Fig. 6
PR130 expression antagonises ATM phosphorylation. a Schematic of PPP2R3A knockout using CRISPR-Cas9 technology. Humanised S. pyogenes Cas9 nuclease was expressed in HCT116 cells with two guide RNAs (gRNA) to introduce two DSB into the PPP2R3A gene (684 bp apart) as described in the Methods section. The usage of two cleavage sites increased the chances to create a successful knockout. b HCT116ΔgRNA and HCT116ΔPR130 (clone #16) cells were cultured with 2 µM MS-275 and/or 1 mM hydroxyurea (HU) for 24 h. ATM, pATM (S1981), p53, pp53 (S15) and PR130 were detected by western blot. β-Actin served as loading control. Numbers indicate densitometric analysis of pATM signal relative to HU-treated sample of each cell line and normalised to β-Actin signal (n = 3). c HCT116 cells were transiently transfected with indicated siRNAs. After 24 h, cells were treated with 1 mM HU and 2 µM MS-275 (24 h). Protein detection was performed by immunoblot (n = 2). d After 20 h of treatment with HU (1 mM) and MS-275 (2 µM), okadaic acid (OA, 25 nM) was added for further 4 h. Co-immunoprecipitations (IP) with anti-pS1981-ATM antibody, rabbit pre-immune serum (pre) or no antibody (no AB) were analysed for pATM and PR130 by western blot. Input (IN) represents 6% of the lysates used for IP. The right panel shows results that were obtained with the same strategy, including individual treatment with HU and MS-275. Data represent results from three independent experiments that were carried out in a similar manner. e IP performed with PR130 antibody using lysates from untreated and MS-275-treated HCT116. Precipitates were probed with pan-acetyl-lysine (ac-Lys) and PR130 antibody. Input (IN) is 6% of the lysate used for immunoprecipitation (n = 2). Numbers indicate densitometric analysis of ac-Lys and PR130 signal relative to untreated cells. f HCT116 cells were transfected with HA-PR130 or vector control (pcDNA3.1) for 24 h and subsequently treated with MS-275 (2 µM, 24 h). Phosphatase activity of the HA-precipitates against phospho-S1981-ATM peptide or a threonine phosphopeptide (positive control) were measured in vitro as liberated pmol phosphate/min. Data are presented as mean ± SEM (n = 3). The western blot verifies precipitation of HA-tagged PR130
