Fig. 3: METTL3 targets NLRP3 mRNA to mediate pyroptosis in HBx-expressing HepG2 cells.

A–E HepG2 cells were transfected with pcDNA3.1-HBx (1 μg/mL, 24 h) to construct the HBx-expressing hepatocytes, followed by treatment with or without CY-09 (10 μM, 24 h), while CY-09 was used as a specific inhibitor of NLRP3 activity. A Expression levels of NLRP3, ASC, Caspase-1, GSDMD, and IL-1β proteins were detected by WB. B Representative IF images showing co-localization of GSDMD-N (red) and Dio (green) were captured by confocal microscopy (Left). Scale bar, 10 µm. Quantification of the relative intensity of GSDMD-N/Dio is shown in a bar graph (Right). C–E Levels of LDH (C), IL-1β (D), and IL-18 (E) in supernatants. F–L HBx-expressing HepG2 cells were transfected with shMETTL3 (1 μg/mL, 24 h) to knock down METTL3, while shNC was transfected as a negative control. F Levels of METTL3 mRNA and METTL3 protein in cells knocked down by serial shMETTL3 (#1-#3). G The level of NLRP3 mRNA was detected. H Expression levels of METTL3, NLRP3, and pyroptosis-related proteins. I Representative IF images showing co-localization of GSDMD-N (red) and Dio (green) in cells (Left), while the quantification is shown in a bar graph (Right). Scale bar, 10 µm. J–L Levels of LDH (J), IL-1β (K), and IL-18 (L) in supernatants. M–O A pB-METTL3 recombinant plasmid was constructed and transfected to construct the stable METTL3 overexpression (OE) in HepG2 cells, while pB-NC was used as a control. M Levels of METTL3 mRNA and METTL3 protein in cells. N The level of NLRP3 mRNA was detected. O Expression levels of NLRP3 and pyroptosis-related proteins in cells. P–U METTL3-overexpressing HepG2 cells were pre-treated with or without CY-09 (10 μM, 24 h) to inhibit NLRP3 activity. P Expression levels of METTL3, NLRP3, and pyroptosis-related proteins. Q Representative images showing the flow cytometry quantification of caspase-1 activity related to pyroptosis. R Representative IF images showing co-localization of GSDMD-N (red) and Dio (green) (Upper), while the quantification is shown in a bar graph (Lower). Scale bar, 10 µm. S–U Levels of LDH (S), IL-1β (T), and IL-18 (U) in supernatants. Data are presented as mean ± SD. *, P < 0.05, compared to the control group. #, P < 0.05, compared to the corresponding group.