(a and b), Suppression of shCRAD-induced cell hyperproliferation by β-catenin inhibition in IECs. FHC (shCtrl and shCRAD) cells were treated with iCRT14 for 14 days and stained with crystal violet (a). Cell proliferation was quantified by measurement of absorbance at 590 nm (b; n = 3 independent experiments). (c-e), Engrailed-LEF1 inhibits CRAD depletion-induced IEC hyperproliferation. 1,000 cells of CCD-841CoN were seeded onto tissue culture plate. Cells were transfected with indicated plasmids. Eng-LEF1 was used as a dominant-negative mutant blocker of β-catenin-mediated gene transactivation53,54. 48 h after transfection, cells were incubated in 37 °C tissue culture CO2 chamber for 14 days. Colony forming was visualized by crystal violet staining (c) and quantified (d and e; n = 3 independent experiments). (f-h), CRC cell growth inhibition by CRAD expression. CRC cells (Vec [control] and CRAD expressing) were analyzed for cell counting (f; n = 3 independent experiments), crystal violet staining (g), and quantification (h). (i-o), β-catenin rescues CRAD-induced CRC cell growth inhibition. CRC cells were transfected with CRAD or-catenin plasmids and analyzed for crystal violet staining (i,k,m), quantification (j, l, n), and cell counting (o). (p and q), CRC cell growth inhibition by CPI motif-containing CRAD mutants. CRAD (full length, ΔCPI, and M1-M4)-transfected CRC cells were analysed for quantification of cell proliferation. HCT116 (p); SW620 cells (q). Representative images are shown; Error bars: mean +/− S.D.; NS: not significant (P > 0.05); Two-sided unpaired t-test.