Figure 2: Genetic inhibition of PKM2 suppresses inflammasome activation.

(a) Western blot analysis of PKM2 expression in BMDMs and PMA-differentiated THP1 cells after knockdown of PKM2 by specific shRNA for 48 h. (b,c) LPS (500 ng ml⁻¹, 3 h)-primed BMDMs (b) and PMA-differentiated THP1 cells (c) were treated with various 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)). Extracellular levels of IL-1β, IL-18 and HMGB1 and cellular levels of caspase-1 were assayed using ELISA or activity assay kit (n=3, *P<0.05, ANOVA LSD test). (d,e) In parallel, TNF levels were assayed with ELISA (d); IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) and the precursors of IL-1β (pro-IL-1β) and caspase-1 (pro-caspase-1) in lysates of BMDMs were assayed using western blot (e). (f) Native gel electrophoresis was performed using whole-cell extracts from LPS treatment alone (500 ng ml⁻¹, 3 h) or LPS (500 ng ml⁻¹, 3 h)-primed BMDMs after treatment with ATP (5 mM, 30 min) or poly(dA:dT) (1 μg ml⁻¹, 8 h). (g) Shikonin (5 μM) did not increase PKM2-shRNA-mediated inhibition of IL-1β release in LPS (500 ng ml⁻¹, 3 h)-primed BMDMs following treatment with ATP (5 mM, 30 min). (h) Q-PCR analysis of gene expression in indicated BMDMs following treatment with LPS (500 ng ml⁻¹) for 3 h (n=3, *P<0.05, t-test). All quantification data expressed as means±s.e.m of three independent experiments. Western blot data are representative of two independent experiments.