Fig. 6: METTL3/YTHDF3 m6A axis degrades PTX3 mRNA during M2 macrophage activation.
a Heatmap profiling downregulated genes in human METTL3-deficient THP1-derived macrophages. b Western blot showing protein levels of YTHDF3 in both Mettl3 KO mice BMDMs (n = 3 animals) and METTL3-deficient THP1-derived macrophages (n = 3 cells), respectively. c RT-qPCR analysis of the abundance of YTHDF3 in monocyte-derived macrophages from 55 childhood allergic asthma and 50 healthy controls. The detailed minimum, median, maximum, 25th, 75th percentile (box), and 5th and 95th percentile (whiskers) of box plots were provided in the Source data file. d RT-qPCR (left) analysis of M2-associated markers mRNA levels (n = 4 cells), and ELISA (right) detection of the IL-10 secretion levels (n = 4 cells for control group and n = 5 cells for knockdown groups) in THP1-derived macrophages transfected with YTHDF3 siRNAs pools (200 nM), followed by IL-4 stimulation. e Elevated levels of p-AKT and p-STAT6 in YTHDF3-deficient THP1-derived macrophages were detected by Western blot (n = 3 cells). f RT-qPCR (n = 3 cells) and ELISA (n = 3, 6, 4 cells for Ctrl LV+siNC, METTL3 LV+siNC, METTL3 LV+siYTHDF3 group, respectively) showing M2 activation-associated markers expression in THP1-derived macrophages overexpressing METTL3, with or without YTHDF3 knockdown. g YTHDF3 RIP-analysis of PTX3 m6A level in control or METTL3-deficient THP1-derived macrophages (n = 7 cells for control group and n = 8 cells for knockdown group). Relative enrichment was normalized by input. h The increase of PTX3 mRNA (n = 3 cells) and secretion levels (n = 3 cells for control group and n = 5 cells for knockdown group) in YTHDF3-deficient THP1-derived macrophages was quantitated by qPCR and ELISA. i Western blot showing up-regulated PTX3 protein levels in YTHDF3-deficient THP1-derived macrophages (n = 3 cells). j Effect of YTHDF3 knockdown on PTX3 mRNA stability in THP1-derived macrophages treated with actinomycin D (n = 4 cells). k Dual-luciferase reporter assays demonstrating the effect of YTHDF3 on PTX3 3ʹUTR reporters with either WT or mutated m6A site (n = 4 cells). Statistical analysis of the data was performed using two-sided unpaired t test (b, d, h, i), Mann–Whitney test (c), 1-way ANOVA (f right), and 2-way ANOVA (e, f left, g, j, k) followed by either Tukey’s or Sidak’s multiple comparison tests. Data are presented as means ± SEM from one of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s, not significant.
