Fig. 2

Association between Slug expression and EMT/CSC properties in Em Ca cells. a Upper left: Hec6 cells were seeded at low density with or without STK2. The cell numbers are presented as means ± SDs. P0, P3, P5, and P7 indicate 0, 3, 5, and 7 days after cell passage, respectively. Upper right: western blot analysis for the indicated proteins from Hec6 cells cultured in STK2. Lower: cell cycle analysis of Hec6 cells cultured in STK2 for 6 days by flow cytometry. Con, control. b Aldefluor analysis of Hec6 cells cultured in STK2 (lower) and its control (upper) for 5 days. Cells negative for ALDH activity (treated with ALDH inhibitor DEAB) are located in the area to the far left of each plot, and the positive cells are in black gates (R1 and R2). The percentage of live single cell population contained in each gate is shown. DEAB, diethylaminobenzaldehyde. c Left: phase-contrast images of spheroids of Hec6 cells cultured in STK2 for 2 weeks. Right: the number of spheroids is presented as means ± SDs. Con, control. d Left: phase-contrast images of two independent Hec6 cell lines stably overexpressing Slug (H6SL#8 and #21) and mock-transfected cells. Right: western blot analysis for the indicated proteins from H6SL#8 and #21 cells and the mock. e Upper: H6SL#8 cells were seeded at low density. The cell numbers are presented as means ± SDs. P0, P3, P5, P7, and P9 indicate 0, 3, 5, 7, and 9 days after cell passage, respectively. Lower: cell cycle analysis of H6SL#8 and mock cells at 7 days by flow cytometry. f Aldefluor analysis of H6SL#8 (lower) and mock (upper) cells. Cells negative for ALDH activity (treated with ALDH inhibitor DEAB) are located in the area to the far left of each plot, and the positive cells are in black gates (R1 and R2). The percentage of live single cell population contained in each gate is shown. DEAB, diethylaminobenzaldehyde