Fig 2: MTHFD2 functions through the PI3K/AKT signaling pathway in bladder cancer. (A) Western blot analysis of the activation status of PI3K/AKT signaling under different concentrations of 740Y-P stimulation in T24-shRNA2 cells. (B) Comparison of the activation status of PI3K/AKT signaling between T24-shRNA2 and T24-NC cells with 740Y-P stimulation. Cell proliferation analyzed by (C) Cell Counting Kit-8 and (D) colony formation assay. The bar charts show the proliferative and colony-forming capacity of cells at day 4 and 14, respectively. (E) Cell apoptosis (sum of early and late apoptotic cells) was analyzed by flow cytometry in T24-shRNA2 and T24-NC cells with 740Y-P stimulation. (F) Western blot analysis of PD-L1 expression in T24-shRNA2 and T24-NC cells with 740Y-P stimulation. *P<0.05, **P<0.01, ***P<0.001. 7-AAD, 7-aminoactinomycin D; APC, allophycocyanin; IHC, immunohistochemistry; NC, negative control; OD, optical density; p-, phosphorylated; PD-L1, programmed death-ligand 1; shRNA, short hairpin RNA.

Fig 3: Elevated MTHFD2 expression is associated with a poor prognosis in BC. Association of MTHFD2 expression with (A) TNM-T stage and (B) AJCC stage in TCGA-BC cohort, and (C) TNM-T stage and (D) AJCC stage in TMA cohort. Association of MTHFD2 expression with overall survival in (E) TCGA-BC cohort and (F) TMA cohort. Forest plot of (G) univariate and (H) multivariate Cox regression analysis in TCGA-BC cohort. **P<0.01. AJCC, American Joint Committee on Cancer; BC, bladder cancer; MTHFD2, methylenetetrahydrofolate dehydrogenase 2; TCGA, The Cancer Genome Atlas; TMA, tissue microarray.

Fig 4: MTHFD2 is associated with immune infiltration in BC. (A) MTHFD2 expression was negatively correlated with tumor purity, and positively correlated with the infiltration of CD8+ T cells, macrophages, neutrophils and dendritic cells in TCGA-BC dataset, as determined by TIMER analysis. (B) Association of MTHFD2 expression with several immune cells in TCGA-BC dataset, as determined using CIBERSORT. (C) MTHFD2 expression was strongly related with the expression of immune checkpoint molecules CD274, CTLA4, HAVCR2, LAG3, PDCD1, PDCD1LG2, TIGIT and SIGLEC15 in TCGA-BC dataset. (D) MTHFD2 expression was significantly positively correlated with TMB in TCGA-BC dataset. (E) Association of MTHFD2 expression with the TME presented as ESTIMATE, Immune and Stromal scores in TCGA-BC dataset. MTHFD2 expression was significantly and positively correlated with (F) CD8 and (G) PD-L1 in TMA. *P<0.05, **P<0.01, ***P<0.001. BC, bladder cancer; MTHFD2, methylenetetrahydrofolate dehydrogenase 2; PD-L1, programmed death-ligand 1; TCGA, The Cancer Genome Atlas; TMA, tissue microarray; TMB, tumor mutational burden; TME, tumor microenvironment.

Fig 5: MTHFD2 related pathway analysis in BC. Analyses between patients with low- and high-MTHFD2 expression from TCGA-BC cohort: (A) Volcano plot presents DEGs; red dots indicate upregulated genes, blue dots indicate downregulated genes and grey dots indicate genes with no significant difference. The top 20 enriched KEGG pathways in (B) upregulated DEGs and (C) downregulated DEGs. The top 20 enriched GO functions in (D) upregulated DEGs and (E) downregulated DEGs. (F) Top 20 enriched pathways in GSEA using Hallmark gene sets. (G) Enrichment result of PI3K-AKT-mTOR signaling in GSEA-Hallmark. (H) Correlation between MTHFD2 and PI3K-AKT-mTOR signaling analyzed by ssGSEA. Analyses of RNA-seq data between T24-shRNA2 and T24-NC cells: (I) A heatmappresents DEGs. (J) Top 20 enriched GO functions. (K) Top 20 enriched KEGG pathways. (L) Network of significantly enriched KEGG pathways presented by Cytoscape. BC, bladder cancer; DEG, differentially expressed gene; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; MTHFD2, methylenetetrahydrofolate dehydrogenase 2; NC, negative control; RNA-seq, RNA-sequencing; shRNA, short hairpin RNA; ssGSEA, single-sample GSEA; TCGA, The Cancer Genome Atlas.