Fig 2: METTL3 silencing inhibits cell invasion, migration, and EMT in OS cell lines. (A) METTL3 mRNA expression in OS cells and NHOst cells by RT-qPCR. (B) the transfection efficiency of sh-METTL3 determined using RT-qPCR. (C) migration of OS cells determined using wound healing assay. (D) invasion of OS cells determined using Matrigel invasion assay. (E) EMT process of OS cells determined using immunofluorescence staining. All the experiments were repeated at least three times, and data are represented as mean ± SD. These data were analyzed by one-way/two-way ANOVA followed by Tukey's post-tests. *p < 0.05, **p < 0.01.

Fig 3: TRAF6 overexpression abrogates the repressive effects of METTL3 silencing on the tumorigenic and metastatic properties of OS cells. (A) tumor images and tumor growth curves. (B) tumorigenic activity assessed by measuring tumor weight. (C) cell metastasis efficiency assessed by measuring lung light radiation intensity. (D) histological inspection was measured by HE staining. (E) TRAF6 and EMT markers in lung tissues of mice determined using western blot. All the experiments were repeated at least three times, and data are represented as mean ± SD. These data were analyzed by two-way ANOVA followed by Tukey's post-tests. *p < 0.05.

Fig 4: METTL3 is elevated in OS and correlates with dismal prognosis. (A) METTL3 expression in tissues of metastatic and non-metastatic OS patients by RT-qPCR. (B) the expression of other methyltransferases in OS tissues by RT-qPCR. (C) survival analysis of patients differentially expressing METTL3 analyzed using Kaplan-Meier. (D) METTL3 expression differences between patients at different Enneking stages. (E) immunohistochemical detection of METTL3 between OS tissues from metastatic/non-metastatic patients. (F) m6A mRNA expression differences in OS tissues by m6A analysis. All the experiments were repeated at least three times, and data are represented as mean ± SD. These data were analyzed by unpaired t test. *p < 0.05.

Fig 5: TRAF6 expression is affected by METTL3 in a m6A-dependent manner in OS cells. (A) differentially expressed genes in HOS cells examined using transcriptome sequencing. (B) identification of METTL3 downstream genes by RNA-seq and MeRIP-seq. (C) m6A modifications in OS cells after METTL3 knockdown examined using RIP experiments. (D) construction of MT TRAF6 by modifying the m6A shared sequence. (E) protein expression of MT TRAF6 in METTL3 downregulated cells examined using western blot. (F) the m6A modification of TRAF6 by METTL3 examined using dual-luciferase assay. (G) TRAF6 mRNA expression in cells after DAA treatment using RT-qPCR. All the experiments were repeated at least three times, and data are represented as mean ± SD. These data were analyzed by two-way ANOVA followed by Tukey's post-tests. *p < 0.05.