Research Paper Volume 15, Issue 10 pp 4429—4443

Figure 4. Effects of NOX2 down-regulation on the inhibitory effects of metformin on inflammation and KGN cell pyroptosis. (A) Cells were transduced with PBS, 100 nM si-NC, or si-NOX2 for 48 h. The transfection efficiency was evaluated using Western blot analysis; (B–G) Cells were pretreated with 10 μM LPS for 24 h or transfected with si-NOX2 for 48 h and then incubated with 20 μM metformin for another 12 h; (B) Intracellular levels of ROS were measured using commercial kits; (C) Levels of LDH in the cell culture medium were examined using an LDH cytotoxicity detection kit; (D) Caspase-1 activity was assessed using a colorimetric caspase-1 activity assay kit; (E) The levels of IL-1β, IL-18, IL-6, and TNF-α in the cell culture supernatant were determined by ELISA. (F) RT-PCR detected the mRNA levels of NLRP3, ASC, caspase-1, and GSDMD; (G) The protein levels of NLRP3, pro-caspase-1, cleaved-caspase-1, ASC, and GSDMD-N were detected by Western blot; Data are represented as the means ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns. not significant.