Keratinocyte-Secreted Factors Modulate Fibroblast Signaling

The effect of keratinocyte-conditioned media (CM) on TGFβ1 and CTGF expression in normal human dermal fibroblasts (NHDF) was performed as follows. In cases where CM was collected from transfected cells, conditioned media was collected from transfected keratinocytes 24 hours after transfection with FOXO1 siRNA or scrambled control siRNA (Dharmacon) using GenMute siRNA Transfection Reagent (SignaGen Labs). Cells were incubated with 5–10 nM siRNA with transfection reagent for six hours and transferred to standard medium for 16 hours. Cells were transferred to fresh media for the collection of conditioned media. To assess the impact of conditioned media on fibroblasts or human bone marrow-derived mesenchymal stem cells (hBMSCs) the conditioned media was diluted 1:1 with α-MEM medium supplemented with 1% FBS. Therefore, the fibroblasts were incubated in low-serum (total 0.5% FBS) when stimulated with keratinocyte conditioned media. For antibody neutralization studies, anti-TGFβ139 (link) (10 μg/ml, R&D Systems) or anti-CTGF40 (link) blocking antibody (10 μg/ml, Santa Cruz) was added 1 hour prior to incubation of keratinocyte-conditioned media with NHDF or hBMSCs for 3 days.