Fig 2: TGF-ß1 enhances cell senescence and laminin (LM) deposition induced by H2O2. Senescent HLE B-3 cells [or cell basement membranes (BMs)] were cultured in medium with H2O2 (400 µM) for 96 h. (A-G) HLE B-3 cells were treated with H2O2 only, or in combination with LY2109761 (5 µM) or SB431542 (10 µM) for 72 h. (A-C) Percentage of SA-ß-gal-positive cells (A), protein expression of GLB1 (B) and immunofluorescence analysis of p21 (C) in HLE B-3 cells. (D) Total LM in HLE B-3 cells, as detected by ELISA. (E) Immunoblot analysis of LM subunits and MMP-9 in HLE B-3 cells. (F) Total LM in HLE B-3 cell BM, as detected by ELISA. (G) Immunofluorescence analysis of LMa4 (green) and LMß3 (green) in HLE B-3 cell BMs (Scale bars: 100 µm). (H) HLE B-3 cells treated with TGF-ß1 (15 ng/ml) for 96 h. Protein expression levels of TGF-ß1, p21, MMP-9 and LM subunits in HLE B-3 cells analyzed via IB. Data were shown as mean ± SD and were analyzed using paired t-test. *, p<0.05; **, p<0.01.

Fig 3: Accumulation of d-galactose and BMSC dysfunction in a rat chronic hypoxia model. a New-born Sprague-Dawley rats were housed in normoxic or hypoxic chambers for 3 weeks, respectively (n = 9 per group). Peripheral blood samples were obtained, and the d-galactose concentrations were measured. b Stools from the rats described in a were subjected to qPCR to assess the Lactobacillus content. c BMSCs from a were cultured to passage 3, and cell lysates were prepared to assess the levels of p53, p27, p21, p16 and ß-actin by western blotting. d Western blotting was used to assess the protein levels of GALM, GALK1, GALK2, aldose reductase, GLB1 and ß-actin in the rat liver. The data are the mean ± SD from three independent experiments, and statistical significance was analysed using Student’s t-test (**P < 0.01)

Fig 4: Premature senescence model of human lens epithelial cells (HLE B-3) induced by H2O2. Cells of the control group were cultured in medium only, whereas cells of senescent group were cultured in medium with H2O2 for 96 h. (A) Viabilities (left) of HLE B-3 cells treated with different concentrations of H2O2 (0–600 µM) for 96 h, as measured via an MTT assay. Morphologic changes (right) of HLE B-3 cells following a 96 h exposure to 400 µM H2O2. (B) Percentage of SA-ß-gal-positive cells in HLE B-3 cells treated with different concentrations of H2O2 (0–600 µM) (left). SA-ß-gal activity as measured by cell staining (right). (C) Immunoblot analysis of GLB1, p21 and P53 in HLE B-3 cells. (D) Immunofluorescence analysis of p21 (green) in HLE B-3 cell nuclei. (Scale bars: 100 µm). Data were shown as mean ± SD and are representative of 3 independent experiments.

Fig 5: (a) Western blot of Src, p-Src, and PAI-1 protein expression levels under different conditions. (b) Quantitative analysis of figure (a). (c) Western blot of GLB1, P16, P21, and P53 expression levels under different conditions. (d) Quantitative analysis of figure (c). (e) Western blot of FP receptor, Src, p-Src, and PAI-1 protein expression levels under different conditions. (f) Quantitative analysis of figure (e).