Figure 5: PKM2-dependent glycolysis promotes EIF2AK2 phosphorylation.

(a) LPS-primed BMDMs were treated with ATP (5 mM, 30 min) in the absence or presence of shikonin (1 and 5 μM). p-EIF2AK2 was assayed (n=3, *P<0.05 versus ATP group, ANOVA LSD test). (b,c) Knockdown of PKM2 (b) and PDK1 (c) by shRNA inhibited ATP- (5 mM, 30 min) and poly(dA:dT) (1 μg ml⁻¹, 8 h)-induced p-EIF2AK2 in LPS (500 ng ml⁻¹, 3 h)-primed BMDMs (n=3, *P<0.05 versus control shRNA group, ANOVA LSD test). (d) Western blot analysis of IL-1β in culture supernatants (SN) and the precursors of IL-1β (pro-IL-1β) in lysates of LPS-primed BMDMs following treatment with lactate (5 mM, 3 h) and ATP (5 mM, 30 min). (e) LPS-primed BMDMs were treated with lactate (5 mM, 3 h) in the absence or presence of C16 (1 and 5 μM). IL-1β and HMGB1 release and p-EIF2AK2 expression were assayed (n=3, *P<0.05 versus ATP group, ANOVA LSD test). (f) Knockdown of EIF2AK2 by shRNA inhibited lactate (5 mM, 3 h)-induced IL-1β, IL-18 and HMGB1 release and caspase-1/11 activation in LPS-primed BMDMs (n=3, *P<0.05 versus control shRNA group, ANOVA LSD test). (g) Inhibition of EIF2AK2 by C16 or shRNA or knockout of caspase-1 limited MDP (200 ng ml⁻¹, 8 h) or flagellin (200 ng ml⁻¹, 8 h))-induced cytokine release in LPS-primed BMDMs (n=3, *P<0.05, ANOVA LSD test). (h) Double knockout of caspase-1 and caspase-11 inhibited lactate (5 mM, 3 h)-induced IL-1β release in LPS-primed BMDMs (n=3, *P<0.05, ANOVA LSD test). (i) A-438079 (1 μm) suppressed ATP (5 mM, 30 min), but not lactate (5 mM, 3 h)-induced p-EIF2AK2 expression in LPS-primed BMDMs. (j) Knockout of NLRP3 and AIM2 inhibited lactate (5 mM, 3 h)-induced IL-1β and HMGB1 release and caspase-1/11 activation in LPS-primed BMDMs (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.