Loss of MMP7 leads to a decrease in transformation in cells lacking PKP3.

<p><b>(A)</b> mRNA prepared from HCT116 derived PKP3 knockdown cells transfected with the vector control (shpkp3-2 + vec) or the MMP7 knockdown construct (shpkp3-2 + shMMP7-1 and shpkp3-2 + MMP7-2) was used as a substrate for reverse transcriptase followed by real time PCR reactions using oligonucleotides specific for MMP7. All expression was normalized to the levels of GAPDH. The fold change is graphed on the Y-axis and the clone name is on the X-axis. Note that MMP7 levels are lowered in the double knockdown clones as compared to the vector control. The standard errors are plotted and student’s t test was performed (* indicates a p value <0.01). <b>(B)</b> 75μg of a whole cell extract (WCE) was resolved on 12% SDS PAGE gels followed by Western blotting with antibodies specific to PKP3. Note that PKP3 levels are lower in clones with a PKP3 knockdown. Western blots for β actin served as a loading control (upper panels). 100μg of acetone precipitated cell supernatants were resolved on 12% SDS PAGE gels followed by Western blotting with antibodies specific to MMP7. Note that MMP7 levels are higher in supernatants prepared from the PKP3 knockdown cells as compared to the vector controls and the levels are lower in the double knockdown clones. The same blot was stained with Ponceau stain to demonstrate equal loading of proteins (lower panels). <b>(C)</b> Scratch wound healing assays were performed on the HCT116 derived vector control (vec), PKP3 knockdown clones (shpkp3-1 and shpkp3-2), shpkp3-2 derived vector control clone (shpkp3-2+vec) and shpkp3-2 derived MMP7 knockdown clones (shpkp3-2+shMMP7-1 and shpkp3-2+shMMP7-2) as described. <b>(D)</b> Matrigel invasion assays were performed in Boyden’s chambers for HCT116 derived vector control cells, PKP3 knockdown clones and the double knockdown clones. The number of cells observed in ten random fields of the membrane for each clone was determined as described in Materials and Methods, representative images of for each clone are shown. The mean and standard deviation of three independent experiments are plotted. Note that loss of PKP3 leads to an increase in invasion as compared to the vector control and this phenotype is reversed in the double knockdown clones. <b>(E)</b> Soft agar colony formation assay was performed with the HCT116 derived vector control and pkp3 knockdown clones, the shpkp3-2 derived double knockdown clones and the shpkp3-2 derived vector control clone. The mean and standard deviation of three independent experiments is plotted. <b>(F)</b> 1x10⁶ cells of the shpkp3-2 derived vector control or double knockdown clones were injected sub-cutaneously into nude mice and allowed to develop tumors. The table shows the number of mice injected with the respective clones and the number of mice among them which were able to develop tumors. Wherever indicated the p value was calculated using a student’s t-test.