Fig. 3: Silencing of ZAK gene in mesenchymal cells reverses EMT phenotypes and attenuates bone metastasis

a Changes in the expression of ZAK and EMT-associated genes by ZAK knockdown in mesenchymal cells as determined by Western Blots. b Immunofluorescence staining patterns of vimentin and E-cadherin in MDA-MB-231 cells expressing shCrtl or shZAKs. Cell nuclei were stained with DRAQ5. Scale bars = 50 μm. c Representative migration photos of the above cells determined by Boyden Chamber assay. Quantification of migration is shown as means (SD from three independent experiments). ***P < 0.001 (One-way ANOVA followed by Tukey’s test). d Proliferation curves of the above cells grown in regular (left) or nutrition reduced media (right). Mean values ± SD from three independent experiments are shown. *P < 0.05, **P < 0.01 and ***P < 0.001 (Two-way ANOVA followed by Bonferroni post-tests). e Dose-response curves of the above cells treated with graded concentrations of paclitaxel. IC50 values were obtained by using logistic nonlinear regression analyzing model of MicroCal Origin 8.5 software. Error bars denote SD from two independent experiments, each done in quadruplicate. **P < 0.01 (One-way ANOVA followed by Tukey’s post hoc test). f MDA-MB-231-shCtrl cells and MDA-MB-231-shZAK2 cells were injected into left cardiac ventricle of immunedeficient mice (n = 6), respectively. Representative 2D bioluminescence imaging photos of the two groups at different time points are shown in the left panel. Right graph represents fold changes of bioluminescence values at each time point, which were calculated using Day 0 values as baseline and are plotted as Means ± SD g Representative 3D bioluminescence plus micro CT images of the above shCtrl mice and shZAK-2 mice on Day 28. Quantification of 3D bioluminescent sources is shown on right panel. Additional data related to Fig. 3 are in Supplementary Figures S3 and S4