Figure 1.

Antagonistic effect of melatonin against OIS-induced senescence of human ovarian surface epithelial cells. (A) HOSEpiCs were subjected to the retrovirus-mediated gene transfer of H-RasV12 or of an empty vector as a control and treated with melatonin at 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM concentrations. Representative senescence-associated β-galactosidase staining (SA-β-gal) and 4′,6-diamidino-2-phenylindole (DAPI) staining images of HOSEpiCs and percent SA-β-gal-positive cells; scale bar, 50 μm. (B) Ratio of telomere to single copy gene expression (T/S) of the HOSEpiCs and the relative telomere lengths computed from T/S. (C) Growth curves of HOSEpiCs treated as described in (A). Number of cells from each group counted at the indicated time points. (D) Representative immunofluorescence images of BrdU-labelled and DAPI-counterstained HOSEpiCs transfected with an H-RasV12 or an empty control vector, where indicated, treated with (+) or without (−) 1 μM melatonin for 8 days. Histogram of BrdU-positive cells treated as described above. (E) The senescent markers p53, p21, and p16 were analyzed by Western blotting. The results are presented in the scatter plot. β-actin was used as the loading control. Statistical values are presented as the mean ± standard error of the mean of three independent experiments. A P < 0.05 was considered statistically significant using one-way analysis of variance. ∗, P < 0.05.