Figure 2. D-gal treatment induced premature senescence of GBM cells. (A) Representative images of C6 cells (in the bright field) treated with D-gal at 0 mM, 28 mM, 111 mM and 222 mM for 8 d. (B) Immunostaining analysis of PH3 (green) in control cells and in C6 cells treated with 222 mM D-gal for 8 d. (C) Quantitative analysis of the percentage of PH3+ cells over total cells as shown in (B) (n=15). (D) Representative images showing β-galactosidase staining in C6 cells treated with D-gal at 0 mM, 28 mM, 111 mM and 222 mM concentrations for 2, 4, 6 and 8 d. (E) Quantification of the percentage of β-galactosidase+ C6 cells over total cells as shown in (D) (n=15). Scale bars, 20 μm. Data shown are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.