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. 2015 Dec 8;14(24):3851–3863. doi: 10.1080/15384101.2015.1106760

Figure 5.

Figure 5.

Cell cycle changes after inhibition or activation of the H3K56ac regulatory pathway. (A, B) Inhibition of sirtuin activity in HeLa cells (AGK-2, nicotinamide) induced an increase in H3K9ac and truncated form of H3K56ac without influencing ASF1A levels. The SIRT1 activator resveratrol induced an increase in H3K9ac levels together with ASF1A. C646, a p300/CBP inhibitor, reduced H3K56ac levels. No changes in H3K56 levels were observed after DNA replication inhibition by aphidicolin. (C) Increased and decreased H3K56ac levels are both connected to cell cycle perturbations in HeLa cells. The graph represents the proportion of cells in the different cell cycle stages. (D) Increased and decreased H3K56ac levels are both connected to the inhibition of DNA replication in HeLa cells. The cells were treated with EdU for 2 h. The chart represents the ratio of the EdU-positive, EdU-negative and mitotic cells. After 24 h of treatment with resveratrol and C646, the number of EdU-positive cells was reduced. At least 500 cells were analyzed for each inhibitor. (E) The number of H3K56ac foci (red) in the EdU-positive (green) and -negative cells was similar after SIRT2 inhibition. The cells were treated with AGK-2 for 24 h. No noticeable difference between the EdU-positive (green) and -negative cells was observed. H3K56ac localized to mitotic chromosomes after sirtuin inhibition. After 24 h of SIRT2 inhibition, H3K56ac (red) colocalized with the mitotic DNA (blue). (F) A delay in the cell cycle progression in HeLa cells after inhibition or activation of the H3K56ac regulatory pathway. HeLa cells were synchronized using a double thymidine procedure. After release from the thymidine block, the cells were treated with different inhibitors for 12 h. Cell cycle progression was measured by staining with PI and flow cytometry. Twelve hours is sufficient time to reach G1 phase in the control cells treated with DMSO. SIRT1-3 inhibition by AGK-2 does not affect cell cycle progression. A cell cycle delay was observed after sirtuin inhibition by nicotinamide. G1/S and S phase arrest was observed after the HeLa cells were treated with resveratrol and C646. Aphidicolin, a DNA replication inhibitor, was used as the G1/S phase arrest control.