Fig. 4.

Mettl14 regulates Myd88 via m6A modification and IL-6 through p65. THP-1 cells were treated with NC, siM14-1, siM14-2, overexpressed (OE) Mettl14 or OE Myd88 before treatment with LPS (500 ng/ml). a Heat map showing the DEGs in the siMettl14 group and NC group. n = 3 per group. b Results of a GO analysis performed on the DEGs. c Results of a KEGG pathway analysis performed on the DEGs. d The expression of Myd88 mRNA was detected by qRT-PCR. n = 3 per group. The data are expressed as the means ± SDs. P values were determined by one-way ANOVA with Fisher’s LSD post hoc test. **P < 0.01; ***P < 0.001. e–f Myd88 protein levels were detected by immunofluorescence (e) and western blotting (f). Scale bar = 50 μm. g At 24 h after transfection, THP-1 cells were treated with cycloheximide (CHX). IL-6 protein was detected by western blotting. h IL-6 mRNA levels was measured by qRT-PCR. n = 3 per group. The data are expressed as the means ± SDs. P values were determined by one-way ANOVA with Fisher’s LSD post hoc test. **P < 0.01; ***P < 0.001. i qRT-PCR analysis of Myd88 mRNA stability following treatment with actinomycin D (ActD, 2 μM). n = 3 per group. The data are expressed as the means ± SDs. P values were determined by one-way ANOVA with Fisher’s LSD post hoc test. **P < 0.01. j RIP analysis of the interaction of m⁶A with Myd88 mRNA. n = 3 per group. The data are expressed as the means ± SDs, Student’s t test. *P < 0.05; ns, not significant. k The localization of p65 (green) was observed with immunofluorescence, and the nuclei (blue) were stained with DAPI. Scale bar = 50 μm. l The nuclear translocation of p65 was analyzed, and its localization in the cytosol and nucleus was separately quantified using western blotting. m ChIP assay results showing the ability of p65 proteins to bind IL-6 promoters. n = 3 per group. The data are expressed as the means ± SDs. P values were determined by one-way ANOVA with Fisher’s LSD post hoc test. *P < 0.05; ***P < 0.001; ****P < 0.0001