Fig. 7.

Knockdown of MASTL severely impairs the proliferative, and migratory capacity of cancerous cell lines. a Western blot analysis of MDA-MB-231 cells stably expressing doxycycline inducible bicistronic vectors containing GFP and either shMASTL or scramble controls (shCont). Cells were treated for 72 h with either vehicle, or 1 µg/mL doxycycline (+DOX). Representative blots and quantification from three independent experiments shown (mean ± SEM, n = 3, two-way ANOVA multiple comparisons, n.s = not significant; **p < 0.01). b IncuCyte Cell proliferation assay. Total cell number (nuclei measured by H2B-mCherry), expressed relative to starting time (mean ± SEM shown, n = 3). Two-way ANOVA with multiple comparisons. c Timelapse single cell analysis was used to quantify average mitotic length (NEBD to anaphase). Mean ± SEM is shown, *p < 0.05, **p < 0.01. d Cells were seeded into matrigel and maintained in complete media ± DOX for 7 days. Colonies were fixed and stained for DNA (DAPI, cyan), filamentous actin, F-actin (Phalloidin, red), and shRNA expression was checked by GFP (green) expression. Scale bar 50 µm. e The length of stellate projections, number of projections and average central colony size were quantified from a minimum of 30 colonies from three biological replicates (n.s. not significant, two-way ANOVA, ****p < 0.0001). f Schematic representation of Organotypic invasion assay, where MDA-MB-231 cells are seeded on top of a organotypic collagen matrix and allowed to invade for 14 days toward a chemotactic media gradient. g Representative images of the α-cytokeratin IHC stained organotypic collagen matrix, MDA-MB-231 cells stained brown. Scale bar 50 µm. h Quantification of 30 random fields from three independent experiments for each condition were scored. Shown are the percentage of the cells relative that invaded into the collagen relative to the cells that failed to invade (mean ± SEM, two-way ANOVA with multiple comparisons, ns = not significant, ****p < 0.0001)