Figure 3

RIG-I contributes to IL-1β production in EV-A71-infected macrophages in an NLRP3-independent manner. (A) The NLRP3 KD THP-1 cells were primed with PMA for 48 h for differentiation. The primed cells were subsequently transfected with siRIG-I and scrambled siRNA for 48 h and then infected with EV-A71 (M.O.I. = 2). The expression levels of NLRP3, RIG-I, EV-A71 VP0, and β-actin were examined by Western blot. (B) The supernatants were harvested at 12 h p.i.. The IL-1β levels were determined by ELISA. (C) THP-1 macrophages were transfected with siNLRP3, siRIG-I, and siNLRP3 + siRIG-I for 48 h, respectively. The transfected cells were subsequently infected with EV-A71 at the M.O.I. of 2. The expression levels of RIG-I, NLRP3, EV-A71 VP0, and β-actin were examined by Western blot. IL-1β levels in the supernatants were determined by ELISA. (D) Lysates prepared from mock- or EV-A71-infected PMA-primed THP-1 cells were subjected to immunoprecipitation with anti-ASC or anti-NLRP3 antibodies attached to sepharose. Whole lysates and immunoprecipitates were analyzed by immunoblotting with anti-NLRP3, anti-RIG-I, and anti-ASC. The expression of β-actin was used as an internal control. Data are expressed as mean value \(\pm\) SD (***p < 0.001, Student’s unpaired T-test).