Fig. 3

Knockdown of neuregulin (NRG1) reduces macrophage-induced transendothelial migration. a Western blot (top) showing knockdown of NRG1 protein expression in MDA-MB 231 by NRG1-targeted shRNA constructs(shRNA1, shRNA2) but not by empty vector control (shControl) after culture in the presence of doxycycline (+) compared to culture in the absence of doxycycline (−) or parental (231). Quantitation of knockdown (bottom) shown as the relative expression of NRG1 in the various lines with versus without doxycycline. Results presented as mean and SEM of NRG1 protein expression in doxycycline-treated cells divided by expression in untreated cells in three independent experiments; *p < 0.05, t test. b Cells containing the vector control or shRNA constructs were treated with doxycycline for 5 days and assayed for transendothelial migration after 24 h in the absence or presence of macrophages. Data are means and SEM from three independent experiments; **p < 0.01, t test. c Transendothelial migration of cells containing the shRNA1 construct but not induced (No Dox) was compared to doxycycline-treated cells (Dox). Data are means and SEM from three independent experiments; *p < 0.05, t test. d shRNA1 cells transduced with empty rescue vector control (Control) or vector containing NRG1 rescue construct (NRG1) were cultured in the absence or presence of doxycycline (Dox). Treatment with doxycycline blocked the macrophage enhancement of in vitro transendothelial migration (iTEM) in the control vector line, but expression of the NRG1 construct rescued macrophage enhancement. Data are means and SEM from three independent experiments; *p < 0.05 by t test