Fig. 5

Inhibitory effect of TEPA on cell migration and morphology in TNBC (MDA-MB-231) and NB (SH-SY5Y) cells. a Representative image (n = 3) of transwell migration assays performed on MDA-MB-231 cells treated with 0, 4, and 8 mM of TEPA. b Transwell cell invasion assay for MDA-MB-231 cells treated with 0 and 8 mM of TEPA. c Representative morphology alteration of MDA-MB-231 cells treated with TEPA. Images show how TEPA treatment caused a transition from a mesenchymal to an epithelial-like phenotype. Images were taken at 100 × magnification using an ECLIPSE Ni-E light microscope. The graph shows the average cell size (µm2) relative to the control. Significance was determined by One-way ANOVA where ** and **** represent p < 0.001 and p < 0.0001, respectively. d, e Inhibitory effect of TEPA on cell migration induced by TGF-β in a scratch assay. After generating artificial gaps, TNBC and NB cells were pre-treated with TEPA (4 mM for TNBC and 2 mM for NB) for two hours and then with 15 ng/ml TGF-β for MDA-MB-231 and 10 ng/ml TGF-β for SH-SY5Y in complete media. Wound closure was monitored by IncuCyteS3 at 0 and 14 h for TNBC and 0 and 36 h for SH-SY5Y. Significance was determined by One-way ANOVA with p-value < 0.0001 for both MDA-MB-231 and SH-SY5Y cells. f Analysis of MMP-9, MMP-14, and vimentin at protein levels. Western blot analysis of MMP-9, MMP-14, and vimentin following treatment of MDA-MB-231, and SH-SY5Y, DIPG007, and DIPG010 cells with TEPA (0, 4 and 8 mM for MDA-MB-231 and DIPG010, and 0 & 2 mM for SH-SY5Y and DIPG007) for 24 h. GAPDH has been used as a positive and an endogenous control. Down, Expression analysis by quantitative PCR. The alteration of MT1X gene expression following treating MDA-MB-231, SH-SY5Y, and DIPG007 cells with TEPA for 24 h were analyzed by Real-Time PCR. MT1X is a surrogate marker which its expression is proportional to the intracellular copper concentration. GUSB was used as an internal control