Fig. 4: Sox11 is necessary for TGF-β1 induced pEMT and collective invasion.

A Structures of Sox11 WT and KO loci. The red arrowheads mark the target sites for two sgRNAs used to delete a large part of the Sox11 open reading frame (ORF). UTR: untranslated region. B Whole-mount phase contrast microscopy of 931-TKA and 1308-TKA organoid lines treated with TGF-β1 for 72 h. The parental lines and clonally derived organoids treated with non-targeting (NT) sgRNAs (931-TKA: NT6; 1308-TKA: NT2) have WT Sox11 genes. KO lines carry biallellic deletions in Sox11 (931-TKA: KO3 and KO17; 1308-TKA: KO47 and KO56). Scale bar: 100 µm. Representative pictures from one of three independent biological replicates are shown. C Representative images of transwell invasion assays with Sox11 WT and mutant organoid lines as indicated. Organoids were seeded in 3 mg/ml Matrigel in the upper chambers of transwell inserts and treated with TGF-β1 for 72 h. Invasive organoid cells that had passed through the Matrigel layer and crossed the transwell bottom membrane were visualized by crystal violet staining. D Quantification of transwell assays with Sox11 WT and mutant organoid lines. Areas of transwell membranes covered by invaded organoid cells as exemplarily shown in (C) were measured by ImageJ (n = 3). Statistical significance was assessed by one-way ANOVA, ns: not significant; **: p value < 0.01; ***: p value < 0.001. E RNA expression levels of epithelial (Cdh1, Ephb3) and mesenchymal markers (Itga5, Fn1, Snai1, Zeb1) in the indicated Sox11 WT and mutant organoid lines treated with solvent or TGF-β1 for 72 h were determined by qRT-PCR and normalized to those of Eef1a1 (n = 3). The box plots show the 26th to 75th percentiles of the data and the median. Dots represent results of individual measurements. Rel. exp.: relative expression. One-way ANOVA; ns: not significant; *: p value < 0.05; **: p value < 0.01; ***: p value < 0.001. F Protein expression levels of epithelial markers (E-cadherin, Ephb3) and mesenchymal markers (integrin α5, fibronectin, Snail1, Zeb1) in the indicated Sox11 WT and mutant organoid lines treated with solvent or TGF-β1 for 72 h were determined by western blot analyses. Phosphorylated Smad2/3 (pSmad2/3) and total Smad2/3 amounts were analyzed to show TGF-β pathway activation. Detection of Gsk3β served to control equal loading. MW: molecular weight standards in kDa. Images are representative examples from one of three independent biological replicates.