Fig. 8: 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.