Figure 4: PKM2 contributes to glycolysis in caspase-1-deficient macrophages.

(a) LPS (500 ng ml⁻¹, 3 h)-primed WT (caspase-1^+/+) and caspase-1^−/− BMDMs were treated with inflammasome activators (ATP (5 mM, 30 min), poly(dA:dT) (1 μg ml⁻¹, 8 h), MDP (200 ng ml⁻¹, 8 h) or flagellin (200 ng ml⁻¹, 8 h)). The levels of PEP and lactate were assayed using a commercial kit (n=3, *P<0.05, ANOVA LSD test). (b) Inhibition of PKM2 using shikonin (5 μM) or shRNA significantly inhibited ATP- (5 mM, 30 min) or poly(dA:dT) (1 μg ml⁻¹, 8 h)-induced PEP and lactate production in LPS (500 ng ml⁻¹, 3 h)-primed caspase-1^−/− BMDMs (n=3, *P<0.05, ANOVA LSD test). (c) In parallel, ATP-induced interaction between NLRP3 and PYCARD and poly(dA:dT)-induced interaction between AIM2 and PYCARD were assayed with immunoprecipitation (IP). (d,e) Knockdown of PKM2 did not increase LDH release and ΔΨm loss in LPS (500 ng ml⁻¹, 3 h)-primed caspase-1^−/− BMDMs following treatment with ATP (5 mM, 30 min) or poly(dA:dT) (1 μg ml⁻¹, 8 h) (n=3, *P<0.05, ANOVA LSD test). All quantification data expressed as means±s.e.m of three independent experiments. Western blot data are representative of two independent experiments.