Figure 10.

Silencing mPGES-1 prevents colon cancer cells from disrupting pericytes. HT29 human colon cancer cells were transfected with control- or PTGES (mPGES-1 gene)-targeting siRNA for 48 h. (a) Cell lysates were examined by RT-qPCR to analyze mPGES-1 expression in HT29 cells. The PTGES mRNA level was normalized to cyclophilin A. Fold change relative to the control is shown. (b) ELISA was performed to measure the concentration of PGE2 in the conditioned media. Student t-test, ****p < 0.0001. (c) HBVPs were incubated either with fresh culture medium as a control condition (–) or with conditioned medium (CM) from control (siCtrl) or PTGES-silenced HT29 cells. Western blot was performed to analyze N-cadherin, Cx43 and R-Ras expression 48 h later. (d) HBVPs were incubated with DMSO or PGE2 (100 nM) for 72 h, or treated with conditioned medium from control- or PTGES-silenced HT29 cells for 48 h. The cells were detached, and the adhesion of these cells to new culture plate was determined by crystal violet staining and absorbance at 595 nm. The data shown is a representative of three independent experiments performed in quadruplicates. One-way ANOVA test, **p < 0.01; ****p < 0.0001. (e, f) HBVPs (green) were incubated either with fresh culture medium as a control, or with conditioned medium from control- or PTGES-silenced HT29 cells for 48 h prior to coculturing with HUVECs (red) on culture plates. Cells were stained for N-cadherin (e) or Cx43 (f) 18 h later. Scale bar 50 μm.