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