Fig. 3

GR Ser134 is required for TGFβ1-mediated migration of TNBC cells. a Phosphorylation of GR at Ser134 by 10 ng/mL of TGFβ1 in wt-GR and S134A-GR MDA-MB-231 CRISPR models was examined by Western blot. Densitometry values of the pS134-GR levels are indicated relative to vehicle control. b Proliferation of MDA-MB-231 cells expressing either wt-GR or S134A-GR was examined using MTT growth assays. The mean of three biological replicates ± SD is shown. c MDA-MB-231 cells expressing either wt-GR or S134A-GR clone #1 were treated with Veh, 10 ng/mL TGFβ1, or 10% FBS overnight and then analyzed by scratch wound assay. Cells were exposed to respective treatments during the course of the migration assay to stimulate migration. The fraction of wound area closure was determined by tracing images in ImageJ. The mean of three field images from each of the three biological replicates is shown ± SD. Significance was assessed by two-way ANOVA and Tukey post hoc for comparison within groups (**, p < 0.01 and ****, p < 0.0001). d Transwell migration assays were used to test the migratory activity of wt-GR and S134A-GR (clone #1 and clone #2) MDA-MB-231 cells with either vehicle or TGFβ1 (10 ng/mL) as the chemoattractant in the bottom chamber. Cells were allowed to migrate for 18 h. The mean of three biological replicates is shown ± SD. Significance was assessed by two-way ANOVA and Tukey post hoc for comparison within groups (****, p < 0.0001). e Soft-agar colony formation assays were used to test the effect of TGFβ1, Dex, RU486, and their respective combinatorial treatment on MDA-MB-231 cells expressing either wt-GR or S134A-GR (clone #1 and clone #2). The mean of four field images from each of the three biological replicates is shown ± SD. Significance was assessed by two-way ANOVA and Tukey post hoc for comparison within groups relative to Veh wt-GR cells (****, p < 0.0001). f Secondary tumorsphere of wt-GR or S134A-GR MDA-MB-231 cells. Cells were treated with either Vehicle or 1 μM Dex. Data are presented as the average ± SD of three biological replicates. Significance was assessed by two-way ANOVA and Tukey post hoc for comparison within groups (****, p < 0.0001). g Transwell migration assays were used to test the migratory activity of U2OS cells expressing either wt-GR or S134A-GR with either vehicle or 10 ng/mL TGFβ as the chemoattractant. 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 < 0.0001). h Secondary tumorsphere of wt-GR or S134A-GR U2OS cells. No treatment was added. Data are presented as the average ± SD of three biological replicates. Statistical significance was assessed by unpaired Student’s t test (*, p < 0.05)