Figure 4: Thy-1 in CAFs regulates the CAF-induced EMT and EMT-associated aggressive behavior in MKN28 cancer cells. (A) Treatment with Thy-1-specific siRNA (si-Thy-1) significantly reduced Thy-1 expression in both two primary CAF clones (CAF02 and CAF04). ***, p<0.001, relative to control siRNA (si-Control). (B) Transwell migration and invasion of MKN28 cells co-cultured with the CAF cells treated with si-Thy-1 or si-Control. Cell migration and invasion were performed as described in the “Materials and Methods”. Note that both the migratory and invasive capacities were markedly reduced in the MKN28 cells co-cultured with the CAF cells treated with si-Thy-1, compared to the cells co-cultured with the CAF cells treated with si-Control. Scale bar: 200 μm. ***, p<0.001, relative to the CAF cells treated with si-Control. (C) Expressions of EMT markers in MKN28 cells co-cultured with the CAF cells treated with si-Thy-1 or si-Control. The results showed that expression of the epithelial marker E-cadherin was significantly increased by 3- to 6-folds in MKN28 cells after co-cultured with Thy-1 knockdown CAF cells, compared to the cells co-cultured with si-Control-treated CAF cells. In addition, after co-cultured with Thy-1 knockdown CAF cells, MKN28 cells showed a marked reduction of the expression of the mesenchymal markers, vimentin and Slug, by ~42-95% and 89-90%, respectively. Thy-1 knockdown in CAFs did not significantly affect the expression of Snail and MMP-9 in MKN28 cells, indicating a targeted regulation of Thy-1 for the E-cadherin transcription repressors in the CAF-induced EMT process. ***, p<0.001, relative to si-Control-treated CAF cells.