Fig. 9

Paxillin is required for ATP- and Nigericin-induced NLRP3 inflammasome activation. a, b BMDMs prepared from C57BL/6 mice bone marrow were infected by lentivirus that express sh-Paxillin for 3 days. LPS-primed BMDMs were stimulated by MDP (50 μg/ml) for 6 h, ATP (5 mM) for 30 min, poly (dA:dT) (5 μg/ml) for 6 h, or Salmonella for 4 h. c, d LPS-primed BMDMs were stimulated by ATP (5 mM) for 30 min, Nigericin (2 μM) for 2 h, MSU (100 μg/ml) for 6 h, or Alum crystals (200 mg/ml) for 6 h. e, f BMDCs prepared from C57BL/6 mice bone marrow were infected by lentivirus that express sh-Paxillin for 3 days. LPS-primed BMDCs were stimulated by ATP (5 mM) for 30 min, Nigericin (2 μM) for 2 h, or MSU (100 μg/ml) for 6 h. IL-1β levels in supernatants were determined by ELISA (a, c, e). Mature IL-1β (p17) and cleaved Casp-1 (p10) in supernatants as well as Paxillin, pro-IL-1β, and pro-Casp-1 in lysates were determined by Western blot (b, d, f). g, h PBMCs isolated from healthy individuals were infected by lentivirus that express sh-Paxillin for 2 days. Before stimulation, PBMCs were treated with LPS (1 μg/ml) for 6 h, and PBMCs were then stimulated by ATP (5 mM) for 30 min, Nigericin (2 μM) for 2 h, MSU (100 μg/ml) for 6 h, Alum crystals (200 mg/ml) for 6 h, or MDP (50 μg/ml) for 6 h. Paxillin in lysates was determined by Western blot (g). IL-1β levels in supernatants were determined by ELISA (h). i, j THP-1 cells stably expressing sh-NC or sh-Paxillin were generated and differentiated into macrophages, which were then treated with ATP (5 mM) for 2 h, Nigericin (2 μM) for 2 h, or MSU (100 μg/ml) for 6 h. IL-1β levels in supernatants were determined by ELISA (i). Mature IL-1β (p17) and cleaved Casp-1 (p22/p20) in supernatants and Paxillin, pro-IL-1β, and pro-Casp-1 in lysates were determined by Western blot (j). Data shown are means ± SEMs; ***p < 0.0001. ns, no significance