Figure 7.

Knockdown of Snail1 and LSD1 expression suppresses cell migration. (A) An E-cadherin promoter luciferase construct was co-expressed with or without Snail1, non-target control (NTC) or LSD1 siRNA in MCF7 cells. After 48 h, luciferase activities were normalized and determined (mean±s.d. of three separate experiments). Luciferase activities for each sample were statistically analysed by Student's t-test (P<0.01). ^*P<0.001; #, no significance. (B) LSD1 or NTC siRNA was expressed in isogenic MCF7 and Snail1/MCF7 cells. After 48 h, a scratch (‘wound') was induced in a cell monolayer and cell culture was continued for an additional 48 h. Images were obtained at the beginning and at the 48 h time point to monitor the cell migration for the closure of the wound. The percentage of cell migration was calculated on the basis of the migration of MCF7 cells (experiments were conducted at least twice in duplicate). Statistical analysis was performed by Student's t test (P<0.01). ^*P<0.001; #, no significance. (C) Snail1 (SN), LSD1, CoREST and NTC siRNA were co-expressed with the E-cadherin promoter luciferase construct in HCT116, PC3 and MDA-MB231 cells. After 48 h, luciferase activities were normalized and determined (mean±s.d. of three separate experiments). (D) Snail1 (SN), LSD1 and NTC siRNA were expressed in HCT116, PC3 and MDA-MB231 cells as described in (C). After 48 h, a scratch (‘wound') was induced in a cell monolayer and cell culture was continued for 48 h to measure cell migration as described in (B). The statistical analysis for the migration of PC3 cells is shown on the bar graph, whereas statistical results for HCT116 and MDA-MB231 cells are shown in Supplementary Figure S12A. Representative images from PC3 cells are shown in Supplementary Figure S12B. (E) PC3 cells were treated as described above and expressions of LSD1, Snail1, N-cadherin, vimentin, ZO-1 and Actin were examined by western blotting.