Fig. 2. Loss of SOX4 does not induce a loss of EMT.

A FACS plots to determine E-Cad^LO cells by FACS in tumors. Tumors were isolated and subjected to a FACS protocol as described in materials and methods. FACS plots show E-cadherin antibody staining on Y-axis and E-cadherin CFP-reporter expression on X-axis. E-Cad^LO cells are found in the left-bottom gate. Plots are representative pictures for each of the three groups (control, SOX4^KO1, SOX4^KO2). B Quantification of E-Cad^LO cells for control and SOX4^KO tumors shown as percentage of parental population (single cells). Data is represented as average ± SD. ANOVA using Dunnett test for multiple comparisons indicated non-significant differences (p > 0.05) of SOX4^KO1 and **p < 0.01 for SOX4^KO2. C FACS plots of E-Cad^LO cells in organoids in vitro in similar analysis as 2(A). Plots for each of the three groups (control, SOX4^KO1, SOX4^KO2) are representative pictures corresponding to one experiment. Experiment was performed four times. D Quantification of E-Cad^LO cells for control and SOX4^KO organoids in vitro shown as percentage of parental population (all single tumor cells). Data is represented as average ± SD. ANOVA using Dunnett test for multiple comparisons indicated non-significant differences (p > 0.05) of SOX4^KO lines compared to control. E Principal Component Analysis of three SOX4^KO organoid lines and two control organoid lines. F Differential gene expression analysis between control and SOX4^KO organoids lines. Gray dots indicate genes; red dots indicate significant genes (adjusted p-value < 0.1). G Heatmap showing differentially expressed genes between control and SOX4^KO organoids. 304 genes are significantly downregulated and 635 genes are significantly upregulated in SOX4^KO organoids. H Gene-set enrichment analysis (GSEA) representing the enrichment of four different EMT gene sets ([8], Hallmark EMT in GSEA [42, 43]) in the bulk RNA-seq expression dataset.