Mimetics do not inhibit migration of U251 cells after shRNA-mediated knock-down using L1 shRNA-SH1. U251-scrambled and U251-SH1 cells were seeded in 96-well plates. After 24 h, monolayers were scratched, immediately imaged, and then treated with different concentrations of (a) anagrelide (c) 2-hydroxy 5-fluoropyramidine and (e) mestranol (1, 10 and 100 μM). Cells were imaged every 24 h up to 96 h. Migration of cells treated with mimetics is not inhibited compared to scrambled shRNA-treated cells from 48 h to 96 h. Data show mean + SEM. * p< 0.05, ** p < 0.01 and *** p < 0.001 difference to the respective scrambled shRNA (Scr) control, as determined by one-way ANOVA with Tukey’s post hoc test. (b,d,f) Representative images of U251 scrambled and SH1 cells treated with 100 μM mimetics. Scale bar for all images, 150 μm as in upper panel scrambled.