FIGURE 5.

Senescence-associated β-galactosidase staining of ARPE-19 cells treated with oxidants and inhibitors of BMP4 or p38 signaling. The oxidant stressed with or without inhibitor-treated ARPE-19 cells was incubated with SA-β-galactosidase staining buffer for 24 h at 37 °C. Senescent cells were photographed with a phase-contrast microscope and counted as percentage of SA-β-galactosidase-positive cells. A, ARPE-19 cells without stressors or inhibitors; B, ARPE-19 without stressor but with Chordin-like 1; C, ARPE-19 without stressor but with SB203580; D, ARPE-19 cells treated with 150 μm H₂O₂; E, ARPE-19 cells treated with 150 μm H₂O₂ and Chordin-like 1; F, ARPE-19 cells treated with 150 μm H₂O₂ and SB203580; G, ARPE-19 cells treated with 30 μm tBH; H, ARPE-19 cells treated with 30 μm tBH and Chordinlike 1; I, ARPE-19 cells treated with 30 μm tBH and SB203580. The images shown here demonstrate that stressed cells show higher percentage of senescence (SA-β-galactosidase-positive cells; D and G) than controls (A). In the presence of the BMP4 antagonist, Chordin-like 1, or a phospho-p38 inhibitor, SB203580, in the medium, the numbers of senescent RPE cells were reduced to base-line level (E, H, F, and I). J, tBH and H₂O₂ induced 65 and 48% cell senescence, respectively, whereas the control and stressor plus inhibitor groups showed less than 5% background cell senescence (^**, p < 0.001).