Fig 2: Detection of molecular changes in NSF cells versus SF cells using RT2 profiler qPCR arrays. (A) SW620, DLD-1 and HCT116 SF-and NSF-cells were subjected to sphere forming conditions and mRNA was isolated and analyzed with RT2 PCR profiling to obtain expression patterns for 160 adherens junction, extracellular matrix and adhesion molecules. Heat maps indicating the level of expression representing are shown (red: high expression, yellow: intermediate expression, white low or absent). Full dataset available in Supplementary Table 1. (C–E) Quantification of CDH1, CDH3 and CTNNA1 mRNA expression of the data from (A). Maximal fold changes (FC) between SF and NSF are indicated in red. (F) E-(CDH1), P-cadherin (CDH3) and a-catenin (CTNNA1) protein expression was assessed by Western blotting. GAPDH is shown as loading control. (G) Western blot analysis of a-catenin as well as E- and P-cadherin expression in the DLD1-NSF subclones #1–6. GAPDH and a-tubulin are shown as controls.

Fig 3: Effect of double versus single knockdowns in HCT116 and DLD-1 cells. (A,C) Quantification of projected spheroid versus cell assemblage areas of after 48 h (cell covered areas). Boxes represent the interquartile range; the horizontal line in the box indicates the median; whiskers extend to min and max. P-values are indicated and non significant comparisons are labeled with ns. HCT116-NTC (n = 12), HCT116-CDH1-KD (n = 12); HCT116-CDH3-KD (n = 10), HCT116-CDH1/3-KD (n = 13); DLD-1-NTC (n = 17), DLD-1-CDH1-KD (n = 12), DLD-1-CDH3-KD (n = 15), DLD-1-CDH1/3-KD (n = 17), DLD-1-CTNNA1-KD (n = 8). (B,D) Knockdown efficiency was determined by Western blotting.

Fig 4: Phenotypes induced by loss of E-cadherin, P-cadherin and a-catenin by RNA interference. (A,D,G) Phase contrast micrographs of cells cultivated under spheroid forming conditions for 48 hours. Cell covered areas are encircled with a white dotted line for clarity. Scale bars: 100 µm. (A) CDH1 knockdown in HCT116 and DLD-1 cells. (D) CDH3 knockdown in DLD-1 and SW620 cells. (G) CTNNA1 knockdown in HCT116 and SW620 cells. (B,E,H) Quantification of projected spheroid versus cell assemblage areas of after 48 hours (cell covered areas). Boxes represent the interquartile range; the horizontal line in the box indicates the median; whiskers extend to min and max. P-values are indicated and non-significant comparisons are labeled with ns. (B) HCT116-NTC, HCT116-CDH1-KD n = 4 each; DLD-1-NTC, DLD-1-CDH1-KD n = 6 each; E DLD-1-NTC, DLD-1-CDH3-KD n = 6 each; SW620-NTC, SW620-CDH3-KD n = 6 each. H HCT116-NTC, HCT116-CTNNA1-KD n = 6 each; SW620-NTC n = 6, SW620-CTNNA1-KD n = 5. (C,F,I) Knockdown efficiency was determined by Western blotting.