Fig 2: PPP1CA–Rif1 dephosphorylates H3T11 to regulate telomere silencing and cellular senescence in mammals.a RT-qPCR analysis of the transcription of telomere-proximal genes (C1S, CCND2, CDL163L1) in control (shCtrl) and PPP1CA-knockdown (shPPP1CA) HUVECs. b ChIP-qPCR analysis of PPP1CA occupancy at telomere-proximal genes in HUVECs. c ChIP-qPCR analysis of the occupancy of H3pT11/H3 at telomere-proximal genes in control (shCtrl) and PPP1CA-knockdown (shPPP1CA) HUVECs. d Immunoblot analysis of H3pT11 in control (shCtrl) and Rif1-knockdown (shRif1#1, shRif1#2) HUVECs. e Co-IP and reciprocal IP showing the interaction between PPP1CA and Rif1. f ChIP-qPCR analysis of PPP1CA occupancy at telomere-proximal genes in control (shCtrl) and Rif1-knockdown (shRif1) HUVECs. g ChIP-qPCR analysis of H3pT11/H3 occupancy at telomere-proximal genes in control (shCtrl) and Rif1-knockdown (shRif1) HUVECs. h RT-qPCR analysis of the transcription of telomere-proximal genes in control (shCtrl) and Rif1-knockdown (shRif1) HUVECs. i Knockdown of PPP1CA and Rif1 significantly increased telomere length. j Effect of PPP1CA knockdown on the senescence of HUVECs as determined by SAHF formation (DAPI staining) and SA-β-gal staining. Right panel: quantification of the number of SAHF-positive and SA-β-gal positive HUVECs. k RT-qPCR analysis of the transcription of P21, P16, IL1A, IL1B, IL6 and IL8 in shCtrl and shPPP1CA HUVECs. l Effect of Rif1 knockdown on the senescence of HUVECs as determined by SAHF formation (DAPI staining) and SA-β-gal staining. m RT-qPCR analysis of the transcription of P21, P16, IL1A, IL1B, IL6 and IL8 in shCtrl and shRif1 HUVECs. For a–d, f–m, data represent the mean ± SE of three biological independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: FBXW7 vs. CDC7 synthetic lethality is RIF1 dependent. (A) Western blot showing that RIF1 levels are increased in the FBXW7mutant cells. Uncropped blot scans are presented in Fig. S1C. Quantification was conducted by band densitometry and normalization to the loading controls. (B) Western blot analysis of MCF10A FBXW7‐24 cells, transfected with a SMARTpool or individual siRNAs targeting RIF1, showing efficient downregulation of the target protein. (C, D). Dose response curves of MCF10A p53mutant and FBXW7‐24 cells to CDC7i (XL413) upon RIF1 depletion. siRIF1_1 oligo was not used in these experiments as it does not lead to RIF1 depletion. The data points represent mean and standard deviation of n = 3 experiments; statistical analysis was done by two‐way ANOVA P < 0.001.

Fig 4: Loss of ASF1 rescues end resection defect caused by BRCA1 deficiency.A, RPE1 FLAG-Cas9 P53−/− BRCA1−/− LentiV2 and sgASF1A + sgASF1B cells were untreated (NT) or treated with 10 Gy of IR and released for 6 h. Cells were fixed and then immunostained with γH2AX and RAD51 antibodies. B, statistical quantification of γH2AX foci formation from (A). Data are represented as mean (±SD) (n = 3). ns (Student's t test). C, statistical quantification of RAD51 foci formation from (A). Data are represented as mean (±SD) (n = 3). ∗∗p < 0.01 (Student's t test). D, RPE1 FLAG-Cas9 P53−/− BRCA1−/− LentiV2 and sgASF1A + sgASF1B cells were untreated (NT) or treated with 10 Gy of IR and released for 6 h. Cells were fixed and then immunostained with RPA2 and RIF1 antibodies. E, statistical quantification of RPA2 foci formation from (B). Data are represented as mean ± SD (n = 3), ∗∗∗p < 0.001, Student's t test. F, statistical quantification of RIF1 foci formation from (B). Data are represented as mean (±SD) (n = 3). ns (Student's t test). G, Western blot to determine the siRNA knockdown efficiency in U2OS DR-GFP reporter cell lines transfected with indicated siRNAs. H, U2OS DR-GFP reporter cell lines transfected with indicated siRNAs and I-SceI plasmids were collected for flow cytometry analysis to determine HR repair efficiency. Quantification data are represented as mean (±SD) (n = 3). ∗∗p < 0.01, ∗∗∗p < 0.001 (Student's t test). DR, direct repeat; HR, homologous recombination; IR, ionizing radiation; ns, not significant; RPA, replication protein A.

Fig 5: TEADs interacts with DNA repair proteins. A, bar graph representing the abundance (average sum PSMs) of canonical binding partners, YAP, TAZ, and VGLL4 binding to TEAD equally in HEK293 cells. B, as described in (A) but for Detroit 562 cells. C, co-immunoprecipitation assay comparing MYC-tagged WT TEAD3 and MYC-tagged NLS mutant (NLSm) TEAD3. Figure is representative of three or more replicates. WCL, whole cell lysate. D, co-immunoprecipitation of MYC-tagged WT TEAD3 after treatment with 20 μg/ml bleomycin (BLM) for the indicated time points. Figure is representative of three or more replicates. ∗indicates a nonspecific band detected along with RIF1. RIF1, Rap1-interacting factor 1; RIF1, Rap1-interacting factor 1; TEADs, TEA domain family members 1 to 4; TAZ, transcriptional co-activator with PDZ-binding motif; PARP1, poly(ADP-ribose) polymerase 1; XRCC6, X-ray repair cross-complementing protein 6; XRCC5, X-ray repair cross-complementing protein 5; YAP, Yes-associated protein.