Fig. 3

SLC6A8 is upregulated by p65/NF-κB in hypoxic TNBC cells. a Schematic illustration of the predicted binding sites for p65/NF-κB in the Slc6a8 promoter region recognized from JASPAR database. b Pearson correlation analysis of Slc6a8 and p65/NF-κB expression based on data from TCGA database. c MDA-MB-231 cells were transfected with pGL3-vector, pGL3/Slc6a8 promoter wide-type (WT) reporter and pGL3/Slc6a8 promoter mutated (MUT) ETV4, FOS, p65/NF-κB or TP53 binding sites reporter under normoxia or hypoxia for 24 h, and luciferase activity assay was performed to assess Slc6a8 transcript activities under normoxic and hypoxic conditions. d-f qRT-PCR and western blot to show p65/NF-κB knockdown (d) decreased SLC6A8 expression both in RNA (e) and protein (f) levels in hypoxic MDA-MB-231 and BT549 cells. g Luciferase assay weas performed to check Slc6a8 transcript activities in p65/NF-κB parental or silenced MDA-MB-231 and BT549 cells cultured in normoxia or hypoxia condition for 24 h. h ChIP assay was conducted to measure the binding of p65/NF-κB to the Slc6a8 promoter region in MDA-MB-231cells cultured in normoxia and hypoxia by p65/NF-κB and IgG antibodies, and qRT-PCR was performed to determine the binding fragment of Slc6a8 promoter precipitated by p65/NF-κB antibodies, presented as enrichment folds normalized to normoxia. Data were presented as mean ± SD (^**P < 0.01, ^***P < 0.001)