Fig 2: Glucose effects of TET1 activation and OGT inhibition in TNBC lines. a Comparison of total cellular TET activity using a 5-hydroxymethyl cytosine (5hmC) assay from TNBC cells grown in low vs. high glucose. MDA-MB-231 cells were cultured in low glucose (1.0 g/L) or high glucose (4.5 g/L) media for 3 days prior to nuclear extraction and 5hmC analysis. Bars indicate SEM of 3 technical replicates per condition, with 3 biological replicates in MDA-MB-231 cells. b Comparison of TARDBP mRNA levels by qRT-PCR between high and low glucose conditions. Media containing high glucose (4.5 g/L) was added for 24 h following growth in low glucose media (1.0 g/L). b TARDBP mRNA levels in OSMI-4 treated (10 µM, 24 h) TNBC cells in high glucose conditions were measured by qRT-PCR, normalized to non-treated controls. Bars indicate SEM of 3 biological replicates, with 3 technical replicates for each cell line. c Comparison of the average OGT inhibition data between low (see Fig. 2k) and high glucose in three TNBC cell lines (HCC70, MDA-MB-468, and MDA-MB-231). d Western blot analysis of TARDBP with OSMI-4 treatment in high glucose media (N = 2, representative blot shown)

Fig 3: Obesity-associated hyperglycemia drives cellular OGT and O-GlcNAc levels in TNBC tumors. a Timeline for DIO induction and tumor studies. b Glucose in blood samples from control and high-fat diet at 5 and 12 weeks. 12 per group. c–e OGT, TET1, and TARDBP levels, respectively, in TNBC cell line MDA-MB-468-derived tumors harvested from DIO (N = 3) and lean control mice (N = 3). The qRT-PCR analysis of each sample was performed in triplicate and means normalized. f Chemoenzymatic labeling of total O-GlcNAc levels in TNBC tumors isolated from DIO vs. lean mice, N = 1. Mouse weight on diet is shown in Additional file 1: Online Resource 6

Fig 4: OGT is upregulated in TNBC tumors and cell lines. a, b Whole genome expression analyses of human patient samples targeting OGT mRNA expression in TNBC, matched breast tissue, and other subtypes of breast cancer. Line equals median, means were compared. Non-tumor samples, N = 8; TNBC/basal-like samples, N = 81; HER2-enriched, N = 53; luminal A, N = 208; luminal B, N = 110. NS indicates no significance (p > 0.05). c, d qRT-PCR analysis of OGT and OGA in non-tumor breast (MCF 10 A), two hormone-expressing BC lines, and three TNBC cell lines. Bars indicate standard error of the mean (SEM) from 3 independent experiments. e Western blot analysis of OGT and OGA protein expression in non-tumor MCF 10 A and tumor lines, with hormone status indicated. f, g Densitometry analysis of OGT and OGA expression in 6 cell lines, displayed relative to the MCF10A OGT band and normalized to actin. N = 1. h Western blot analysis of total cell O-GlcNAc (CST O-GlcNAc MultiMAb antibody) in 6 breast cell lines. N = 1. i Densitometry for total O-GlcNAc levels in 6 breast cell lines. j Dose-response for total cellular O-GlcNAcylation with OSMI-4 in MDA-MB-468 cells, 48 h treatment. Western blot performed with anti-O-GlcNAc MultiMAb. Bars indicate SEM of 3 biological replicates, with 3 technical replicates for each cell line. k TARDBP mRNA analysis by qRT-PCR upon OGT inhibition with OSMI-4- (10 µM, 24 h in low glucose media). Data is normalized to vehicle-treated controls. l Hypothesis for OGT inhibition impact on the TET1-driven target gene TARDBP based on published TET1 pathway [ref. 13]. m Western blot analysis of TARDBP protein expression in OSMI-4 treated (10 µM, 48 h in low glucose (1.0 g/L) media) or control (DMSO) treated TNBC cell lines. N = 2. n Densitometry analysis of TARDBP expression with OSMI-4 treatment, displayed relative to the untreated cells and normalized to tubulin

Fig 5: Knockdown and overexpression studies confirm the novel OGT pathway organization. a TNBC cells were treated with SmartPool OGT siRNA (4 targeting sequences) or a control, non-targeting SmartPool siRNA for 48 h. TARDBP mRNA and protein levels were measured. b TET1 knockdown with SmartPool siRNA (4 targeting sequences) in MDA-MB-231 cells in low glucose (LG, 1.0 g/L) or high glucose (HG, 4.5 g/L) media. Cells were maintained in each respective media for 3 days prior to knockdown. Bars indicate the SEM of 3 technical replicates, but a single biological replicate was performed so significance was not determined. c TNBC cells were treated with SmartPool TARDBP siRNA sequences and OGT mRNA and protein levels were measured. Data is normalized to non-silencing siRNA-treated controls. d Adenoviral vectors containing mRNA for GFP control or human TET1 were used to transiently overexpress the corresponding gene product for 24 h before mRNA was analyzed. Bars indicate the SEM of 3 technical replicates, 2 biological replicates performed. e, f TNBC cells overexpressing GFP or TET1 were treated with OSMI-4 for 24 h and the mRNA and protein levels of TET1 and TARDBP were analyzed. Bars indicate the SEM of 3 technical replicates, 2 biological replicates performed. g Densitometry analysis of TARDBP and OGT knockdown in three TNBC cell lines. Western blots are representative of at least 2 biological replicates per condition. All cells were maintained in low glucose = 1.0 g/L media for at least 3 days prior to analysis. h Densitometry analysis of overexpression-mediated TET1 and TARDBP immunoblot levels with OGT inhibition in MDA-MB-231 cells. Western blots are representative of at least 2 biological replicates per condition. All cells were maintained in low glucose (1.0 g/L) media for at least 3 days prior to analysis