Figure 4.

C12-iE-DAP induced NOD1 activation also enhancesin vitroadhesion and migration of colon cancer cells. (A) Representative images of HT29 adhering to collagen I, IV and fibronectin. Bright intensities represent RFP-expressing HT29 cells. There is a marked increase in adhesion upon C12-iE-DAP stimulation compared to DMSO control and ML130 co-incubation. (B) Quantification of adhesion using crystal-violent-colorimetric method shows significant increase in HT29 adhesion to fibronectin (2-fold), collagen I (2-fold) and IV (1.5-fold) compared to baseline. These increases are abolished with the co-incubation of ML130. (C) MC38 adhesion to collagen I and fibronectin under DMSO, C12 and ML130 + C12 treatments are done to ensure that the effect of NOD1 on HT29 is not specific to cell line of one species. (D) There is a significant increase in MC38 adhesion to collagen I (1.5-fold) and fibronectin (1.5-fold) compared to baseline. These increases are again abolished with the co-incubation of ML130. (E) Similarly, C12-mediated NOD1 activation also increases simple migration of RFP-expressing HT29 cells in a Boyden chamber model. (F) Quantification of migration across the Boyden membrane shows a 2-fold increase in NOD1 activated cancer cells compared to DMSO and ML130 co-incubation (P < 0.0001). (G) Scale bar measures 1mm and applies to all microscopy images in Fig. 4. Error bars represent SEM. A minimum of 3 repeats each with 8 replicates are done for each ECM and 3 replicates for migration assays. All comparisons are made with respect to the DMSO control. Only significant comparisons are labelled. “***” denotes P < 0.0001