Fig. 7
PR130 modulates cell cycle control during replicative stress. a HCT116ΔgRNA and HCT116ΔPR130 (clone #3) cells were treated with 2 µM MS-275 and/or 1 mM hydroxyurea (HU) for 24 h. Indicated proteins and their phosphorylation were analysed by western blot; β-Actin served as loading control. b Densitometric analysis of CHK1 phosphorylation (S317) and total CHK1 levels after 24 h following protein detection via western blot. Data were normalised to the respective loading controls. The amount of (phosphorylated) proteins was normalised to that of HU-treated cells. Results represent the mean ± SD (n = 8; two-way ANOVA, ****P < 0.0001). c HCT116ΔgRNA (left panel) and HCT116ΔPR130 (clone #3; right panel) cells were treated with 1 mM HU and 2–5 µM MS-275 for 28 h. Cells were stained with PI and analysed using flow cytometry. Data are mean ± SD (n = 3). d Cells were incubated as stated in a. Immunoblots were carried out for WEE1, pHDAC2, pCDK1 and HSP90 as loading control. Numbers indicate densitometric analysis of western blot signals relative to untreated HCT116ΔgRNA cells and normalised to HSP90 (n = 3). e Western blot analysis of p21 and β-actin (loading control). Signals of p21 are displayed relative to untreated controls and normalised to β-actin. f Increasing doses of 1–5 mM HU stall the cell cycle of HCT116 cells more pronouncedly in early G1/S phase. HU was applied for 24 h (n = 3). g HU (1–5 mM) was applied to HCT116 cells for 24 h. Western blot was done as indicated. Numbers indicate densitometric analysis of pCHK1, p21 and pCDK1 signals relative to untreated control and normalised to loading control (n = 3)
