Sc 65398

Fibroblasts, keratinocytes and melanocytes from the same donors were isolated (Larsson et al, 2005 (link)). Skin constructs consisting of a fibroblast/collagen matrix in the bottom with a layer of melanocytes and keratinocytes allowed to form an epidermis-like layer with cornified keratinocytes covering the construct were prepared according to Li et al, 2011 . Ex vivo skin (biopsies of 4 mm diameter) was obtained from excess skin from reduction plastic surgery of breast. The biopsies were placed in inserts with reconstructed skin medium II (Li et al, 2011 ). After exposure to UV, the samples were fixed, embedded in paraffin and subjected to immunohistochemical staining according to standard methods using a monoclonal anti-mouse primary antibody FAP-α (1:100, sc65398, Santa Cruz Biotechnology) and polyclonal anti-rabbit primary antibody tyrosinase (1:50, ab175997, Abcam, Cambridge, UK).

For the detection of the intracellular distribution of FAP, MPRIP, and YAP, cells (1 × 10⁴ cells/well) were seeded into six-well glass-bottom plates, fixed in 4% paraformaldehyde for 15 min, and then permeabilized with 0.2% Triton X-100 (PBS) for 10 min. Nonspecific binding sites were blocked with 1% BSA in PBS for 1 h. Cells were treated with a primary antibody specific for FAP (sc-65398, Santa Cruz Biotechnology), MPRIP (14396, Cell Signaling Technology, Beverly, MA) or YAP(14074, Cell Signaling Technology, Beverly, MA) overnight at 4°C. Thereafter, the cells were incubated with Alexa Fluor647-goat anti-mouse IgG (H + L) and Alexa Fluor488-goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, 115-605-003, 111-545-003). Herein, 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime Institute of Biotechnology, Shanghai, China) was used to stain nuclei before capturing images. The images were acquired using a confocal microscope (Zeiss, Oberkochen, Germany). The red fluorescence indicated FAP expression, the green fluorescence indicated MPRIP or YAP expression, and the blue fluorescence indicated the nuclei.