Figure 5.

Carbonyl stress and cellular senescence in RPTECs. A: Phase-contrast light micrographs with SABG staining of RPTECs at passage 3 with or without etoposide exposure and at passage 10 show senescence of cells at passage 3 with etoposide exposure and at passage 10. Glutaraldehyde fixation was done before staining. SABG staining was performed by incubation for 5 hours without CO₂. Original magnification = ×100. Scale bars: 100 μm. Blue staining indicates positive cells. B: Quantitative analysis of SABG-positive cells per total cells in each field. Most RPTECs were senescent at passage 10 or with etoposide exposure. *P < 0.01 versus day 0 at passage 3. C: Transcript levels of p53, p21^WAF1/CIP1, and p16^INK4A of RPTECs at passage 3 with or without etoposide exposure and at passage 10 determined by real-time quantitative RT-PCR. All showed significant elevation compared with cells at passage 3 without etoposide exposure. Values of those at passage 3 were set as 1. *P < 0.01; **P < 0.05 versus each at passage 3; ^†P < 0.05 versus p21^WAF1/CIP1 at passage 10. D: Protein immunoblot of p53 of whole lysate of RPTECs at passage 3 with or without etoposide exposure and at passage 10 confirmed the results of real-time quantitative PCR analysis. Cells were lysed in urea buffer under reducing conditions. Actin served as a control. E: Densitometric quantification of p53 immunoblot of RPTECs at early passage (P3) with or without etoposide exposure and at late passage (P10). The level of cells at early passage without etoposide exposure was set as 1. *P < 0.01 versus cells at passage 3 without etoposide exposure. F: Immunofluorescent micrographs for the detection of CEL showed carbonyl stress in senescent RPTECs. Only cells at passage 3 with etoposide exposure and at passage 10 exhibited a cytosolic positive signal. Cells were fixed with methanol 50%:acetone 50%. Green indicates anti-CEL (fluorescein isothiocyanate); blue stain identifies nuclei (Hoechst 33258 dye). Original magnification = ×400. Scale bars: 100 μm.