Fig 1: METTL3 regulates osteoarthritis progression in OA rats via mediating ADAMTS12. OA rats were induced by the ACL-T method and then treated with METTL3 inhibitor SAH. Rats were grouped as sham, ACL-T, and ACL-T + SAH. (A) HE, PAS, and safranin O-fast green of rat cartilage tissues in each group. n = 5 (B and C) OARSI score and micro-CT assays were performed in each group. n = 5. (D–F) RT-qPCR, western blot, and IHC assays for METTL3, ADAMTS12, and STAT1 levels in rat cartilage tissues in each group. n = 5. (G) The binding relationship between STAT1 and ADAMTS12 promoter region in rat cartilage tissues was tested by ChIP assay. n = 5. (H) The binding relationship between STAT1 mRNA and IGF2BP2 in rat cartilage tissues was tested by RIP assay. n = 5. *P < 0.05; **, P < 0.01; ***, P < 0.001
Fig 2: Increased m6A RNA methylation and METTL3 expression in RIF endometrial tissues. a The levels of m6A RNA methylation in the endometrial tissues from RIF patients (n=9) and normal control women (n=8) were evaluated by the m6A RNA Methylation Assay Kit. b The m6A levels in the endometrial tissues from RIF patients (n=6) and health control women (n=6) were evaluated by dot blotting assay. c The mRNA levels of major m6A methyltransferases (METTL3, METTL14, RBM15, WTAP and VIRMA) and demethylases (FTO and ALKBH5) in the endometrial tissues from RIF patients (n=13) and normal control women (n=13) were detected by qRT-PCR. d The protein levels of METTL3 in the endometrial tissues from RIF patients (n=8) and normal control women (n=4) were detected by Western blotting. *P<0.05, **P<0.01, ***P<0.001compared with the controls
Fig 3: METTL3 regulates IL-1β-treated chondrocytes by modulating ADAMTS12. Rat chondrocytes were transfected with NC, shMETTL3, shMETTL3 + OE-NC, or shMETTL3 + OE-ADAMTS12 followed by IL-1β stimulation. (A-B) RT-qPCR and western blot for METTL3 and ADAMTS12 expressions in IL-1β-treated rat chondrocytes. n = 3. (C) IF assays for METTL3, STAT1, and ADAMTS12 levels in treated rat chondrocytes. n = 3 (D) TUNEL staining of rat chondrocyte. n = 3 (E) Apoptosis of rat chondrocyte detected by flow cytometry. n = 3. *P < 0.05; **, P < 0.01; ***, P < 0.001
Fig 4: m6A motifs in MYC 3´UTR promotes the translation of MYC mRNA.A m6A peak distribution in MYC mRNA in MCF7 (left panel) and MDA-MB-231 (right panel) visualized in IGV. Input reads are represented in darker colors and the enriched RNA immunoprecipitated in yellow (MCF7) or red (MDA-MB-231). The amplified region by qPCR is depicted with a red line below MYC gene body. B RT-qPCR of m6A RNA immunoprecipitation (MeRIP) showing the enrichment of m6A in MYC relative to GAPDH in MCF7 (left) and MDA-MB-231 (right). C Relative level of SELECT products specific to m6A site in MYC 3´UTR, using total RNA from DMSO treated or STM2457 treated MDA-MB-231 cells. D RT-qPCR analysis of MYC after FLAG-METTL3 immunoprecipitation performed in control cells (+Dox) or in cells overexpressing Tet-off FLAG-METTL3 (-Dox) in MDA-MB-231 cells. E RT-qPCR analysis of MYC mRNA (upper panel) and western blot for MYC (lower panel) in MDA-MB-231 upon STM2457 treatment. βACTIN is used as loading control. F RT-qPCR analysis of MYC mRNA after treatment with actinomycin D at the time points 0, 10, 30 and 60 min in MDA-MB-231 control and treated with STM2457. G Relative Renilla luciferase activity of the psiCHECK2-MYC 3´UTR in MDA-MB-231 cells treated with DMSO (control) or with STM2457 for 48 h. Control cells were transfected with psiCHECK2 empty vector. Renilla luciferase activity was measured and normalized to Firefly luciferase. Data are mean ± SEM; n = 3 or 4; ****p < 0.0001; ***p < 0.001; *p < 0.05. In A, D, E, and G P-values were determined by two-tailed t-test; in C P-values were determined by one-tailed t-test. H Western blot showing the overexpression of SRSF11 in MDA-MB-231 in comparison to MCF10-A and MCF7. HDAC1 is used as loading control. I Western blot assessing the expression of SRSF11 in MDA-MB-231 upon STM2457 treatment. βACTIN is used as loading control. J Overlaps between AS events of knockdown of METTL3 in MCF7 and MYC-associated AS events (left panel); P-value < 0.0001. GO analysis of the common genes between AS events between knockdown of METTL3 in MCF7 and MYC-associated AS events (right panel); P-value < 0.05. K Overlaps between AS events of knockdown of METTL3 in MDA-MB-231 and MYC-associated AS events (left panel); P-value < 0.0001. GO analysis of the common genes between AS events in knockdown of METTL3 in MDA-MB-231 and MYC-associated AS events (right panel); P-value < 0.05.
Fig 5: HOXA10 overexpression rescues METTL3-impaired embryo attachment in vitro. a The mRNA levels of METTL3, HOXA10, ITGB3 and EMX2 in the METTL3-overexpressing Ishikawa cells with or without HOXA10 overexpression were analyzed by qRT-PCR. b The protein levels of METTL3, HOXA10, ITGB3 and EMX2 in the METTL3-overexpressing Ishikawa cells with or without HOXA10 overexpression were analyzed by Western blotting. c The levels of m6A RNA methylation in the METTL3-overexpressing Ishikawa cells with or without HOXA10 overexpression were evaluated by the m6A RNA Methylation Assay Kit. d The levels of m6A in the METTL3-overexpressing Ishikawa cells with or without HOXA10 overexpression were evaluated by dot blotting assay. e A vitro model of a confluent monolayer of Ishikawa cells co-cultured with BeWo spheroids was used to evaluate the embryo attachment. Bar 100 μm. The data are the average of three independent experiments (n=3). *P<0.05, **P<0.01, **P<0.001 versus the indicated group
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