Figure 5.

Wogonin induced senescence-associated heterochromatin foci in senescent cells. (a) Hut-102 and Jurkat cells were treated with or without 20 μM wogonin for 5 d; then, the cells were fixed and permeabilized, and nuclei were stained with DAPI (blue). SAHF formed in wogonin-treated Hut-102 and Jurkat cells. Representative confocal images for wogonin-induced SAHF formation in cells from confocal laser scanning microscopy are shown (original magnification ×1000; immersion objective ×100/×100 with immersion oil type). Images are representative of three independent experiments. (b) The percentage of positive cells with the DNA agglutination points (DAPI foci) was counted (with 20 cells counted per field). Columns represent the mean from three parallel experiments (mean ± SEM). ^∗∗p < 0.01, ^∗∗∗p < 0.001, compared with the control group. (c) Hut-102 and Jurkat cells were treated with or without 20 μM wogonin for 5 d. The expression of the SAHF marker protein H3K9me3 in control and wogonin-treated Hut-102 and Jurkat cells was performed by western blot. β-Actin was used as loading controls. (d) Relative protein expression level of H3K9me3 was determined. Columns represent the mean from three parallel experiments (mean ± SEM). ^∗p < 0.05, ^∗∗p < 0.01, compared with the control group. (e) Hut-102 and Jurkat cells were treated with or without 20 μM wogonin for 5 d; then, the cells were fixed, permeabilized, and stained with an antibody against H3K9me3 (green), while nuclei were stained with DAPI (blue). Immunofluorescent images showed the distribution of H3K9me3 in Hut-102 and Jurkat cells (original magnification ×1000; immersion objective ×100/×100 with immersion oil type). Images are representative of three independent experiments.