Figure 3. Succinylation at K498 of PKM2 sensitizes cells to oxidative damage.

A. H₂O₂ and Mena treatment decrease succinylation level of PKM2. 293T cells were transfected with Flag-PKM2 and treated with 500 uM H₂O₂ for 1h or 50uM Mena for 30min as indicated. The succinylation levels of PKM2 were determined by western blot. B. H₂O₂ and Mena induced oxidative stress decreases PKM2 activity. 293T cells were transfected with Flag-PKM2 and treated with 500 uM H₂O₂ for 1h or 50uM Mena for 30min, followed by immunoprecipitation and enzyme activity was measured and mean values of relative enzyme activity of triplicate experiments with standard deviation (±SD) are presented. C. H₂O₂ and Mena induced oxidative stress decreases wild-type PKM2 activity, but not K498E and K498R mutants. 293T cells were transfected with indicated plasmids, followed by treatment of 500 uM H₂O₂ for 1h or 50uM Mena for 30min, proteins were immunoprecipitated, and PKM2 activity was assayed. Relative enzyme activities of triplicate experiments ± SD are presented. D. A549 cells stably expressing succinylation mimetic K498E mutant reduces NADPH production. Stable cells verified in Figure 4A were prepared, and NADPH was measured using a NADPH kit. Error bars represent ±SD for triplicate experiments. E. The effect of over expression of PKM2 on ROS levels can be reversed by co-expression of SIRT5, but not on K498E and K498R mutants. 293T cells were transfected with indicated plasmids and DCF staining was performed to measure ROS levels. Error bars represent ±SD for triplicate experiments. PKM2 and SIRT5 protein levels were analyzed by western blot in Supplementary Figure 3A. F. Succinylation mimetic K498E mutant sensitizes cells to oxidative damage. A549 cells expressing PKM2 wild type or K498E mutant were exposed to different concentrations of H₂O₂ as indicated in the figure for 24h, and the viability of cells was measured by trypan blue exclusion. Error bars represent ±SD for triplicate experiments. G. SIRT5 can reverse the sensitization of cells to ROS caused by over expression of PKM2, but not K498E and K498R mutants. 293T cells were transfected with indicated plasmids and exposed to different concentrations of H₂O₂ as indicated in the figure for 24h. The viability of cells was measured by trypan blue exclusion. Error bars represent ±SD for triplicate experiments. H. H2O2 and Mena treatment increase the binding of PKM2 to SIRT5. 293T cells were treated with 500 uM H₂O₂ for 1h or 50uM Mena for 30min as indicated. The interaction between PKM2 and SIRT5 was determined by immunoprecipitation followed by western blot.