Fig. 6

The senescence properties were determined in lean hASCs cultured on different hASC-derived matrixes. a Representative images of lean hASCs cultured on different hASC-derived matrix for SA-β-Gal staining and immunostained with antibody against γ-H2AX. Histogram showing the difference of the positive area percentage of SA-β-Gal and the relative intensity of γ-H2AX normalized to DAPI each nuclear of cells. The number of fields for SA-β-Gal staining was 14–23 in each group, respectively. n = 3 independent experiments from lean hASC-derived matrix, n = 3 different donors from obese hASC-derived matrix. Scale bars, 100 μm. The number of cells for the expression of γ-H2AX was 519 in lean hASC-derived matrix and 407 in obese hASC-derived matrix. Scale bars, 25 μm. b The relative mRNA levels of cell cycle inhibitors genes, SASP-related genes, and GLB1 in lean hASCs cultured on different ECMs were determined by quantitative RT-PCR. n = 3 independent experiments from lean hASC-derived matrix, n = 3 different donors from obese hASC-derived matrix. Fold represents that the relative expression of each gene was normalized against lean hASC-derived matrix from each independent experiment. c Representative images of lean hASCs cultured on the anti-TGF-β1 neutralizing antibody and control IgG1-treated obese hASC-derived matrix for SA-β-Gal staining and immunostained with antibody against γ-H2AX. Histogram showing the difference of the percentage of SA-β-Gal-positive area in each field, the percentage of SA-β-Gal-positive area was normalized against IgG1 group from each independent experiment. The number of fields was 16–24 for SA-β-Gal staining in each group, respectively. n = 3 different donors per group. Scale bars, 100 μm. Histogram showing the relative intensity of γ-H2AX normalized to DAPI each nuclear of cells. The number of cells for the expression of γ-H2AX was 491 in IgG1 group and 429 in anti-TGF-β1 neutralizing antibody group. Scale bars, 25 μm. Fold represents that the relative expression of each gene was normalized against control IgG1 group from each independent experiment. d The relative mRNA levels of cell cycle inhibitors genes, SASP-related genes, and GLB1 in lean hASCs cultured on different ECMs were determined by quantitative RT-PCR. Fold represents the relative expression of each gene was normalized against IgG1 group from each independent experiment. n = 3 different donors per group. Significant difference by unpaired two-tailed paired t test. Data are presented as the means ± SEM. hASC-matrix, hASC-derived matrix