Fig. 6

Phosphorylation of GR Ser134 is critical for MAPK signaling. a Gene Set Enrichment Analysis and associated MAPK gene heat map reveal a dramatic loss of expression of genes important for MAPK signaling in TNBC cells expressing S134A-GR compared to wt-GR. b Representative Western blot analysis of pS134-GR, total GR, p-p38, and total p38 protein levels (loading control) in MDA-MB-231 cells expressing either wt-GR or S134A GR clone #1, and c either shcontrol or sh-14-3-3ζ . Cells were treated with 10 ng/mL TGFβ1 at the indicated timepoints. Representative experiments of at least three independent repeats are shown. d WT-GR or S134A GR clone #1 and clone #2 were treated with 10 ng/mL TGFβ1 to confirm previous findings with regard to p38 MAPK phosphorylation. Densitometry values for the p-p38 levels are shown relative to vehicle control in wt-GR. e MAP3K5 protein expression is shown for wt-GR, S134A clone #1, and S134A clone #2. Densitometry values for the MAP3K5 levels are shown relative to wt-GR. f TCGA normalized data was log2 transformed for TNBC patients. Correlation between MAP3K5 and GR was calculated by Pearson’s correlation. g Western blot analysis of pS134-GR, total GR, p-p38, and total p38 in MDA-MB-231 cells pretreated with either 10 μM selonsertib (MAP3K5) or DMSO control for 30 min followed by either vehicle control or 10 ng/mL of TGFβ1 for 1 h. Total p38 serves as a loading control. Densitometry values for pS134-GR and p-p38 levels are indicated relative to vehicle control of each cell line. h Transwell migration assays were used to test the migratory activity of MDA-MB-231 cells in response using either vehicle or TGFβ1 (10 ng/mL) as the chemoattractant in the bottom chamber. Additionally, cells were treated with either 10 μM SB203580 (SB) or 10 μM selonsertib (Sele) in both the upper and lower chambers. Cells were allowed to migrate for 18 h. The mean of the percentage of three biological replicates is shown ± SD. Significance was assessed by Two-way ANOVA and Tukey post hoc for comparison within groups (***, p < 0.001,****, p < 0.0001)