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. 2011 Dec;179(6):2810–2821. doi: 10.1016/j.ajpath.2011.08.023

Figure 6.

Figure 6

Attenuation of the senescent phenotypes of RPTECs at early passage with etoposide exposure by overexpression of GLO1. A: GLO1 activity assay measured at 24 hours after transfection of each vector confirmed successful overexpression of the transgene. Cells were lysed with sodium phosphate buffer with 1% Triton X-100 surfactant. *P < 0.01 versus empty vector. B: Phase-contrast light micrographs with SABG staining of RPTECs at passages 3 to 4 transfected with empty vector or GLO1-containing vector with or without etoposide exposure showed amelioration of senescence by GLO1. Original magnification = ×100. Scale bars: 200 μm. Blue stain indicates SABG-positive cells. C: Quantitative analysis of SABG-positive cells per total cells in each field. *P < 0.01 versus cells without etoposide exposure and transfected with empty vector. D: Relative transcript levels of p53, p21WAF1/CIP1, and p16INK4A of RPTECs with etoposide exposure and transfected with plasmid vector determined by real-time quantitative RT-PCR. All showed a significant decrease in GLO1 transfectants, which demonstrated that GLO1 protected RPTECs against cellular senescence. Values of those transfected with empty vector were set as 1. *P < 0.01; **P < 0.05 versus cells transfected with empty vector. E: Protein immunoblot of p53 of whole lysate of RPTECs with or without etoposide exposure and transfected with plasmid vectors. p53 level in cells treated with etoposide exposure was ameliorated by GLO1 introduction. Actin serves as a control. F: Densitometric quantification of p53 immunoblot of RPTECs with or without etoposide exposure and transfected with plasmid vectors. The level of those transfected with empty vector without etoposide exposure was set as 1. *P < 0.01; **P < 0.05 versus cells without etoposide exposure and transfected with empty vector. G: Immunofluorescent micrographs of RPTECs with or without etoposide exposure and transfected with plasmid vectors for the detection of CEL accumulation. Cytosolic positive signals in cells exposed to etoposide were decreased by GLO1 introduction, demonstrating that overexpression of GLO1 ameliorated carbonyl stress. Green indicates anti-CEL (fluorescein isothiocyanate); blue stain identifies nuclei (Hoechst 33258 dye). Original magnification = ×400. Scale bars: 100 μm.