Fig. 1.

HDACi impairs checkpoint signalling upon replicative stress. a HCT116 cells were stimulated with 1 mM hydroxyurea (HU) and 2 µM MS-275 for 24 h. Western blot analyses of whole-cell lysates were performed to detect ATM and ATR as well as their phosphorylated forms (pATM S1981; pATR T1989). β-Actin served as loading control. Numbers indicate densitometric analysis of signals relative to HU-treated samples and normalised to β-Actin (n = 3). b Densitometric evaluation of ATM phosphorylation at S1981 and ATM levels (after 24 h) following protein detection via immunoblot. Data were normalised to the loading controls. The respective amounts (phosphorylated) proteins are compared to those of HU-treated cells. Results represent the mean ± SD (n = 3; one-way ANOVA; ****P < 0.0001). c Cells were treated with 1 mM HU and 2 µM MS-275 for 6-24 h. Whole-cell extracts were blotted to two membranes to detect phosphorylated and total levels of ATM, CHK1 (pCHK1 S317) and CHK2 (pCHK2 T68) by immunoblot. HSP70 and mSIN3A are loading controls. Numbers indicate densitometric analysis of signal relative to HU-treated cells and normalised to the respective loading control (n = 3). d Densitometric analysis of phosphorylated (S317) and total CHK1 levels after 24 h detected via western blot. Data were normalised to the respective loading controls. Results display relative amounts of pCHK1/CHK1 compared to HU-treated cells as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA (****P < 0.0001). e Cells were either pre-incubated with 2 µM MS-275 or were left untreated for 24 h and subsequently exposed to 10 J/m² UVC. Plates were harvested 0, 1, 2 and 3 h after irradiation. Indicated proteins were detected via western blot. HSP70 and β-Actin served as loading controls