Research Paper Volume 14, Issue 5 pp 2131—2147

Figure 1. Senescent human fibroblasts express markers of senescence. IMR-90 fibroblasts were induced to senesce by doxorubicin (300 nM, 24 h) and SA-β-Gal staining was performed on day 9 after doxorubicin treatment. (A) Representative images of SA-β-Gal stained senescent (S) and non-senescent (NS) cells. (B) Quantification of SA-β-gal-positive cells in NS and S IMR-90 cells. Four fields were quantified per well (n=6) with a total of 7713 and 2666 cells counted for NS and S cells, respectively. (C) Immunofluorescence was performed to detect γ-H2AX in NS and S IMR-90 cells, 10 days after exposure to vehicle or doxorubicin, respectively. Representative images of NS and S cells stained for γ-H2AX (green) and Hoechst (blue). (D) The percentage of cells with 3 or more γ-H2AX foci/cell (γ-H2AX+ cells) was scored from a total of 780 NS and 387 S cells. The results are presented as mean % of cells with 3 or more foci/cell. (E–G) mRNA levels of cell cycle regulators p16 and p21, Lamin B1, and various SASP factors, IL-6, IL-8, and IL-1α assessed through Quantitative Realtime PCR in NS and S IMR-90 cells (n=3). All results are presented as a mean and error bars represent ±SEM. Statistical analysis performed using unpaired t test. *p < 0.05, **p < 0.01, and ***p < 0.001.