Figure 3.

SRM depletion enhances erdafitinib efficacy via HMGA2. A, Intracellular polyamine metabolites levels measured by HPLC of WT and SRM KO MGH-U3 cells. CAD, cadaverine; SPM, spermine. B, Intracellular putrescine (PUT) and spermidine (SPD) levels measured by ELISA of MGH-U3 cells treated with 10 μmol/L MCHA or DMSO for 96 hours. C, Western blotting with the indicated antibodies in WT and SRM KO MGH-U3 cells. GAPDH was used as an internal control. D, Heatmap depicting the differentially expressed proteins between WT and SRM KO cells (left) or WT and SRM KO cells treated with 100 nmol/L erdafitinib for 96 hours (P < 0.005; right). Each group contained three independent replicates. Scale bar, log₂-fold change. E, Venn diagram showing the lapping of the downregulated collection in group as depicted in D. Analysis pipeline was performed to identify proteins regulated by SRM KO: (i) 93 proteins were identified after overlapping; (ii) 20 proteins were selected with high abundance (WT count >6,000). Er, erdafitinib. F, Western blotting with the indicated antibodies in WT and SRM KO MGH-U3 or SW780 cells. GAPDH was used as an internal control. G, RIP assays in MGH-U3 cells using eIF5A and IgG antibody. The precipitate was subjected to Western blotting with the antibody against eIF5A. The eIF5A-enriched mRNAs relative to the IgG-enriched value was calculated by qRT-PCR. Scale bar, fold change. H, Western blotting with the indicated antibodies in WT and SRM KO MGH-U3 cells and those transfected with scramble or HMGA2. GAPDH was used as an internal control. I, Cell Counting Kit-8 assay revealed the cell viability of WT and SRM KO MGH-U3 cells and those transfected with scramble or HMGA2 treated with 100 nmol/L erdafitinib. J, Colony formation assay in the indicated MGH-U3 cells with erdafitinib treatment. K and L,In vivo growth curve (K) and representative of xenograft tumors (L) formed by subcutaneous injection of WT and SRM KO MGH-U3 cells and those transfected with HMGA2 into the right flanks of nude mice treated with erdafitinib (15 mg/kg; 5 × 10⁶ cells per mouse; n = 6 for each group). M, IHC staining of Ki67 on WT and SRM KO MGH-U3 xenografts treated as in K. Scale bar, 50 μm. Data are presented as the means ± SD from three independent experiments. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student t test).