Fig 2: IL-6 induces a feedback activation of METTL3 nuclear functions via promoting its deacetylation.a IL-6 levels from the supernatants of the indicated breast cancer cell lines were measured by ELISA (n = 3 biologically independent experiments). b QRT-PCR quantification of IL-6 mRNA levels in the indicated breast cancer cell lines (n = 3 biologically independent experiments). c IP and IB analysis of acetyl-K177 METTL3 levels in the indicated breast cancer cell lines. d Based on results from Supplemental Fig. 6a, METTL3 protein levels in each fraction were quantified, the percentage of nuclear or cytosolic METTL3 to total METTL3 levels in WCL was calculated. e Representative immunofluorescence for Myc-METTL3 (red) and DAPI (blue, cell nuclei) in METTL3 reconstituting MCF-7 cells treated with rIL-6 (upper panel) (n = 3 biologically independent experiments). Scale bars, 10 μm. Quantification of nuclear METTL3 percentage is presented as mean ± SD (lower panel). From left to right: ***P = 0.0001, ns P = 0.19, ns P = 0.59, respectively, by two-sided t test. f Lysates of METTL3 reconstituting MCF-7 cells treated with rIL-6 for 12 h were subjected to IP and IB analysis. g Relative NAD+ levels were measured by HPLC-MS in MCF-7 cells infected with the indicated lentiviruses and treated with rIL-6 for 1 h (n = 3 biologically independent experiments). From left to right: ***P = 0.0003, ns P = 0.0543, **P = 0.0068, respectively, by two-sided t test. h LC-MS/MS quantification of the m6A/A ratio in polyadenylated RNA isolated from the indicated MCF-7 cells with rIL-6 treatment for 24 h (n = 3 biologically independent experiments). From left to right: **P = 0.0059, **P = 0.0019, ns P = 0.29, **P = 0.0035, ns P = 0.20, ns P = 0.085, ns P = 0.061, respectively, by two-sided t test. i LC-MS/MS quantification of the m6A/A ratio in polyadenylated RNA isolated from METTL3 reconstituting MCF-7 cells treated with rIL-6 for 24 hours (n = 3 biologically independent experiments). From left to right: **P = 0.0035, **P = 0.0029, ns P = 0.33, ns P = 0.97, ns P = 0.26, respectively, by two-sided t test. j Migration and invasion assays were conducted in METTL3 reconstituting MCF-7 cells treated with rIL-6 (left panel) (n = 3 biologically independent experiments). Scale bars, 50 μm. Quantification is presented on the right panel. From left to right: **P = 0.0031, *P = 0.037, ###P = 0.0007, ***P = 0.0002, **P = 0.0071, ###P = 6.41e-05, respectively, by two-sided t test. *compared to WT. #compared to WT + rIL-6. k m6A-MeRIP-qPCR analysis of the indicated m6A substrates normalized to input in METTL3 reconstituting MCF-7 cells subjected to rIL-6 treatment for 24 h (n = 3 biologically independent experiments). From left to right: **P = 0.0019, **P = 0.0034, *P = 0.0156, ns P = 0.23, **P = 0.0053, *P = 0.017, **P = 0.01, ns P = 0.22, *P = 0.012, *P = 0.013, **P = 0.0017, ns P = 0.80, **P = 0.004, **P = 0.0035, ***P = 0.0007, ns P = 0.95, respectively, by two-sided t test. l QRT-PCR quantification of the indicated mRNAs in METTL3 reconstituting MCF-7 cells treated with rIL-6 for 24 h (n = 3 biologically independent experiments). From left to right: *P = 0.026, **P = 0.0073, **P = 0.0028, ns P = 0.16, ***P = 0.0005, **P = 0.0022, **P = 0.0037, ns P = 0.60, ***P = 0.0007, ***P = 0.0006, *P = 0.0205, ns P = 0.43, ***P = 0.0002, **P = 0.0072, **P = 0.0021, ns P = 0.40, respectively, by two-sided t test. m Lysates of METTL3 reconstituting MCF-7 cells treated with rIL-6 for 24 h were subjected to IB analysis. All data are represented as mean ± SD. All P values were calculated by Student’s t test. Source data are provided as a Source Data file.

Fig 3: METTL3/KHDRBS3 promotes the adenosine methylation of miR-125a-5p. (A) Graph showing the ELISA assay of METTL3 (#ABX381419, CliniSciences, Nanterre, France) and FTO (#K4921, Biovision, Nanterre, France) expression level in H1975 and H1975NivoSat cells. (B) The presence of METTL3 and KHDRBS3 proteins was analyzed by western blot in immunoprecipitation products made with antibodies directed against METTL3 and IgG (control) and protein extracts from H1975 and H1975NivoSat cells. (C) The transfection impact of H1975NivoSat cells with siRNA control (siRNA-A) and siRNA directed against KHDSBR3 and METTL3 on the expression level of miR-125a-5p and 411-5p (RT-qPCR), KHDRBS3 and METTL3 transcripts (RT-qPCR), the level of enrichment in m6A of miR-125a-5p and 411-5p. (D) Impact of chemical (Ravoxertinib, 100 nM, 72 h) and biological (siRNA) inactivation of ELK1 on the expression level of the KHDRBS3 transcript and the enrichment level of ELK1 on the KHDRBS3 promoter. DMSO and siRNA-A were used as control.

Fig 4: METTL3 acetylation impedes cancer metastasis via fine-tuning its nuclear and cytosolic functions.Proposed model for pathophysiological stimuli-mediated METTL3 acetylation/deacetylation and its functional implications.

Fig 5: Adenosine methylation of miR-125a-5p regulates its repressive function toward VSIG3/IGSF11 and influences cellular immunogenicity. (A) RT-qPCR analysis of expression changes of genes involved in cell immunogenicity. (B) Venn diagram highlighting the existence of 2 miRNAs (miR-125a-5p and muR-181c-5p) having a modification of m6A or m5C and being described as targeting IGSF11. (C) Graphs illustrate the impact of transfection of wild-type and adenosine methylation forms of miR-125a-5p on IGSF11 at the protein and mRNA level and on their binding to GW182 within H1975 cells. (D) Graphs illustrate the impact of Sinefungin (SFG) on the expression and adenosine methylation level of miR-125a-5p, on the interaction of this miRNA with GW182, and on the protein and mRNA expression levels of IGSF11 in H1975NivoSat cells. H1975 cells were used as control. (E) Immunogenicity of H1975NivoSat cells was compared with the one of H1975NivoSat cells treated with Sinefungin or anti-IGSF11 through the analysis of their PBMC-induced lysis. H1975 cells were used as control.