Figure 6.

NOD1 knockdown (KD) cell lines abrogate the pro-metastatic effects of NOD1 stimulation by C12-iE-DAP. (A) Subcultures of HT29 are created using shRNA with puromycin selection. On immunoblotting, the level of NOD1 remaining in Sh1, Sh3 and Sh4 are 48% ± 10%, 60% ± 6% and 94% ± 14%, respectively compared to 100% ± 12% for the non-targeting control (NTC) (P = 0.0255). (B) Wildtype and knockdown HT29 cells lines are subjected to a static in vitro collagen I adhesion assay to determine their phenotype when stimulated with C12. Sh1 fails to respond to C12 stimulation and demonstrates low adhesion comparable to control (P < 0.0001). In contrast, Sh4, which has the same level of NOD1 as NTC, is still able to mediate full C12 response. (C) To ensure that the observed knockdown of NOD1 in Sh1 is not due to non-specific effects of lentiviral particles, we also use siRNA to transiently reduce NOD1 expression, and obtain similar results of NOD1 reduction (49% ± 9% remaining) compared to siControl (P = 0.0443). (D) siRNA knockdown adhesion to collagen I is subsequently performed. The results are similar to those of Sh1 adhesion with siNOD1 lacking a response to C12 stimulation (P < 0.0001). (E) Furthermore, through in vivo hepatic adhesion assay (n = 5 mice/condition) showed a 2-fold increase in for wildtype + C12, but not for DMSO or NTC controls or Sh1 cell lines (P < 0.0001). (E) Representative images are shown for in vivo hepatic adhesion assay. Bright intensities represent either CSFE labeled (wildtype HT29) or RFP tagged (NTC and KD HT29) cells. Scale bar measures 1 mm. Error bars represent SEM. All comparisons are made with respect to the DMSO, siControl, or NTC control. Only significant comparisons are labelled. “***” denotes P < 0.0001, “****” denotes P < 0.00001