Fig 2: METTL3‐PBX1 axis elevated BH4 induced GC cells metabolism reprogramming. (A) The de novo biosynthesis pathway of BH4 in eukaryotic cells and the function site of GCH1 and SPRi3. (B) ELISA assay measured the BH4 levels in cell lysis solution (normalized by total protein) in AGS cells (WT, catalytic mutant METTL3‐overexpressing, and the blank control). (C) ELISA assay measured the BH4 level in AGS cells (METTL3 knockdown and negative control). (D) ELISA assay measured the BH4 levels in cell lysis solution of AGS cells with PBX1 knockdown and negative control. (E) GO analysis of different genes in BH4‐treated AGS cells. (F) mRNA levels of NOS2‐3 in METTL3 overexpressed AGS cells and corresponding controls, as measured by qPCR. (G) ELISA quantified the NO levels of cell lysis solution in AGS cells overexpressing METTL3 (normalized by total protein). (H‐I) mRNA levels of NOS2‐3 (H) and NO levels (I) in METTL3 knockdown AGS cells and corresponding controls. (J) mRNA levels of NOS2‐3 in AGS cells treated with BH4 or distilled deionized water (blank), as measured via qPCR. (K) ELISA quantified the NO levels in AGS cells treated with BH4 or blank. (L) The volcano plot showed the different metabolites of AGS cells on BH4 treated. The data are presented as mean ± SEM of three independent experiments. *, P <0.05; **, P <0.01; ***, P <0.001; ns, not significant. Abbreviations: BH4, tetrahydrobiopterin; SPRi3, SPR inhibitor 3; NOS, nitric oxide synthase; NO, nitric oxide; ELISA, enzyme‐linked immunosorbent assay

Fig 3: METTL3‐mediated m6A modification of PBX1 mRNA maintains stability. (A) Venn plot showed the overlap genes in RNA sequence and m6A sequence analysis. (B) The mRNA levels of the overlap genes in Figure 2A in AGS cells with METTL3 overexpression or knockdown and their negative controls. (C) Western blotting images revealed downregulation of PBX1 on METTL3 knockdown in AGS and MKN1 cells. (D) IHC images of PBX1 expression in xenograft tumor tissues with METTL3 knockdown and the negative control. Scale bar: 200 μm. (E) RIP‐qPCR assay using METTL3‐specific antibody and IgG control antibody to detect the enrichment of METTL3 binding to PBX1 mRNAs. (F) MeRIP‐qPCR analysis was employed to demonstrate METTL3‐mediated PBX1 m6A modifications. The m6A modification of PBX1 was depleted following the knockout of METTL3. (G‐H) The levels of PBX1 mRNA in cells with METTL3 knockdown (G), METTL3 mutant cells (H), and their corresponding control AGS cells, after treatment with actinomycin D (2 μg/mL) at the indicated time points were detected by qPCR. (I) Immunofluorescence images of METTL3 and PBX1 in tumor and normal adjuvant tissues from GC patients. Scale bar: 100 μm. The data are the mean ± SEM of three independent experiments. **, P <0.01; ***, P <0.001; ns, not significant.Abbreviations: RIP, RNA immunoprecipitation; Me‐RIP, m6A‐modified RNA immunoprecipitation; GC, gastric cancer; PBX1, pre‐B‐cell leukemia homeobox 1

Fig 4: METTL3‐mediated BH4 synthesis promoted GC progression. (A) CCK‐8 assay showed the proliferation ability of METTL3 knockdown and negative control cells treated with artificial BH4. (B) Quantification of migration and invasion cells of METTL3 knockdown and negative control cells treated with artificial BH4. (C) CCK‐8 assay showed the proliferation ability of METTL3 overexpressing cells treated with SPRi3 and negative controls. (D) Quantification of migration and invasion of AGS cells overexpressing METTL3 treated with SPRi3. (E) Image of xenograft tumors of MGC803 cells with METTL3 overexpression and blank control. SPRi3 (300 mg/kg) was injected in the surroundings of METTL3‐overexpressing tumors and inhibited tumor growth. DMSO was employed as a negative control. (F) The volume of tumors in Figure 6E at different time points. (G) The BH4 levels in 12 GC tissues and paired normal gastric mucosa were detected by ELISA assay. (H) IHC staining of METTL3, PBX1, and GCH1 in clinical specimens of GC patients (upper panel, Scale bar: 200 μm). Kaplan‐Meier OS analysis of METTL3, PBX1, and GCH1 expression in patients with GC (n = 117). (I) The graphic illustration of METTL3 inducing BH4 synthesis via METTL3‐PBX1‐GCH1 axis. The data are presented as mean ± SEM of three independent experiments. *, P <0.05; **, P <0.01; ***, P <0.001. Abbreviations: BH4, tetrahydrobiopterin; SPRi3, SPR inhibitor 3

Fig 5: m6A‐mediated PBX1‐regulated GC cells proliferation and metastasis. (A) Kaplan‐Meier survival curves of OS and PFS based on PBX1 expression in TCGA database using the online bioinformatics tool Kaplan‐Meier Plotter. (B) CCK‐8 assays revealed knockdown of PBX1 with siRNAs inhibited proliferation ability of GC cells. (C) Knockdown of PBX1 in GC cells suppressed migration and invasion ability as indicated by transwell assay. Cell numbers per field were calculated representatively. (D) PBX1 protein level was increased following METTL3 overexpression and was rescued by PBX1 siRNAs. The protein levels were measured by western blotting assay (left panel), and the grayscale was measured by ImageJ software. (E) CCK‐8 assay showed that the proliferation ability increased on METTL3 overexpression, and this effect was rescued by PBX1 knockdown. (F) Quantification of migration and invasion cells on METTL3 overexpression transfected with PBX1 siRNAs or the negative controls in transwell assays. (G) PBX1‐overexpressed plasmids were transfected into METTL3‐knockdown AGS cells and the corresponding controls. Western blotting analysis revealed the expression efficiency (left panel), and the grayscale was measured by ImageJ software. (H‐I) CCK‐8 and transwell assay revealed that PBX1 overexpression rescued the suppression of proliferation (H), migration, and invasion (I) caused by METTL3 knockdown. The data are presented as mean ± SEM of three independent experiments. *, P <0.05; **, P <0.01; ***, P <0.001. Abbreviations: OS, overall survival; PFS, progression‐free survival; TCGA, The Cancer Genome Atlas Program; CCK‐8, cell counting kit 8