Figure 3. Prolonged treatment of HDFs with Y-27632 causes a CAF-like phenotype. (A–H) HDFs were treated with PBS or 10 μM Y-27632 for 1, 3, 6 and 9 days. HDFs were collected at the indicated times, and mRNA expression levels of several cancer associated fibroblast phenotype related genes as indicated were analyzed using RT-qPCR. The fold changes of these genes in Y-27632 treated HDFs relative to the control (expression level as 1) are shown. (I) and (J) HDFs were treated as described for (A–H), and cell culture media were collected at the indicated times for ELISA assays of protein levels of TNC (i) and PDFGRα (J). The fold changes of TNC and PDFGRα in Y-27632 treated HDFs relative to the control at 3 days (expression level as 1) are shown. (K) HDFs were cultured in epidermal medium with PBS or 10 μM Y-27632 for 3 days, then the medium was replaced without adding Y-27632 for another 3 days, after which the conditioned media were collected and used to culture SCC13 cells. The SCC cells were cultured with the conditioned media for 8 days, and then were assessed every two days for the analysis of cell growth using a CCK8 kit. All experiments were repeated at least 4 times, *P<0.05, **P<0.01 when compared to the corresponding control.