Fig 2: METTL3 increased in hepatic IR and interference of METTL3 attenuated IR induced by high fat. PT-qPCR analysis of all writers (Alkb5 and Fto) and erasers (Mettl14, Mettl3, Virilizer, Wtap) mRNA expression in livers from HFD mice (red) and control mice(black) (A). Representative western blot analysis of all writers and erasers expression in HepG2 cells between FFA group and Control group (A). Representative western blot analysis and quantification of Mettl3 expression in HepG2 cells between FFA group and NC group (C). Glucose consumption (D) and glucose uptake (E) analysis in HepG2 cells, it was divided into six groups, disposed with or without insulin; with or without FFA; with or without Mettl3 SiRNA. Representative western blot analysis and quantification of Glut2 expression in HepG2 cells disposed with insulin (10−7 mmol/L insulin for 4 h) between METTL3 SiRNA group and Control group (F)

Fig 3: Paclitaxel treatment increases m6A levels and METTL14 expression in the DRG.(A) Timeline of paclitaxel treatment, behavioral assessment, and tissue collection in rats. (B–D) Paclitaxel induced long-lasting notable mechanical allodynia, thermal hyperalgesia, and cold pain in rats (at least 6 rats per group, 2-way ANOVA followed by Šidák’s post hoc test). PWT, paw withdrawal threshold; PWL, paw withdrawal latency. (E) Representative dot blot images and quantification showing the m6A level in the bilateral L4–L6 DRGs in rats 10 days after treatment with saline or paclitaxel (PCX) (n = 12 rats per group, Student’s t test). (F) Representative immunoblot images and quantification show the protein levels of FTO, METTL3, METTL14, METTL16, WTAP, and ALKBH5 in the bilateral L4–L6 DRGs in rats 10 days after treatment with saline or PCX (n = 6 rats per group, Student’s t test). (G and H) Quantification and representative immunoblot images show the mRNA and protein levels of METTL14 in the bilateral L4–L6 DRGs in rats 0–14 days after treatment with PCX (n = 4 rats per group, 1-way ANOVA followed by Dunnett’s post hoc test for mRNA and least significant difference post hoc test for protein). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 4: m6A methylation modulates intestinal epithelial innate defense against C. parvum infection. (A) Knockdown of Alkbh5 or Fto in IEC4.1 cells. Cells were transfected with the CRISPR/Cas9 KO(h) for Alkbh5 or Fto and the HDR plasmids. Stable transfected cells were cloned and confirmed by real-time PCR and Western blot analysis. Gapdh was also blotted for control. (B) Knockdown of Alkbh5 or Fto in IEC4.1 cells decreased the infection burden of C. parvum infection. Knockdown of Alkbh5 or Fto increased m6A RNA methylation in IEC4.1 cells, as measured by m6A RNA methylation quantitation assay and dot blot. IEC4.1 cells and cells deficient with Alkbh5 or Fto were then exposed to C. parvum infection for 24 h. IEC4.1 cells transfected with the empty vector, marked as IEC4.1-WT (wild type) were used as the control. Infection burden of C. parvum was quantified by measuring parasite cpHsp70 or cp18s using real-time PCR. (C) Knock-in of Alkbh5 or Fto in IEC4.1 cells. Cells were transfected with the CRISPR/Cas9 KO(h) for active Alkbh5 (Alkbh5-KI) or Fto (Fto-KI) vectors. Stable transfected cells were cloned and confirmed by Western blot analysis. (D) Overexpression of Alkbh5, but not Fto, increased the infection burden of C. parvum in IEC4.1 cells. Cells stably expressing Alkbh5 or Fto were exposed to C. parvum infection for 24 h and infection burden of C. parvum was quantified. (E) Knockdown of Alkbh5 via siRNA decreased the infection burden of C. parvum in IEC4.1 cells. Cells were treated with the siRNA to Alkbh5 for 24 h and exposed to C. parvum infection for additional 24 h. Cells treated with the non-specific scrambled siRNA were used as the control and infection burden of C. parvum was quantified. (F) Knockdown of Alkbh5 via siRNA decreased the infection burden of C. parvum in 2D intestinal epithelial monolayers. Monolayers were cultured and treated with the siRNA to Alkbh5 for 24 h and exposed to C. parvum infection for additional 24 h. Cells treated with the non-specific scrambled siRNA were used as the control and infection burden of C. parvum was quantified. Data represent three independent experiments. *p<.05 vs cells transfected with the empty-vector control (as IEC4.1-WT in A, B, D) or cells treated with the control-siRNA (in E, F).

Fig 5: Global m6A levels and METTL14 expression are increased in ALI mice.Mice were injected intraperitoneally with LPS (15 mg/kg) or an equal volume of saline. A, B m6A dot-blot assays of lung tissues from Ctr and ALI mice. C A colorimetric assay measured m6A mRNA methylation in lung tissues of each group. D RT-qPCR analysis and (E, F) Western blot analysis of m6A regulators (METTL3, METTL14, METTL16, WTAP, FTO, and ALKBH5) in lung tissues of Ctr and ALI mice. G–L The immunofluorescence co-staining analysis of METTL14 (green) and CD68 (a macrophage marker, red) (G, H), F4/80 (a macrophage marker, red) (I, J), or Siglec-F (a resident alveolar macrophage marker, red) (K, L) in mouse lungs. The graphs depict mean ± SD, n = 6 mice per group.