantitative PCR determines VEGFA mRNA expression in day 4 senescent ARPE cells treated with HIF1α inhibitor KC7F2 or HSP90 inhibitor IPI-504. The data in each quantitation figure were collected from three independent experiments, the two-tailed unpaired t-test was used for statistical analysis, ^*P < 0.05, ^***p < 0.001. (G) Immunoblot of HIF10α protein in day-4 senescent ARPE-19 cells treated with IPI-504 at 0, 0.1, 1 and 5 μM. β-actin was used for protein loading control. (H) Densitometry quantitation of HIF1α vs. β-actin in G, the data shown are mean ± SD. The two-tail unpaired t-test was used for statistical analysis (n = 3). ^*P < 0.05; ^***P < 0.001. (I) Immunoblot HIF1α and GAPDH proteins in senescent ARPE-19 cells treated in media containing PBS (sham, lane 1), IPI-504 alone (lane 2), IPI-504 +chloroquine (lane 3) and IPI-504 + MG132 (lane 4).