Fig 2: IFIT1 was methylated by METTL3 and METTL14 and recognized by IGF2BP2/3. A. PCR analysis of IFIT1 mRNA levels in HCT116 cells after transfection with METTL3 siRNAs (n=3, mean± SD, One-way ANOVA). B. PCR analysis of IFIT1 mRNA levels in HCT116 cells after transfection with METTL14 siRNAs (n=3, mean± SD, One-way ANOVA). C. Western blot assays detecting IFIT1 protein expression in HCT116 cells after transfection with METTL3 siRNAs. D. Western blot assays detecting IFIT1 protein expression in HCT116 cells after transfection with METTL14 siRNAs. E. PCR analysis of IFIT1 mRNA levels in HCT116 cells after transfection with pcDNA3.1_METTL3 and pcDNA3.1_METTL14 (n=3, mean± SD, two-tailed Student's t-test). F. Western blot assays detecting IFIT1 protein expression in HCT116 cells after transfection with pcDNA3.1_METTL3 and pcDNA3.1_METTL14. G. The detection of half-life(t1/2) of IFIT1 mRNAs in HCT116 cells transfected with control siRNAs and METTL3 siRNAs using real-time PCR, linear regression analysis. H. The detection of half-life(t1/2) of IFIT1 mRNAs in HCT116 cells transfected with control siRNAs and METTL14 siRNAs using real-time PCR, linear regression analysis. I. The detection of half-life(t1/2) of IFIT1 mRNAs in HCT116 cells with and without METTL3 overexpression using real-time PCR, linear regression analysis. J. The detection of half-life(t1/2) of IFIT1 mRNAs in HCT116 cells with and without METTL14 overexpression using real-time PCR, linear regression analysis. K. PCR analysis of IFIT1 mRNA level in HCT116 cells with IGF2BP1, IGF2BP2 and IGF2BP3 siRNAs transfection (n=3, mean± SEM, One-way ANOVA). L. The detection of half-life(t1/2) of IFIT1 mRNAs in HCT116 cells transfected with control siRNAs and IGF2BP2 siRNAs using real-time PCR, linear regression analysis. M. The detection of half-life(t1/2) of IFIT1 mRNAs in HCT116 cells transfected with control siRNAs and IGF2BP3 siRNAs using real-time PCR, linear regression analysis. N. The direct binding of IGF2BP2 with IFIT1 3’UTR using Agarose electrophoresis and RIP-qPCR assays in HCT116 cells (n=3, mean± SD, two-tailed Student's t-test). O. The direct binding of IGF2BP3 with IFIT1 3’UTR using Agarose electrophoresis and RIP-qPCR assays in HCT116 cells (n=3, mean± SD, two-tailed Student's t-test).

Fig 3: METTL14 activates the Wnt/β‐catenin signalling pathway (Wβ‐CSP) in CM cells. (A) The β‐catenin expression of the Wnt signalling pathway was examined by WB analysis in METTL14 stably‐transfected OCM1 and MUM‐2B cells. (B) The expression of critical members of the Wnt signalling pathway was validated by WB in METTL14 stably‐transfected OCM1 and MUM‐2B cells.

Fig 4: The anomaly of neurogenesis was rescued by overexpression of Mettl14 in primary NPCs from Arhgef2−/− mice (at E13.5)(A) The protein expression of Npdc1 and Cend1 was examined by western blot. n = 3–4 per genotype, ∗p < 0.05, ∗∗p < 0.01, one-way ANOVA.(B) The size of cortical neuro-spheres was measured at 72 h after transfection. A 2.3-fold increase of sphere size was noted by loss of Arhgef2, which could be recovered by HBLV-m-Mettl14-Zsgreen. Green: Zsgreen. Scale bar, 100 μm, n = 4 per genotype (3 measurements for each group), ∗∗p < 0.01, one-way ANOVA.(C) Knockout of Arhgef2 significantly decreased βIII-tubulin+ cells (early neuron) and increased βIII-tubulin-Ki67+ proliferating cells (precursors), and the proportion could be rescued after Mettl14 overexpression. Cells were seeded at a density of 5 × 104/cm2 and were observed at day 4 after transfection. βIII-tubulin, marker for early neuron, green; Ki67, marker for proliferating cells, red; DAPI, blue. Scale bar, 50 μm. n = 6 per genotype, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA. Data are represented as mean ± SD.

Fig 5: The METTL14/LINC02159/FOXC2 signal upon MTRX1133 treatment in the tumourigenic properties of KRASG12D mutated CRC. A: Western blotting to detect FOXC2 expression in the indicated cells. B: Cell proliferation was detected by the CCK-8 assay. C: The EdU assay was utilised to measure cell proliferation. Scale bar; 50 μm. D: A transwell migration experiment was performed to measure cell migration. Scale bar; 50 μm. E: A transwell invasion assay was conducted to evaluate cell invasion. Scale bar; 50 μm. *P<0.05, **P<0.01, ***P<0.001. Data are presented as mean ± SD.