Fig 2: Cybrid-specific potential targets are differentially expressed in breast tumours. (A) Dysregulated genes in more than 10% of the patients in the TCGA breast cancer dataset,12 of which 3 genes, NDRG1, EXT1, and PVT1, located in the frequently amplified 8q24 cytoband. (B) NDRG1 was also amplified or overexpressed in 30% of the METABRIC dataset.13 (C) The highest levels of NDRG1 expression are observed in the basal subtype in both datasets. TCGA indicates The Cancer Genome Atlas.

Fig 3: GR mediates progesterone action in PR-negative breast cancer cells. A, Western blot analysis of GR upon knockdown of GR in MDA-MB-231 (PR-negative) cells using two siRNAs targeting GR. The cells were treated with (+) and without (−) progesterone. Vinculin was used as an internal protein loading control. The numbers on the blot indicate intensity ratio for GR expression normalized to respective vinculin levels. The analysis is representative of three independent experiments. B, quantitative real-time PCR for SGK1 and NDRG1 in GR-depleted MD-231 cells in the presence of progesterone. The bar plot indicates -fold expression of both the genes in each of the siRNAs, compared with respect to expression in siRNA-control. GAPDH was used as an internal normalization control. The analysis is representative of three independent experiments performed in triplicates. C, cell migration in MD-231 cells expressing siRNA-I targeting GR and siRNA-control, treated with (+) and without (−) progesterone, was performed for 21 h. The bar plot indicates percentage cellular migration of the cells upon depletion of GR compared with siRNA-control in the presence and absence of progesterone, and the analysis is representative of three independent experiments performed in triplicates. p value was calculated using Student's unpaired t test. ***, p < 0.0005; ns, not significant. Error bars indicate S.D.

Fig 4: NDRG1 regulates the activation of multiple cellular kinases and cell migration in T47D cells. A, Western blot analysis depicting knockdown of NDRG1 in T47D cells (PR-positive). sh-NT was used as vector control for NDRG1 expression. β-Actin protein was used as a loading control for Western blotting. Numbers on the blot indicate intensity ratio for NDRG1 expression normalized to respective β-actin levels. The analysis is representative of three independent experiments. B, Western blot analysis of p-EGFR (Tyr-1086), p-AKT (Ser-473), and p-ERK1/2 (Thr-202/Tyr-204) in NDRG1 knockdown clones of T47D cells. β-Actin used as a loading control for Western blotting. The figure is representative of three independent experiments. Numbers on the blot indicate average intensity ratio calculated from all of the three replicate experiments for phosphorylation levels of EGFR, AKT, and ERK1/2, normalized to the respective total protein levels (EGFR, AKT, and ERK2). Western blot analysis is representative of three independent experiments. C, cell migration analysis upon depletion of NDRG1 in T47D breast cancer cells. Cells were monitored by a time-lapse wound healing assay for 20 h. Cell migration from the initial to the 20-h time point was plotted as percentage wound closure (average of the three biological replicate experiments), and the comparison was with respect to sh-NT. The figure is representative of three independent experiments performed in triplicates. p value was calculated using Student's unpaired t test. ***, p < 0.0005; ns, not significant. Error bars indicate S.D.

Fig 5: Silencing NDRG1 in SUM-159 and MDA-MB-231 cells. (A) Western blot analysis of lysates from MDA-MB-231 and SUM-159 cell lines transfected with NDRG1 siRNA at 48, 72, 96, and 120 hours post-transfection. (B) Transfection after 48 hours was validated by immunocytochemistry for depleted NDRG1 protein (brown) in MDA-MB-231 and SUM-159 cells. Blank control represents non-transfected control cells; transfection reagent represents cells in the presence of DharmaFECT 4 transfection reagent only; negative control represents cells transfected with non-targeting control siRNA. (C) Proliferation of SUM-159 (top) and MDA-MB-231 (bottom) cells was determined using the SRB assay at 120 and 72 hours, respectively, post-transfection with NDRG1 siRNA or non-targeting control siRNA. Experiments were performed in triplicate, and data were normalised to control (untreated) cells; statistical significance was computed using 1-way ANOVA. (D) Migration rate of SUM-159 (top) and MDA-MB-231 (bottom) cells was determined by wound healing assay 48 hours after transfection. Relative migration was determined after 24 hours. Data were normalised to control cells; statistical significance was computed using 1-way ANOVA. ANOVA indicates analysis of variance; SRB, sulforhodamine B.