Fig 2: Bioinformatic analysis of NR4A1 expression in human breast cancer and normal breast tissue(A) Analyses of U133A and U133Plus2 datasets in the GENT (Gene Expression Across Normal and Tumor Tissue) Database revealed a significant decrease in NR4A1 mRNA expression in breast cancer tissues versus normal tissues. The case number in each group is indicated. C cancer; N, normal. (B) Analysis of the Metabric dataset with 1986 samples showed a significant decrease in NR4A1 mRNA expression in human breast cancers versus normal breast tissue. The number of samples in each group and the p value are indicated.

Fig 3: TRH stimulates the promoter activity of the NR4A1 gene through TRH receptors.A) A fragment spanning bp -1295 and +152 of the promoter of the human NR4A1 gene fused to a luciferase reporter gene (pA3NR4A1(-1295~+152)-Luc) was transfected into CV-1 cells (CV-1) that express no endogenous TRH receptors (TRHRs) and GH4C1 cells (GH4C1) expressing endogenous TRHRs. In CV-1 cells, 100 nM TRH did not stimulate the promoter activity of the NR4A1 gene, in contrast, it led to a significant increase in GH4C1 cells (255±33% of the control without TRH, n = 3, p<0.05). B) When TRH receptors were expressed in CV-1 cells (CV-1+TRHR), 10-7 M TRH induced a significant increase of NR4A1 promoter activity, suggesting that TRH exhibited stimulation of the NR4A1 gene through TRH receptors. Fold-increase on incubation with 100 nM TRH is represented. The value in GH4C1 cells is also shown as a control. C) The rat POMC promoter containing the region from bp -706 to +64 fused to a luciferase reporter (POMC-Luc) was transfected into At-T20 cells expressing the endogenous POMC gene. The promoter activity was significantly increased about 6-fold by overexpression of NR4A1. D) The level of endogenous POMC mRNA was not altered either by incubation with expression of TR and T3 or by incubation with expression of TRH receptors (TRHRs) and 100 nM TRH in At-T20 cells. E) The POMC promoter activity was not stimulated by incubation with 100 nM TRH even with expression of TRH receptors (TRHRs), suggesting that the effect of TRH occurred in a promoter-specific manner. Data are presented as the mean ± SEM. from three experiments. *, p<0.05; N.S., not significant.

Fig 4: Expression and regulation of NR4A1 by TRH in pituitary thyrotrophs in vivo.The expression of NR4A1 in pituitary thyrotrophs was confirmed by double fluorescent immunohistochemistry. In numbers of cells in the anterior pituitary NR4A1 were expressed and stained as green signals in the nucleus. A) Numbers of cells expressing NR4A1 were also stained with antibody against TSHß (red signals) in the wild-type pituitary (left panel). As shown in the right panel, the intensity and number of the TSHß-immunopositive cells were remarkably decreased in the TRH-deficient pituitary, and at X400, most of the NR4A1 expression was reduced or lost in these cells as seen as black dots (indicated by white arrows). B) As observed in Fig 4B and C, the expression of ACTH (B) and FSH (C) was also observed as red signals in the cytoplasm in the anterior lobe. As expected, these ACTH- and FSH- positive cells expressed NR4A1, but the staining intensity was not altered in the TRH-deficient pituitary (right panel).

Fig 5: IHC analysis of NR4A1 protein expression in the mouse basal-like mammary gland tumors(A) Validation of NR4A1 antibody specificity by using the liver sections prepared from NR4A1 WT (positive control) and knockout (negative control) mice. (B) The schedules for collecting mouse mammary glands and tumors from p53F/F×BRCA1F/F and K14-Cre×p53F/F×BRCA1F/F mice. The early time point was at the mouse age of 4 months. The medium and late time points for K14-Cre×p53F/F×BRCA1F/F mice were the time points when the diameters of their mammary tumors reached 1 and 2 cm, respectively, while these time points for p53F/F×BRCA1F/F mice were the age points that matched each K14-Cre×p53F/F×BRCA1F/F mouse with 1 or 2 cm tumor. (C) Analysis of NR4A1 in normal mammary glands of p53F/F×BRCA1F/F mice and non-tumor mammary glands and different stage tumors of K14-Cre×p53F/F×BRCA1F/F mice by IHC (brown color). The tumor ID numbers are indicated.