Fig. 3
Hyperlactylation of BLM at Lys24 induces EPI resistance. a Scatterplot showing the quantification of lactylated sites in relation to peptide intensities in tumor cells separated from EPI-treated CDXE-resistant and CDXparental models, highlighting lactylated proteins and peptides in the DNA repair pathway. KEGG pathway (b) and GO biological process (c) analyses of upregulated lactylated proteins in EPI-treated CDXE-resistant models. d Radar diagram showing the top 30 lactylated proteins enriched in the tumor resistance pathway in tumor cells separated from EPI-treated CDXE-resistant models; the larger pink circles represent higher log2FC values; blue and purple numbers represent the quantification of protein lactylation in the EPI-treated CDXE-resistant and CDXparental models, respectively. e BLM-knockout E-resistant cells (E-resistant-BKO) were transfected with HA-tagged wild-type (WT), K24R, K31R, or K38R BLM; after transfection, HA-tagged BLM proteins were immunoprecipitated with an anti-HA antibody or an IgG control and analyzed via western blotting with anti-HA and anti-pan-Kla antibodies. f Illustration of BLM-K24 lactylation identified by MS. g Representative immunofluorescence images showing K24-lactylation-specific antibodies (BLM-K24la, red) merged with DAPI (blue) of nuclei in NR-PT, R-PT, and R-RT bladder tumor tissues; scale bar: 50 μm. BLM-K24 lactylation levels were determined by western blot with BLM-K24la antibody in NR-PT and R-PT bladder tumor samples (h) and in parental and E-resistant UM-UC-3 cells (i). j Western blot showing BLM-K24 lactylation levels in E-resistant cells treated with 0.2 μM EPI, 20 mM sodium lactate, and/or 20 mM sodium oxamate, as indicated. k HA-tagged BLM proteins were immunoprecipitated with anti-HA from E-resistant-BKO cells transfected with HA-tagged WT and K24R BLM plasmids; western blot analysis was performed to determine BLM-K24 lactylation levels. l Cell viability assays detecting the viability of E-resistant and E-resistant-BKO UM-UC-3 cells transfected with WT, K24R, K31R, or K38R BLM plasmids after treatment with 0.2 μM EPI; one-way ANOVA followed by Tukey’s test. m The same experimental setup as in k but displaying cell proliferation by colony formation assays; one-way ANOVA followed by Tukey’s test. The graphs represent three replicates per condition (n = 3). ***p < 0.001 represents a significant difference between two groups
