File:Aberrant-Expression-of-Interleukin-1β-and-Inflammasome-Activation-in-Human-Malignant-Gliomas-pone.0103432.g003.jpg

English: (A) Western blot showing induction of intracellular 32 kDa pro-IL-1β and secreted 17 kDa IL-1β following IL-1 stimulation. Addition of ATP and nigericin (Nig.) increased IL-1β processing (decrease of proIL-1 and increase of secreted IL-1) in GBM2 and U87 cells. NLRP3 protein was detected in unstimulated (Ctr) cultures and showed a marginal increase after IL-1 stimulation. ATP and Nig substantially increased NLRP3 protein expression. NLRP3 protein was complexed with ASC (immunoprecipitation with anti-ASC antibody) in all conditions and the complex formation was increased by ATP and Nig. Numbers are densitometric ratios to β-actin for intracellular proteins and densitometry of IL-1β measured in concentrated culture supernatants (see Materials and Methods). Average densitometry data (NLRP3, pro-IL-1β and secreted IL-1β) from two experiments for both GBM2 and U87 are shown in Figure S1. (B) ELISA of GBM2 cells confirm enhanced IL-1β processing by ATP and nigericin. (C) Suppression of NLRP3 expression by siRNA in U87 cells. (D) NLRP3 siRNA-transfected U87 cells show significant reduction in the amount of secreted IL-1β in all three conditions (IL-1, + ATP, + nigericin) compared to control siRNA-transfected cells. Data shown are mean ± SD (n = 3) ***p<0.001, ** p<0.01, *p<0.05 by t-test.