Figure 4

E-protein increases NLRP3 inflammasome response to poly(I:C), depending on K⁺ efflux and ROS but not GSDMD. (A) Western blot analysis of NLRP3 and pro-IL-1β protein levels in BMDMs, primed with 20 ng/ml LPS and treated with poly(I:C) for 16 h. Blots were cut to probe with NLRP3 (from ~ 80 kDa to top) or pro-IL-1β and Actin (from ~ 80 kDa to bottom). (B) Quantification of IL-1β secretion into cell media in Wild type (WT) and Nlrp3^−/− BMDMs. BMDMs were transduced with control and E-protein lentiviruses for 72 h and then primed with 20 ng/ml LPS for 3 h and treated with poly(I:C) for 16 h. IL-1β secretion was assessed via IL-1β ELISA kit. (C) Quantification of IL-1β secretion into cell media in WT and Gsdmd^−/− BMDMs. BMDMs were transduced with control and E-protein lentiviruses for 72 h and then primed with 20 ng/ml LPS for 3 h and treated with poly(I:C) for 16 h. IL-1β secretion was assessed via IL-1β ELISA kit. (D) Quantification of LDH release into cell media in BMDMs, transduced with control and E-protein lentiviruses for 72 h and then primed with 20 ng/ml LPS for 3 h and treated with poly(I:C) for 16 h. For inhibitor experiments, LPS-primed BMDM were preincubated with L-NAC (30 mM) 1 h prior to stimulation with poly(I:C). High K⁺ was supplied by adding 70 mM of K⁺ to extracellular media during stimulation with poly(I:C). LDH release was assessed via LDH kit. (E) Quantification of IL-1β secretion into cell media in BMDMs, transduced with control and E-protein lentiviruses for 72 h and then primed with 20 ng/ml LPS for 3 h and treated with poly(I:C) for 16 h. Same treatment was done as described in D. IL-1β secretion was assessed via IL-1β ELISA. (F) Quantification of TNF-α secretion into cell media in BMDMs, transduced with control and E-protein lentiviruses for 72 h and then primed with 20 ng/ml LPS for 3 h and treated with poly(I:C) for 16 h. Same treatment was done as described in D. TNF-α secretion was assessed via TNF-α ELISA. Data are presented as mean ± SD, which were analyzed by one-way ANOVA coupled with Tukey’s test for multiple comparisons. ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05.