Fig. 3
WDR5 silencing inhibits EMT. a Adhesion to a panel of substrates (collagen—CL, laminin—LM, fibronectin—FN, matrigel—MG) in shLuc or shWDR5 MCF10DCIS cells was considered. Cells were stained by crystal violet and adhesion property was calculated by ImageJ and expressed as ratio of relative cells area of shWDR5 versus shLuc in biological triplicates. Statistical significances among areas were calculated by applying an unpaired Student t test (***P < 0.001). b Morphological changes were evaluated by actin cytoskeleton staining of F-actin in MCF10DCIS cells due to WDR5 silencing by immunofluorescence technique (n = 3). Images show F-actin (green), DAPI (blue), and merged staining of shLuc and shWDR5 cells. c Trajectory plots were obtained by in vitro random migration assay, performed in MCF10DCIS cells. ShLuc and shWDR5 conditions were analyzed by time-lapse microscopy (n = 180 cells in shLuc and n = 209 cells in shWDR5) and data acquired every 10 min over a 24-h time course. d Box plot represents the displacement (μm) of cells as accumulated distance. Significant differences among groups were analyzed by an unpaired Student t test (***P < 0.001). e Effect of WDR5 silencing was evaluated in terms of in vitro FBS-directed cell migration. Images were compared to quantify the percentage of cell migration (mean ± SD; n = 3). Significant differences among groups were calculated by applying an unpaired Student t test (***P < 0.001). f–h Effect of WDR5 silencing on the expression of EMT markers were evaluated by immunofluorescence (f) or western blot (g) in MCF10DCIS cells. Vimentin (VIM), N-Cadherin (CDH2), and SNAI1 and SNAI2 total protein expression was detected in shLuc and shWDR5 cells. Tubulin (Tub) or Vinculin (Vin) were used as normalizers, according to the molecular weight of proteins analyzed. h E-Cadherin (CDH1) level was detected by western blot in membranous (MEB), cytoplasmic (CEB), and total protein lysates. GAPDH was used to assess the amount of total protein lysates in the two samples and as positive control of CEB