Fig 2: Knockdown of METTL3 in prostate cancer alters protein expression. A, Correlation (Spearman ρ = 0.4685, P < 1E-15) between the average mRNA expression (n = 3) and protein expression (n = 3) as determined by shotgun proteomics in the NT shRNA line. B, Significantly (adjusted P value < 0.05) differentially expressed proteins with METTL3 knockdown in the two inducible shRNA lines as compared with the NT shRNA line (n = 3 for each shRNA). Common highly differentially expressed proteins between the two shRNA lines are highlighted with a black border. C, The top 60 differentially expressed proteins seen with METTL3 knockdown in both shRNA lines as compared with the NT control (n = 3 for each shRNA). D, Gene set enrichment analysis of proteins differentially expressed with METTL3 knockdown. Proteins were ranked according to the log of the adjusted P value as determined by t test. Shown are the top 20 results in either direction.

Fig 3: Re-expression of m6A writer restores the expression of UCA1 in KO cells. A–D Re-expression of METTL3 in KO cells restores UCA1 RNA level. A and C the representative western blot showing re-expression of METTL3 in HCT-116 KO clone#13 and DLD-1 KO clone#11. B and D the RNA level of UCA1 was detected by qRT-PCR. E–H Re-expression of WTAP in KO cells rescues UCA1 RNA level. E and G the representative western blot showing re-expression of WTAP in HCT-116 KO clone#28 and DLD-1 KO clone#37. F and H the RNA level of UCA1 was detected by qRT-PCR. Error bars represent SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.005

Fig 4: Methyltransferase‐like 3 (METTL3) induces the proliferation of Fusobacterium nucleatum (Fn)‐associated colorectal cancer in vitro and in vivo. (A–C) Ability of HCT‐116 cells to proliferate following F. nucleatum and/or si‐METTL3 treatment was assessed by (A) EdU, (B) cell cycle analyses, and (C) CCK‐8. (D) Xenograft tumors models were generated, with representative xenograft tumors (D, left panel) being shown. Ki‐67 staining of these xenografts (D, middle panel) were also carried out, and average tumor weights for xenograft tumors (D, right panel) were calculated. Data are means ± SD from triplicate experiments. *p < 0.05. Scale bar, 125 μm.

Fig 5: METTL14 upregulation reversed the effect of high glucose on phospho-Akt (Ser 473), phospho-Akt (Thr 308), HDAC5, TGF-β1, and α-SMA in HK2 cells through PTEN.Data were representative of three independent experiments. A Real-time PCR of FTO, METTL3 and METTL14 mRNA in HK2 cells treated with normal glucose or high glucose. *Means P < 0.05 versus normal glucose group. B Western blot and statistical analyses of FTO, METTL3, and METTL14 protein in HK2 cells treated with normal glucose or high glucose. *Means P < 0.05 versus normal glucose group. C Western blot and statistical analyses of phospho-Akt (Ser 473) and phospho-Akt (Thr 308) in high glucose-cultured HK2 cells transfected with pcDNA3.1, pcDNA3.1-METTL3, and pcDNA3.1-METTL14. *Means P < 0.05 versus pcDNA3.1 group. D Western blot and statistical analyses of HDAC5, TGF-β1, and α-SMA in high glucose-cultured HK2 cells transfected with pcDNA3.1, pcDNA3.1-METTL3, and pcDNA3.1-METTL14. *Means P < 0.05 versus pcDNA3.1 group. E Immunofluorescence of HDAC5 protein in high glucose-cultured HK2 cells transfected with pcDNA3.1 and pcDNA3.1-METTL14. Bar: 25 μm. F Western blot and statistical analyses of PTEN in HK2 cells treated with normal glucose (N), high glucose (H), and high glucose plus MA2 (H + MA2). *Means P < 0.05 versus N group, #means P < 0.05 versus H group. G Real-time PCR of PTEN mRNA in HK2 cells treated with high glucose or high glucose plus MA2. *Means P < 0.05 versus H group. H Western blot and statistical analyses of PTEN, phospho-Akt (Ser 473), phospho-Akt (Thr 308), and HDAC5 in HK2 cells transfected with pGenesil-1 and pGenesil-1-METTL14. *Means P < 0.05 versus pGenesil-1 group. I Western blot and statistical analyses of PTEN in high glucose-cultured HK2 cells transfected with pcDNA3.1, pcDNA3.1-METTL3, and pcDNA3.1-METTL14. *Means P < 0.05 versus pcDNA3.1 group.