Elevated levels of fatty acid metabolites in cisplatin‐resistant cells and loss‐of‐function assays using the PHGDH inhibitor NCT503 combined with erdafitinib. (A) Intracellular concentrations of major metabolites of fatty acid metabolism according to metabolomics analysis. Acetyl‐CoA and malonyl‐CoA are shown as representative glycolytic metabolites (n = 1). (B) Upregulated FASN expression in parental T24, cisplatin‐resistant T24, parental J82, and cisplatin‐resistant J82 cells according to western blotting. Increased expression of FASN was observed in cisplatin‐resistant cells (n = 1). The numbers below the blot indicate intensity. (C, D) Cell proliferation according to trypan blue exclusion assay and XTT assay after NCT503 plus erdafitinib combination treatment (n = 6, *P < 0.083, Bonferroni/Dunn's multiple comparison test). The error bars indicate SEM. (E) Apoptosis assay using flow cytometric analysis (n = 3, *P < 0.0083, Bonferroni/Dunn's multiple comparison test). The error bars indicate SEM. (F) HIF1α expression in gemcitabine‐resistant cells after combination therapy according to western blotting (n = 3). (G) Downregulation of FASN expression in cisplatin‐resistant cells after combination therapy according to western blotting (n = 1). (H) Survival curves for the high and low FASN expression groups using OncoLnc (P = 0.008, Mann–Whitney U test). These experiments were repeated at least three times. CR‐J82, cisplatin‐resistant J82; CR‐T24, cisplatin‐resistant T24; GR‐J82, gemcitabine‐resistant J82; GR‐T24, gemcitabine‐resistant T24.