Fig. 5
PR130 upregulation by MS-275 represses checkpoint kinase signalling. a HCT116 cells were stimulated with 1 mM hydroxyurea (HU) and/or 2 µM MS-275. Wedges signify increasing incubation times (6, 10 and 14 h). Asterisks signify pre-incubation with MS-275 for 1 h. Indicated (phosphorylated) proteins were analysed by western blot. Vinculin served as loading control. b HCT116 cells were cultured with 2 µM MS-275 and/or 1 mM HU for 24 h and incubated with the PP2A inhibitor ocadaic acid (25 nM) for additional 4 h. Phosphorylated forms of ATM, CHK1 and mSIN3A (loading control) were detected by immunoblot. Numbers indicate densitometric analysis of western blot signals relative to HU-treated samples and normalised to loading control. c Relative mRNA levels of PPP2R3A in HCT116 cells evaluated by quantitative RT-PCR. Cells were treated with 1 mM HU and/or 2 µM MS-275 (6–24 h). Results show the mean ± SD (n = 3; one-way ANOVA, ***P < 0.001; ****P < 0.0001). d Cells were cultured with or without increasing amounts of MS-275 (0.5; 1; 2; 5 µM) alone or in combination with 1 mM HU for 24 h. Indicated proteins were detected by immunoblot. Densitometric analysis of PR130 signals is indicated relative to untreated cells and normalised to Vinculin levels (n = 3). e Knockdown HDAC1–3 was performed with siRNAs by transient transfection for 48 h. 1 mM HU was added for further 24 h. PR130 was detected by immunoblotting, quantified relative to untreated siCtrl cells and normalised to mSIN3A levels (loading control). f ChIP was performed to detect acetylated histone H3 and the binding of HDAC1/HDAC2 to the PPP2R3A promoter in presence/absence of MS-275. Acetylation of histone H3 is set as 1. Results show the mean ± SD (n = 3). g Cells were transiently transfected and treated as described in e. Indicated (phosphorylated) proteins were detected by western blot. h HCT116 cells were transfected with pcDNA3.1 or HA-PR130 for 48 h and treated with 1 mM HU and 2 µM MS-275 for additional 24 h. Indicated proteins were detected by immunoblotting. Western blot signals were quantified relative to HU-treated cells and normalised to loading controls (Tubulin, Vinculin; n = 2)
