Research Paper Volume 15, Issue 10 pp 4429—4443

Figure 3. Effects of NOX2 overexpression on the inhibitory effects of metformin on inflammation and KGN cell pyroptosis. (A–C) Cells were pretreated with or without 10 μM LPS for 24 h and then incubated with 20 μM metformin for another 12 h. (A) NOX activity was evaluated using a commercial NOX detection kit; (B) Quantification of NOX2 mRNA expression using RT-PCR; (C) Analysis of NOX2 protein levels using Western blotting; (D) Cells were transduced with PBS, LV-NC, or LV-NOX2 for 48 h. The transfection efficiency was evaluated using Western blot analysis; (E–J) Cells were pretreated with 10 μM LPS for 24 h or transfected with LV-NOX2 for 48 h and then incubated with 20 μM metformin for another 12 h; (E) Intracellular levels of ROS were measured using commercial kits; (F) The mRNA levels of NLRP3, ASC, caspase-1 and GSDMD were detected by RT-PCR; (G) The protein levels of NLRP3, pro-caspase-1, cleaved-caspase-1, ASC and GSDMD-N were detected by Western blot; (H) Levels of LDH in the cell culture medium were examined using an LDH cytotoxicity detection kit; (I) Caspase-1 activity was assessed using a colorimetric caspase-1 activity assay kit; (J) The levels of IL-1β, IL-18, IL-6, and TNF-α in the cell culture supernatant were determined by ELISA. 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.