Fig. 3

Hydrodynamic shear stress (+SS) given as orbital shaking led to the acquisition of epithelial to mesenchymal transition (EMT) and stemness-associated genes in primary epithelial tumor cells isolated from patients with breast cancer. a Schematic illustration of the in vitro fluid SS (laminar SS (LSS), oscillatory SS (OSS), or SS) that was exposed to breast cancer cells derived from chemotherapy-treated patients (CT-PCs). CT-PCs (density, 2 × 10⁵ cells) were isolated as described in “Methods” and subjected to cone-and-plate viscometer-based LSS or OSS and orbital shaker-based +SS in non-coated Petri dishes. b (i) Number of suspension cells during LSS (270 rpm corresponds to 20 dyne/cm²) conditions of CT-PCs at 24 h was measured by trypan blue exclusion assay and error bars represent ± SD calculated from at least three independent experiments. Expression level of SS-induced (Egr1, Ap1 and Epcam; (ii)), stemness marker (Nanog. Oct4B and Sox2; (iii)), and EMT marker (N-Cadherin, Twist and Snail1; (iv)), and epithelial marker (E-Cadherin, Claudin-7, and Cytokeratin-8; v) genes in suspension cells during LSS conditions of CT-PCs in non-coated Petri dishes, analyzed by quantitative real-time RT-PCR. c (i) Number of suspension cells during OSS (67 rpm corresponds to 5 dyne/cm²) conditions of CT-PCs in non-coated Petri dishes after 24 h of culture. Expression level of SS-induced (Egr1, Ap1 and Epcam; (ii)), stemness marker (Nanog. Oct4B and Sox2; (iii)), and EMT marker (N-Cadherin, Twist and Snail1; (iv)), and epithelial marker (E-Cadherin, Claudin-7, and Cytokeratin-8; (v)) genes in suspension cells during OSS conditions of CT-PCs in non-coated Petri dishes, analyzed by quantitative real-time RT-PCR analysis. d Number of cells in suspension without SS culture (-SS) or following SS culture (+SS) counted at 3, 5, 7, and 10 days using trypan blue exclusion assay. e-h Quantitative real-time RT-PCR analysis was performed on CT-PCs harvested at 3, 5, 7, and 10 days after culture to measure the expression of SS-induced (Egr1, Epcam, Klf8 and Klf2) (e), stemness marker (Nanog, Sox2, and Oct4B) (f), EMT marker (N-Cadherin, Twist, Snail1, and Vimentin) (g) and epithelial marker (E-Cadherin, Claudin-7, and Cytokeratin-8 (h) genes. i Western blot analysis was performed on CT-PCs (−) and CT-PCs harvested on 10 days after SS culture (SS) to detect the expression of EMT marker (TWIST and N-CADHERIN) and epithelial marker (E-CADHERIN) proteins. j Surface marker expression of CD24, CD44, and CD133 were analyzed using flow cytometry and presented as percentage (fluorescent⁺ cells/all cells × 100%). Expression of each gene in quantitative real-time RT-PCR analysis was normalized to Gapdh. The data are presented as mean ± SEM and are representative of three independent experiments. Statistically significant differences were tested at p < 0.05