Fig. 5

The impact of CIC-TEX on EMT gene expression. a Tumor cells were seeded in soft agar containing 30 μg/ml TEX, where indicated; mean No of colonies±SD (5 replicates) and representative examples after 3wk of culture; b cell cycle progression (flow-cytometry, PI staining) of wt, CIC and kd cells cultured with/without CIC-TEX; mean % of cells (5 replicates) in G0, G1/S and G2/M; c IPA-based Reactome analysis of transcription factor-, stem cell-, EMT-, transcription-, and EMT-regulating genes that mRNA level is ≥2-fold up- or downregulated in CIC-TEX-treated v6kd and Tsp8kd cells (red), Tsp8kd cells (blue) or CD44v6kd cells (violet). d,e Reactome analysis after IPA coordination of miRNA with predicted mRNA targets (miRNA and targetscan databases) of (d) > 2-fold upregulated miRNA (framed) and (e) downregulated miRNA in CIC-TEX-treated kd cells affecting EMT-related genes in kd cells; in (d) mRNA pathways from upregulated miRNA towards EMT are included; for downregulated miRNA (e) only direct predicted mRNA targets are shown (color code as in c). f Flow-cytometry of EMT markers in A818.4 and kd cells with/without CIC-TEX-treatment (72 h); g confocal microscopy of kd cells with/without CIC-TEX-treatment stained for E- or N-cadherin and counterstained for v6 or Tsp8 (scale bar: 10 μm); h Flow-cytometry of ex vivo analyzed EMT markers in dispersed intrapancreatic v6kd tumors from mice with/without CIC-TEX-treatment; (i) Flow-cytometry of EMT-related transcription factors in A818.4 and kd cells with/without CIC-TEX-treatment (72 h); j Confocal microscopy of kd cells with/without CIC-TEX-treatment stained for EMT-related transcription factors NOTCH and Nanog and counterstained with anti-v6 or anti-Tsp8 (scale bar: 10 μm); f,h,i mean % stained cells±SD (3 assays/tumors); a,b,f,h,i significant differences between wt and kd cells: *, significant differences by CIC-TEX-treatment: s. (List of synonyms: Additional file 1: Table S1). CIC-TEX partly rescue impaired anchorage-independent growth and accelerate kd cell cycle progression. DS, confirmed at the protein level, unraveled a strong impact of CIC-TEX on EMT-related transcription factors mostly in v6kd cells at the mRNA and miRNA level, the latter being particularly engaged in Wnt and NOTCH signaling