Figure 4

ZEB1sh-ET cells are more epithelial, express higher MYB, and are more proliferative than SCRsh-ET controls. (A) (i) Western blotting for ZEB1 and CDH1 in PMC42-ET cells transfected with shRNA variants 1 to 4; (ii) bar graph of band intensity of the Western blot shown in (i). (iii) Expression (MT-PCR) of EMT-related genes, ZEB1sh-ET relative to SCRsh-ET; fold expression shown. Data shown are the average of four independent biologic replicates; Student paired t test was used to determine significance (*), set at P < 0.05; error bars represent SEM. The expression of other EMT-related genes not shown in this figure is shown in Additional file 1, part A. (B) ZEB1sh-ET express higher MYB protein and mRNA (Additional file 3D) and are more proliferative (C). (i) Percentage of ZEB1sh-ET cells in S phase as determined by BrdU staining after release from mitotic arrest with nocodazole; (ii) Growth rate as shown by the SRB growth assay. Results shown (C, part i) are from three independent experiments; error bars represent SEM, and the repeated-measures two-way ANOVA statistical test was used to determine significance, *P < 0.05. Result shown in (ii) is one representative SRB assay, of a total of three, all of which were found to be statistically significant by the repeated-measures two-way ANOVA statistical test. Further replicates are found in Additional file 1B. (D) ZEB1 knockdown in MDA-MB-231 breast cancer cells: (i) characterization of ZEB1 knockdown (shRNA) MDA-MB-231 cells by QRT-PCR; (ii) immunocytochemistry showing that ZEB1 knockdown results in reexpression of E-cadherin at the cell membrane, and MYB nuclear reexpression; magnification, 400×, scale bar, 50 μm.