Fig. 2. Serine alleviates senescence of endothelial cells by targeting PKM2.

a Serine bound to PKM2 as determined by DARTS. HeLa lysates were incubated with or without 300 μM serine and then subjected to pronase (50 μg/ml) digestion for 15 min. The digested products were resolved on SDS-PAGE and detected by Coomassie blue-staining. b PKM2 H464 is required for PKM2 to bind serine as determined by DARTS. The lysates of HUVECs (P10) that stably overexpress WT FLAG-PKM2 and FLAG-PKM2 H464A were incubated with 0–600 μM serine and then subjected to pronase (50 μg/ml) digestion for 15 min. The digested products were analyzed by immunoblots with anti-FLAG antibody. Actin was served as a loading indicator. c Molecular docking showing the interaction between serine (Ser, green color) and PKM2. PKM2 residues that bind serine were labeled. d Immunoblots of PKM2 and PKM1 in HUVECs during senescence. e RT-qPCR analysis of P21 and HMGB1 transcription in control (siCtrl) and PKM2-knockdown (siPKM2) HUVECs. f Effect of PKM2 knockdown on cellular senescence in HUVECs (young, P10; senescent, P20) as determined by SAHF detection and SA-β-gal staining. g Effect of ectopic expression of WT PKM2 and its mutants (PKM2 K367M, H464A) on cellular senescence as determined by SAHF detection and SA-β-gal staining. HUVECs that stably overexpress PKM2 (WT PKM2, K367M, H464A) were transfected with siPKM2 to knock down the endogenous PKM2. The ectopic expressed PKM2 was resistant to siPKM2. h Effect of serine (Ser) on cellular senescence in control (siCtrl) and PKM2-knockdown (siPKM2) HUVECs. HUVECs were cultured in serine-free medium and then treated with 300 μM serine for 24 h. For e, g, h, the HUVECs at P10 were used in the experiments. For e, f, data represent means ± SE; n = 3 independent experiments. For g, n = 4 independent experiments. For h, n = 5 independent experiments. Two-sided t-tests were used for statistical analysis. For b, d, a typical example of three biological replicates is shown.