Fig. 2

Inhibition of inflammasome pathway in COX-2 inhibited E. coli infected uroepithelial cells, compromised activation of immune cells. Expression of (A)NLRP3, (B)ASC, (C)CASPASE1 mRNA after 2 h E. coli infection (n = 4) in T-24 cells. (D) Representative western blot analysis of NLRP3 and Pro-caspase 1 compared to housekeeping gene, GAPDH after 3 h E. coli infection (n = 3). (E) Relative caspase 1 activity assay in T-24 cells after 6 h E. coli infection. Expression of the cytokine IL-1β at the (F) mRNA level (n = 3) and (G) secreted level from the supernatants using ELISA (n = 3) after 2 h E. coli infection in T-24 cells. (H) Expression of IL1b mRNA in the urinary bladders of SC-236 treated mice and after 24 h E. coli infection (n = 6/7). (I) Expression of CD86 in dTHP-1 cells after 2 h stimulation with conditioned media obtained from E. coli infected bladder epithelial cells, T-24 (n = 3) using microscopy. Relative densitometry of CD86 is shown. (J) Measurement of intracellular granzyme B levels in NKL cells after 6 h stimulation with conditioned media obtained from E. coli infected bladder epithelial cells, T-24 (n = 8). Mean fluorescence intensity (MFI) was measured and normalized to control cells. In vitro analysis was performed in either duplicate or triplicate. T-24 cells were treated with 5µM SC-236 for 24 h followed by E. coli infection. Conditioned media was obtained from 5µM SC-236 treated and E. coli infected T-24 cells after 2 h. Data are shown as mean + SEM. Results from mice are presented as median. Significance levels mentioned as *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001