Figure 1.

Establishment of an HMEC model of senescence. A, HMECs ceased proliferation at 35–40 population doublings. HMECs were cultured in M87A medium with medium replacement every 2 days (every day after 50% confluence) and passaged at 80–90% confluence (21). Four independent cultures are shown. B, HMEC viability was unaffected by senescence arrest. Viability of proliferating (<15 population doublings) and senescent (>35 population doublings) cells is shown. HMEC viability was measured by trypan blue staining. C, HMECs acquired enlarged, flattened, and irregular morphology at PD 40. Phase-contrast images and cell sizes are represented at PD 10 and 40. Scale bar, 100 μm. **, p < 2 × 10⁻¹⁶ by Mann–Whitney U test. D, HMECs showed increased activity of SA-β-gal at PD 35 measured by fluorescence signal of C₁₂FDG. SA-β-gal measurements are shown at PD 10 and 35. SA-β-gal activity was calculated as follows: mean of samples labeled with C₁₂FDG − mean of samples without C₁₂FDG. **, p value less than 0.0001 by Student's t test. E, measurement of DNA synthesis by EdU incorporation showed decreased DNA synthesis in senescent cells. **, p < 0.001 by Student's t test. F, immunofluorescence imaging of proliferating and senescent HMECs using Hoechst and membrane dye. HMECs at PD 37 showed an increased number of multinucleated cells (denoted by arrows). Scale bar, 50 μm. *, p < 0.002 by Student's t test. G, immunoblot for p16, p21, PAI-1, and actin with lysates from proliferating and senescent HMECs. Recombinant human p16 protein (40 ng) was used as a positive control in the p16 Western blotting. H, immunoblot for γ-H2AX and actin with lysates from proliferating and senescent HMECs. A lysate from the 293T cell line treated with the DNA-damaging agent doxorubicin (Dox; 1 μm for 24 h) or control was used as a positive control. Actin was used as an equal loading control. Error bars, S.D.