Fig 2: CTLA-4 interacts with PP2A, promotes the translocation of PP2A-A into the cytoplasm, and induces autophosphorylation of ATM at Ser1981 after zeocin treatment. (A–C) Co-IP analysis of the interaction of CTLA-4 with PP2A. HeLa cells were transfected with Myc-CTLA-4-WT and Myc-CTLA-4-MT proteins with the Y201F and Y218F mutations. About 24 h after transfection, the cells were treated with 500 µg/ml zeocin for 1 h and harvested at the indicated times. (D) For endogenous co-immunoprecipitation experiments, CD4+ T cells from B7-1/B7-2-/- mice were lysed. (E) Nuclear and cytoplasmic levels of PP2A-A in HeLa cells were detected using western blotting. HeLa cells were transfected with Myc-CTLA-4 (left panel), and pcDNA and Myc-CTLA-4 (right panel) for 24 h. At the indicated times after cessation of treatment, the cells were lysed and separated into nuclear and cytosolic fractions. GAPDH, a marker of the cytosolic fraction; histone H3.1, a marker of the nuclear fraction. (F) Double immunofluorescence staining was performed to detect the PP2A-A and CTLA-4 proteins and their colocalization in HeLa cells. HeLa cells were transfected with Myc-CTLA-4 for 24 h. The cells were treated with 500 µg/ml zeocin for 1 h, harvested at the indicated time points, and then subjected to immunofluorescence staining using specific antibodies against PP2A-A (green), CTLA-4 (red), and Scale bar, 20 µm. (G) ATM was immunoprecipitated and immunoblotted to detect the levels of P-ATM-S1981, total ATM, and the PP2A-A and PP2A-C subunits. Right panel: HeLa cells were transfected with pcDNA, GFP-CTLA-4-WT, a GFP-CTLA-4-TAIL (tail only) mutant, and GFP-CTLA-4-MT sequences containing Y201F and Y218F mutations. About 24 h after transfection, the cells were treated with 500 µg/ml zeocin for 1 h and harvested at the indicated time points. The CTLA-4 extracellular domain is shown as orange ovals, the transmembrane domain is indicated by gray bars, the CTLA-4 cytoplasmic tail is shown as red bars, the double-mutant Y201F/Y218 is shown with blue lines, and GFP is indicated by green circles. Left panel: CD4+ T cells from B7-1/B7-2-/- and B7-1/B7-2/CTLA-4-/- mice were treated with 500 µg/ml zeocin for 1 h and then released and harvested at the indicated time points.

Fig 3: Comparison of PP2A expression in mouse oocytes. A, Localization of PP2A in mouse oocytes. Immunofluorescent staining of PP2A (red) in the 12-h aged and fresh oocytes. DNA (blue) was stained with Hoechst 33258. D-PBS was used as the negative control instead of the primary antibody. Scale bar = 10 µm. B, Comparison of brightness values of PP2A signal in the 12-h aged and fresh oocytes. *p < 0.05. C, Relative expression of PP2A to ß-actin. Bars with different superscripts indicate significant differences (p < 0.05). D, Western blotting for PP2A-Aa and Aß subunit proteins in MII oocyte. ß-actin was used as a loading control

Fig 4: CTLA-4 activates ATM by anchoring the ATM inhibitor PP2A in the cytoplasm following transient treatment with zeocin in T cells, exacerbating the DNA damage response and inducing apoptosis.

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)