Fig 2: DIO3OS interacts with PTBP1 in the nucleus.a Biotin-RNA pulldown assay using full-length DIO3OS transcript (sense) and the antisense RNA, followed by silver staining. Red box indicates the differential band. MW molecular weight. b Mass spectrometry profile of DIO3OS-binding protein PTBP1 with high score. c Biotin-RNA pulldown assay followed by western blot demonstrating the interaction of DIO3OS and PTBP1. MW, molecular weight. d RNA immunoprecipitation assay with the anti-PTBP1 antibody demonstrating the interaction of DIO3OS and PTBP1 in MCF-7/T47D LTED cells. MW, molecular weight. e Fluorescence assessment of the subcellular colocalization of DIO3OS and PTBP1 in MCF-7/T47D LTED cells. DIO3OS and PTBP1 were detected respectively by RNA FISH (green) and immunofluorescence (red) staining. Scale bars, 10 µm. f Biotin-RNA pulldown assay followed by western blot demonstrating the interaction of DIO3OS and the RNA recognition motifs (RRMs) of PTBP1. Schematic diagram for PTBP1 truncation variants deleting the RRM1, RRM2, RRM3, or RRM4 (upper). Representative western blot for in vitro binding of FLAG-tagged PTBP1 truncation variants with DIO3OS (bottom). MW, molecular weight. g, h Cell number counting (g) and MTT assay (h) of MCF-7 LTED cells transiently transfected with control or PTBP1 siRNAs. i, j ECAR values and calculated glycolytic capacity of MCF-7 LTED cells (i) and T47D LTED cells (j) transiently transfected with control or PTBP1 siRNAs. k Glucose uptake of MCF-7/T47D LTED cells transiently transfected with control or PTBP1 siRNAs. l Lactate production of MCF-7/T47D LTED cells transiently transfected with control or PTBP1 siRNAs. Means ± s.d. of experimental triplicates (d, g–l), one representative experiment out of three that were similar (a–c, e, f) are shown, and P-values were determined using two-tailed Student’s t-test (d), two-sided one-way ANOVA with Dunnett’s multiple-comparisons test (g–l). Source data are provided as a Source Data file.

Fig 3: Functional LDHA-203 is upregulated in LTED cells and AI-resistant breast cancer cells.a PCR and agarose gel of the 5’/3’UTR of LDHA mRNA in parental MCF-7/T47D cells and their LTED derivatives. b Half-life of LDHA mRNA in parental MCF-7/T47D cells and their LTED derivatives was determined by using 10 µg/mL actinomycin D (ActD) at indicated time points. Means ± s.d. of n = 4 (MCF-7 cells) or n = 3 (T47D cells) independent experiments are shown. c Immunoblot and quantification of LDHA protein expression in parental MCF-7/T47D cells and their LTED derivatives. MW molecular weight. d qRT-PCR of the 5’/3’UTR of LDHA mRNA in AI-sensitive or AI-resistant breast cancer tissues. Mean ± s.e.m. of n = 18 biological replicates are shown. E PCR and agarose gel of the 5’/3’UTR of LDHA mRNA in AI-sensitive or AI-resistant breast cancer tissues. n = 8 biological replicates. f, g Cell number counting (f) and lactate production (g) of parental MCF-7/T47D cells transiently transfected with different LDHA variant-expressing plasmids or control vector. h, i ECAR values and calculated glycolytic capacity of parental MCF-7/T47D cells with indicated treatments. j Graphical illustration of the DIO3OS/PTBP1/LDHA glycolysis cascade acting as a switch regulating the glucose metabolism in breast cancer adaptation to LTED by stabilizing LDHA mRNA. Means ± s.d. of experimental triplicates (c, f–i), one representative experiment out of three that were similar (a, e) are shown, and P-values were analyzed by two-tailed Student’s t-test (b–d), two-sided one-way ANOVA with Dunnett’s multiple-comparisons test (f–i). Source data are provided as a Source Data file.

Fig 4: DIO3OS regulates LTED cell proliferation and glycolysis through PTBP1.a Lactate production of MCF-7/T47D LTED cells co-transfected with control LNA/DIO3OS-targeting LNA and control vector/PTBP1 overexpression plasmid. b, c ECAR values and calculated glycolytic capacity of MCF-7 LTED (b) and T47D LTED (c) cells with the indicated treatments. d Lactate production of parental MCF-7/T47D cells transfected with control construct or co-transfected with DIO3OS expression plasmid and PTBP1 siRNAs or control siRNA. e, f ECAR values and calculated glycolytic capacity of parental MCF-7 (e) and T47D (f) cells with the indicated treatments. g–i Cell number counting of MCF-7/T47D LTED cells (g) and parental MCF-7/T47D cells (h, i) with the indicated treatments. j Representative immunofluorescence images and quantification of EdU-incorporated MCF-7 LTED cells with the indicated treatments. Scale bars, 50 µm. k Representative images and quantification of plate clone formation of MCF-7 LTED cells with the indicated treatments. Means ± s.d. of experimental triplicates (a–k) are shown, and all P-values were analyzed using two-sided one-way ANOVA with Dunnett’s multiple-comparisons test. Source data are provided as a Source Data file.

Fig 5: DIO3OS interacts with PTBP1 to upregulate LDHA mRNA stability.a Schematic diagram of alternative splicing (AS) analysis for RNA-seq of MCF-7 LTED cells with or without DIO3OS/PTBP1 knockdown (KD). b Venn diagram of the differentially spliced events in MCF-7 LTED cells transfected with control LNA/siRNA and DIO3OS LNA or PTBP1 siRNA. c Pie chart of twelve types of the differentially splicing events occurred in DIO3OS/PTBP1-knockdown MCF-7 LTED cells. d KEGG pathway enrichment of the differentially spliced genes in DIO3OS/PTBP1-knockdown MCF-7 LTED cells using the DAVID bioinformatics tool are plotted as the -log2 P-value. e LC-MS analysis of lactate production in MCF-7 LTED cells with indicated treatment. f PCR and agarose gel of the 5’/3’UTR of LDHA mRNA in MCF-7 LTED cells with indicated treatment. g qRT-PCR of LDHA mRNA in MCF-7 LTED cells with indicated treatment. h Immunoblot of LDHA expression in MCF-7 LTED cells with indicated treatment. MW molecular weight. i Half-life of LDHA mRNA in MCF-7 LTED cells with indicated treatment was determined by using 10 µg/mL actinomycin D (ActD) at indicated time points. j RNA immunoprecipitation assay with the anti-PTBP1 antibody revealing the interaction of PTBP1 and LDHA variant harboring 3’UTR in parental and LTED MCF-7 cells with indicated treatments. k Schematic diagram of the full length of two different LDHA variant-expressing plasmids, and the luciferase reporter plasmids fused with different 5’/3’UTR sequences of LDHA. l Biotin-RNA pulldown followed by western blot demonstrating the interaction of PTBP1 and different LDHA variants with or without intact 3’UTR. MW molecular weight. m RNA immunoprecipitation assay with the anti-PTBP1 antibody revealing the interaction of PTBP1 and LDHA 5’/3’UTR in MCF-7 LTED cells transfected with different LDHA-UTR-fused luciferase reporter plasmids. n Luciferase reporter assay in MCF-7 LTED with indicated treatment. Means ± s.d. of n = 3 (e, g, j, m, n) or n = 4 (i) independent experiments yielding similar results, one representative experiment out of three that were similar (f, h, l) are shown, and P-values were analyzed using two-tailed Student’s t-test (e, j, m), two-sided one-way ANOVA with Dunnett’s multiple-comparisons test (g, i, n). Source data are provided as a Source Data file.