Fig. 5: PHGDH enhances PKM2-catalyzed H3pT11 and prevents premature cellular senescence.

a Immunoblots of H3pT11 in HUVECs during cellular senescence. Analysis of H3pT11 in control (shCtrl) and PKM2-knockdown (shPKM2) HUVECs as determined by immunoblots (b) and immunofluorescence (c). d Lentiviral-mediated overexpression of WT PKM2 but not PKM2 K367M mutant increased H3pT11 in HUVECs. Analysis of H3pT11 in control (shCtrl) and PHGDH-knockdown (shPHGDH) HUVECs when cultured in serine-free medium as determined by immunoblots (e) and immunofluorescence (f). g Immunoblots of H3pT11 in control (siCtrl) and ATF4-knockdown (siATF4) HUVECs. h In vitro kinase assay showing PHGDH enhanced the activity of PKM2 to phosphorylate H3T11. FLAG-PKM2 was purified from control or PHGDH-overexpression (Myc-PHGDH) HeLa cells with anti-FLAG beads. The in vitro kinase assay was performed with purified FLAG-PKM2 and recombinant purified histone H3. H3pT11 was detected by immunoblots. i In vitro kinase assay showing recombinant His-PHGDH directly stimulated the activity of recombinant His-PKM2 to phosphorylate H3T11. j Effect of serine (Ser) on H3pT11 in control (siCtrl), PHGDH-knockdown (siPHGDH) and PKM2-knockdown (siPKM2) HUVECs. k In vitro kinase assay showing serine (Ser) directly stimulates the activity of recombinant His-PKM2 to phosphorylate H3T11. Overexpression of WT H3 but not H3T11A mutant alleviated cellular senescence of HUVECs as determined by expression of p21 (l), SAHF detection and SA-β-gal staining (m) in HUVECs. HUVECs were infected with lentiviruses to overexpress WT H3 and H3T11A. For b, e, j, data represent means ± SE; n = 3 independent experiments. For m, n = 5 independent experiments. Two-sided t-tests were used for statistical analysis. For a, c, d, f-i, k, a typical example of two biological replicates is shown.