Type 4 collagen

Mesenchymal cells were harvested from the median fin folds of 3 dpf larvae. Zebrafish larvae were anesthetized in a standard tricaine (MS-222) solution (0.4% in breeding water) and median fin folds were dissected. The dissected fins were treated with trypsin solution [2.5 mg/mL trypsin (TRL; Worthington), 1.0 mg/mL BSA (Sigma-Aldrich), 1 mM EDTA in PBS] for 10 min at 28 °C. After trypsin treatment, the fins were shaken with PBS for 5 min at 1,000 rpm at 28°C (repeat shaking at three times) and the cell suspension was recovered into one centrifugation tube. Subsequently, the samples were centrifuged at 100 G for 10 min, and the pellet was resuspended in L15 (Gibco) medium with 10% FBS (Gibco) and spread onto a glass bottom dish. Matrigel, type I collagen, type IV collagen were used for the coating substrates on the dish. Matrigel (Corning) was diluted to 1/50 with PBS and coated on the dish. Type 1 collagen (Nitta Gelatin) and type IV collagen (Corning) was diluted to 1/10 with 0.05 N HCl and coated on the dish. Actinotrichia were isolated from the median fin folds of larvae by the above-mentioned method and corrected in culture dishes. The cultured cells and actinotrichia samples were incubated at 28°C and L15 medium with 10% FBS was changed to a fresh one every day.

The ability of migration and invasion in PSCs and PCCs were performed by counting the number of cells migrated or invaded through a Transwell System (8-mm pore size; Becton Dickinson, Franklin Lakes, NJ). For monoculture or co-culture migration and invasion assays, we seeded cells and treated them as previously described [25 ]. For a collagen-coated invasion assay, membranes were coated with 100 μl collagen type I (354,236; Corning, America), type IV collagen (354,233; Corning), or Matrigel at 20 mg per well. To assess the effects of PSC and PCC supernatant stimulation on migration and invasion, separate batches of cells were cultured with each SN indicated in the figure legends. At 24 h (for migration) and 48 h (for invasion) after cell seeding, migrated or invaded cells were fixed with 70% ethanol, stained with hematoxylin and eosin, and counted in five random fields (× 100 magnification). In some cases, migration and invasion abilities were evaluated by normalization to total cell numbers of each cell type to avoid the effects of proliferated cell numbers on migration and invasion. For all migration and invasion assays, three experiments were independently conducted in triplicate.