Fig. 3

Macrophage IGF2BP2 targets NLRP3 in an m6A-dependent manner, contributing to the apoptosis of RPE cells. (A) m6A enrichment of NLRP3 mRNA in BMDMs by m6A-RIP-qPCR. Results are presented relative to those obtained with immunoglobulin G (IgG). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh), m6A negative control; Myc peak, m6A positive control; n = 3. (B) Immunoblotting of IGF2BP2 in BMDMs after RNA pull down assay using single-stranded NLRP3 RNA with methylated or unmethylated adenosine. (C-D) RT-qPCR of (C) NLRP3 mRNAs and (D) NLRP3mRNA degradation in BMDMs treated with actinomycin D for the indicated times. The residual RNAs were normalized to 0 h; n = 5. (E-F) PCR and Western blot analysis of IGF2BP2 in THP-1 following si-NC or si-IGF2BP2 transfection to verify the knockdown effect. (G) Co-culture model of RPE and macrophages. (H) Analysis of nuclear morphological changes in ARPE-19 cells using Hoechst staining, with yellow arrows indicating damaged cells. (I) Detection of apoptosis in ARPE-19 cells using Annexin V-FITC/PI double staining and flow cytometry. Data were shown as mean ± SEM. *P < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001 versus the WT group by two-way ANOVA