Ectopic NID1 expression induces EMT, migration, and invasion in an autocrine and paracrine manner. (A) Western blot analysis of NID1 protein in concentrated CM from DLD1 cells stably transfected with control (pLEX/cont) or NID1 expression (pLEX/NID1) vector. Coomassie Blue staining was used as a loading control. (B) Cell number of DLD1-pLEX/cont and DLD1-pLEX/NID1 cells at indicated time points. (C) qPCR analysis of EMT-related mRNAs VIM and ZEB1 in DLD1-pLEX/cont and DLD1-pLEX/NID1 cells. (D) Immunofluorescence detection of E-cadherin in DLD1-pLEX/cont and DLD1-pLEX/NID1 cells. Nuclear DNA was detected by staining with 4′,6-diamidino-2-phenylindole. Scale bar: 50 μm. (E) Wound healing assay of DLD1-pLEX/cont and DLD1-pLEX/NID1 cells. The width of scratches in 2 independent wells was analyzed for each state. Results represent the average (%) of wound closure. (F) Invasion assay in modified Boyden chambers: DLD1-pLEX/cont and DLD1-pLEX/NID1 cells were seeded on Matrigel-coated filters. After 48 hours, cells that invaded through the Matrigel were counted after 4′,6-diamidino-2-phenylindole staining. (G and H) qPCR analysis of VIM and ZEB1 in DLD1, HCT15, and HCT116 cells treated with CM from DLD1-pLEX/cont and DLD1-pLEX/NID1 cells for 96 hours. (I) Immunofluorescence detection of E-cadherin in DLD1, HCT15, and HCT116 cells treated with CM from DLD1-pLEX/cont and DLD1-pLEX/NID1 cells for 96 hours. Nuclear DNA was detected by staining with 4′,6-diamidino-2-phenylindole. Scale bar: 50 μm. (J) Wound healing assay of DLD1 cells treated with CM from DLD1-pLEX/cont and DLD1-pLEX/NID1 cells for 30 hours. The width of scratches in 2 independent wells was analyzed for each state. Results represent the average (%) of wound closure. (K) Invasion assay in modified Boyden chambers: DLD1, HCT15, and HCT116 cells were seeded on Matrigel-coated filters with indicated CMs used as a chemoattractant in the lower well. After 48 hours, cells that invaded through the Matrigel were counted after 4′,6-diamidino-2-phenylindole staining. Means ± SD of (B, C, G, and H) n = 3 biological replicates, and (E, F, J, and K) n = 2 biological replicates (each n = 3 technical replicates) are shown. (E, F, and J) Significance was determined using the Student t test and (C, G, H, and K) by 1-way analysis of variance with the Tukey multiple comparison post-test; **P < .01; ***P < .001. cont, control.