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. 2021 Jan 8;11:602016. doi: 10.3389/fimmu.2020.602016

Figure 3.

Figure 3

EMR2-enhanced IL-1β production in THP-1 cells is ASC- and NLRP3-dependent. (A, B) THP-1 cells were transfected with the ASC-specific siRNAs (A) or NLRP3-specific siRNAs (B) then cultured on plates coated without or with 2A1 mAb in the absence or presence of LPS for 24 h. A scrambled siRNA was included as a negative control. IL-1β in supernatant was analyzed by ELISA (top panel), while active caspase-1, ASC and NLRP3 protein expression was verified by WB analyses as indicated (bottom panel). Treatment of cells included: lane 1, cells only; lane 2, LPS alone; lane 3, immobilized 2A1 alone; lane 4, immobilized 2A1 plus LPS. The siRNA knock down efficiency was shown as a ratio determined by quantitative comparison of the density of the ASC and NLRP3 protein bands normalized to the β-actin band. (C, D) The parental (# 1), def-ASC (# 2), and def-NLRP3 (# 3) THP-1 cell lines were cultured on plates coated without or with 2A1 mAb in the absence or presence of LPS for 24 h. Cell supernatant was collected and analyzed for IL-8 and IL-1β by ELISA (C). The expression of relevant proteins (NLRP3, ASC, caspase-1, procaspase-1, IL-1β) in the supernatant and cell lysate was examined by WB analyses as indicated (D). Data are means ± SEM of at least three independent experiments performed in triplicate and analyzed by one-way ANOVA. * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus the THP-1 control group.