Research Paper Volume 13, Issue 17 pp 21547—21570

Figure 5. HSP90 associates with and regulates SA-β-Gal in senescent ARPE-19 cells in vitro. (A) SA-β-Gal staining the passage 3 primary monkey RPE cells treated with H₂O₂ for 2 h followed by recovery in normal media for 5 days. IPI-504 (1 and 5 μM) or TPCA-1 (1 μM) were added to day 4 recovery cells for 24 hours. (B) Percentage of SA-β-Gal positive cells out of total number of cells in A. Numbers were derived as an average from 5 different fields of view from three independent experiments. (C) Immunoblot of SA-μ-Gal protein in proliferating ARPE-19 cells (Con, lane 1) or the senescent ARPE-19 cells treated with IPI-504 at 0, 0.1, 0.5, 1 and 5 μM for 24 hours. (D) MG132 rescued the down-regulation of SA-β-Gal protein by IPI-504 in senescent ARPE-19 cells. (E) Immunoprecipitation assay to determine the interaction between HSP90 and SA-β-Gal protein in control (lane 3) and senescent ARPE-19 cells (lane 4). Lanes 1–2 are cell lysate controls, lane 5 is IgG control. (F) GST-pull down assay to determine the interaction between SA-β-Gal proteins and bacterially purified GST-HSP90 in senescent ARPE-19 cells. Upper panel shows the SA-β-Gal protein in day 4 senescent ARPE-19 cells that co-precipitated with bacterially expressed GST-HSP90α fusion protein (lane 2) or GST protein alone (lane 1). The lower panel is the coomassie blue stain of the bacterially purified GST and GST-HSP 90α.