Fig 6

NLGN1 influences the APC/β-cat pathway. A and C Confocal microscopy of nuclear β -catenin into HuTu 80 (A) and SNU-C2A (C) cells. Cells were immunostained with anti α-catenin (magenta) and anti β-catenin (red) antibodies. Nuclear β-catenin was calculated as indicated in the methods section. The images in A and C are representative of 1 out of 3 reproducible experiments. Scale bar: 20 μm. The graphs in B and D show the fluorescent intensity of nuclear β-catenin and values are expressed as mean ± SD (HuTu 80: n=40; SNU-C2A: n=70). Mann-Whitney test: ** p < 0.01, *** p < 0.001. E and F Western blotting analysis of nuclear β-catenin into HuTu 80 (E) and SNU-C2A (F) control (shCTRL) or NLGN1-downregulated (shNLGN1b and c) cells. Cell lysates were fractionated using the NEPER nuclear and cytoplasmic extraction kit (ThermoFisher) Immunoblottings were carried out using antibodies specifically recognizing β-catenin (95 kDa), β -actin (b 45kDa), as housekeeping, and GAPDH (37 kDa), as a control of the purity of the fractions, and the images are representative of 1 out of 3 reproducible experiments. Graphs in E and F show the densitometry of nuclear, cytoplasmic and total β-catenin, normalized on β-actin used as housekeeping. Fold-change is calculated with respect to shCTRL cells and values are expressed as mean ± SE (n = 3 independent experiments). Kruskal-Wallis test with Dunn’s posttest: *, p < 0.05 **, p < 0.01. G-I qRT-PCR of NLGN1, c-MYC, Cyclin D2, Twist (undetectable in NCI-H716), N-Cadherin, MMP2, L1CAM and LAMC2 expression level in HuTu 80 (G), SNU-C2A (H) and NCI-H716 (I) control (shCTRL) or NLGN1-downregulated (shNLGN1) cells. Fold-change is calculated with respect to shCTRL cells for each gene and values are expressed as mean ± SE (n = 3 independent experiments). Mann-Whitney test, two tailed: *, p < 0.05 **, p < 0.01, ***, p < 0.001