Figure 3.

CT26-KD cells exhibit reduced levels of ERK activation and enhanced expression of vimentin, which are reversed by the co-culture or its simulation. CT26-WT or CT26-KD cells (1.5 × 10⁵ cells) were each cultured alone or in co-culture with RAW 264.7 cells that were seeded in the upper chamber of the inserts at a ratio of 1:1, in serum-starvation medium (final volume 650 μL) for 48 h. Alternatively, single cultures of CT26-WT or CT26-KD cells (1.5 × 10⁵ cells/650 μL) were cultured with or without the addition of recombinant TGFβ (10 ng/mL) or recombinant EMMPRIN (25 ng/mL or 250 ng/mL). Lysates were extracted from the CT26 cells in lysis buffer in the presence of proteinase inhibitors, and the concentrations of phosphorylated ERK1/2 and p38 were determined by ELISA. The ratio pERK/pP38 was calculated for (A) the co-culture (n = 5), (B) the addition of TGFβ (n = 5), and (C) the addition of recombinant EMMPRIN (n = 6). (D–G) CT26-WT and CT26-KD cells (3 × 10⁴ cells each) were incubated as in (A–C), mounted on cover slips, and stained for the expression of the proteins E-cadherin (green) and vimentin (red), and using DAPI (blue) to stain cell nuclei. Bar size is 25 μM. (D) Representative images of the staining in co-culture, quantification of vimentin expression (E) in the co-cultures (n = 6), (F) with addition of TGFβ (n = 5), and (G) with addition of recombinant EMMPRIN (n = 4). Data are presented as means ± SE, and analyzed using a two-way ANOVA followed by Bonferroni’s post-hoc test.