Fig 2: PP2A inhibition transiently promotes cell proliferation leading to evasion from dormancy. (A–C) Immunoblots of cell lysate collected from A375 and A375 BRAFi-resistant cells after 2 and 3 d of siRNA transfection (A), 3 h of drug treatment (B), and time-course treatment with 0.2 µM phendione (C) as indicated in each panel, respectively. siRNAs used for transfection include control siRNA, siRNA targeting the catalytic subunits of protein phosphatases PP1-C (siRNA pool against α, β, and γ isoforms) and PP2A-C (siRNA pool against α and β isoforms). (D) Quantification of MYC protein level from immunoblots after time-course treatment with 0.2 µM phendione in A375 and A375 BRAFi-resistant cells. GAPDH protein level was used as internal control to perform normalization. Data shown are mean ± SEM of three independent experiments. (E,F) Histogram and quantification of fluorescent signal from Vybrant DiD cell-labeling dye detected by flow cytometry. (G,H) Quantification of incorporated bromodeoxyuridine (BrdU) measured by flow cytometry. Data shown are mean ± SD of two independent experiments. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. Unpaired two-tailed Student's t-test was used for statistical analysis.

Fig 3: PP1 and PNUTS are recruited to DNA damage sites in vitro and in vivo. (A) As described in Materials and Methods, beads conjugated with biotin-(dA-dT)70 were incubated in Xenopus egg extracts for 30 min, re-isolated, resolved by SDS-PAGE and analyzed by mass spectrometry. The identified repair proteins and phosphatase subunits are shown, along with the numbers of peptides. A control pull-down was performed in Xenopus egg extracts using beads without oligonucleotides. (B) The biotin-(dA-dT)70 pull-down was performed as in panel A. The extract input, control pull-down and biotin-(dA-dT)70 pull-down were analyzed by immunoblotting for Ku70, PNUTS and PP1γ. (C) HeLa cells transfected with GFP-PNUTS were subjected to laser microirradiation as described in Materials and Methods. The fluorescent signal of GFP and immunofluorescent signal of NBS1 phospho-S343 are shown. (D) HeLa cells were subjected to laser microirradiation. The immunofluorescent signals of PP1β and ATM phospho-S1981 are shown.

Fig 4: A Cdk9-PP1 switch governing Spt5 phosphorylation is conserved in human cells.a Purified, recombinant Cdk9/cyclin T1 phosphorylates wild-type (WT) GFP-PP1γ, expressed in human cells and recovered by anti-GFP immunoprecipitation, but not a Thr311→Ala (TA) mutant variant. Phosphorylation was detected with antibody specific for the carboxy-terminal phosphorylation site (Thr320) in PP1α isoform, analogous to Thr311 of PP1γ. b Inhibition of Cdk9 or Cdk1 diminishes PP1γ-inhibitory phosphorylation in human cells. HCT116 cells were treated with DMSO, a Cdk1 inhibitor (RO-3306), a Cdk9 inhibitor (NVP-2), or both, as indicated. Extracts were analyzed by direct immunoblotting (lanes 1–4), or anti-PP1γ immunoprecipitation (IP) followed by immunoblotting (lanes 5–8), with the indicated antibodies. c Cdk9 inhibition diminishes phosphorylation of Spt5-Thr806 but not Ser2 of the Pol II CTD. HCT116 cells were treated with the indicated concentrations of NVP-2 for 1 h and extracts were immunoblotted with the indicated antibodies. d HCT116 cells were treated with 250 nM NVP-2 for indicated times and extracts were immunoblotted with antibodies specific for Spt5, Spt5-pThr806, and Spt5-pSer666, as indicated. Immunoblot (n = 1) signals were quantified with ImageJ software. e Spt5-derived phosphopeptides containing pSer666 or pThr806 or a control histone H3-derived phosphopeptide containing pSer10, as indicated, were incubated with purified PP1 or lambda phosphatase, as indicated, and phosphate release was measured colorimetrically. Individual data points are shown from three biological replicates (n = 3); error bars indicate ±standard deviation (±s.d.) from mean. f HCT116 cells were transfected with an siRNA cocktail targeting all three PP1 catalytic-subunit isoforms or a scrambled control (C) siRNA, as indicated, and extracts were immunoblotted for the indicated proteins or protein modifications. Experiments were performed twice with similar results (a–c, f). Source data are provided as a Source data file. Uncropped blots can be found in the Supplementary information.

Fig 5: MYC and PP1/PNUTS are co-amplified in cancer and co-bind to promoters of MYC target genes. a MYC and PPP1R10, PPP1CA, PPP1CB, or PPP1CC gene amplification is evident and demonstrates significant co-occurrence in breast invasive carcinoma. b HO15.19 MYC knockout cells (EV) or cells ectopically expressing MYC were lysed and immunoblotted with antibodies to PNUTS, MYC, and actin. Shown are representative images of n = 3. c MCF10A cells were analyzed by chromatin immunoprecipitation (ChIP) using MYC or IgG antibody. ChIP’d DNA was quantified by qPCR for MYC or IgG binding to CAD and PPP1R10 promoters or chr6 (negative control). d MCF10A cells probed with MYC rabbit antibody and/or a PNUTS mouse antibody. The proximity of MYC and PNUTS was assayed using the Duolink Proximity Ligation Assay (PLA) In Situ Red Starter kit (Sigma) as per manufacturer’s instructions. Representative PLA signal (red or white) and nuclear staining (DAPI; blue) are shown for cells probed with anti-MYC alone (top row), anti-PNUTS alone (middle row), or anti-MYC and anti-PNUTS (bottom row). PLA signal was quantified and shown as median number of foci per cell with range (n = 3). *p < 0.05, **p < 0.01, one-way ANOVA with Bonferroni test. Scale bars represent 20 µm. e MCF10A cells analyzed by ChIP with MYC, PNUTS or IgG antibody. ChIP’d DNA was quantified using qPCR for MYC, PNUTS or IgG binding to MYC target genes promoters, CDK4, NCL, and HES1, or chr6 (negative control). f For ChIP-re-ChIP analysis, MCF10A cells lysate incubated with MYC rabbit antibody followed by PNUTS (top panel), or MAX (bottom panel) antibody along with IgG antibody as a control. ChIP’d DNA was quantified similarly as above for ChIP experiment. For c, e, f: shown are % input mean (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA with Bonferroni test. Error bars represent s.d.