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: Elevated expression of MTHFD2 in BC. (A) Pan-cancer analysis of MTHFD2 expression, as determined by TIMER. (B) MTHFD2 expression in BC tissue samples and normal bladder tissue samples from TCGA database. (C) MTHFD2 expression in paired BC tissue samples and normal bladder tissue samples from TCGA database. (D) MTHFD2 expression in BC tissue samples and normal bladder tissue samples from the TMA. (E) MTHFD2 expression in paired BC tissue samples and normal bladder tissue samples from TMA. (F) Immunohistochemical analysis of MTHFD2 in the BC tumor and adjacent normal tissues. (G) Expression levels of MTHFD2 in BC and normal bladder cell lines detected by RT-qPCR. (H) Relative expression of MTHFD2 in BC tissues (n=24) and adjacent tissues (n=24) detected by RT-qPCR. (I) Relative expression levels of MTHFD2 in six paired bladder cancer tissues and adjacent tissues detected by western blotting. *P<0.05, **P<0.01, ***P<0.001 as indicated or vs. adjacent tissues or SV-HUC-1 cells. BC, bladder cancer; MTHFD2, methylenetetrahydrofolate dehydrogenase 2; RT-qPCR, reverse transcription-quantitative PCR; TCGA, The Cancer Genome Atlas; TMA, tissue microarray.

Fig 5: Knockdown of MTHFD2 inhibits the proliferation of bladder cancer cells. The knockdown efficiency of MTHFD2-shRNAs was detected by reverse transcription-qPCR in (A) T24 and (B) UMUC3 cells, and by western blotting in (C) T24 and (D) UMUC3 cells. (E) Cell proliferation was determined by the Cell Counting Kit-8 assay. The bar charts shown the proliferative capacity of cells on day 4. (F) Tumorigenic capacity was determined by colony formation assay. (G) Cell cycle and (H) cell apoptosis were determined by flow cytometry. (I) Expression levels of PD-L1 were determined by western blotting in T24 cells infected with MTHFD2 and NC shRNAs. (J) Images of the subcutaneous tumors formed in the nude mice following injection with T24-shRNA2 and T24-NC cells. (K) Tumor volume and (L) tumor weight were reduced in the T24-shRNA2 group compared with those in the T24-NC group. (M and N) Representative H&E staining, and IHC of (M) MTHFD2 and (N) PD-L1 in tumor xenograft tissues compared between the two groups. *P<0.05, **P<0.01, ***P<0.001 as indicated or vs. T24-shRNA2. 7-AAD, 7-aminoactinomycin D; APC, allophycocyanin; IHC, immunohistochemistry; MTHFD2, methylenetetrahydrofolate dehydrogenase 2; NC, negative control; OD, optical density; PD-L1, programmed death-ligand 1; shRNA, short hairpin RNA; Mock, uninfected T24 or UMUC3 cells.