Echistatin

On reaching 90% confluency, C2C12 cells were incubated in DMEM, supplemented with 2% horse serum (differentiation medium, DM) containing various concentrations of lumican. To block signal transduction pathways, cells were pre-treated with SB203580 (p38 inhibitor, Sigma-Aldrich, St. Louis, MO, USA), TC-I-15 (α2β1 integrin inhibitor, R&D Systems), echistatin (ανβ3 integrin inhibitor, R&D Systems), or integrin α7 neutralizing antibody (Origene, Rockville, MD, USA). After 3 days, the cells were fixed in 4% paraformaldehyde for 10 min, permeabilized in 10 mM sodium citrate buffer containing 0.1% Triton X-100 for 10 min, and blocked with 2% BSA for 1 h. Subsequently, cells were probed overnight with an anti- myosin heavy chain (MyHC) antibody at 4 °C. The cells were incubated with Alexa Fluor 555-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h, and nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for 10 min. Fluorescence images were obtained using an Axio Imager microscope (Carl Zeiss, Oberkochen, Germany) at 100× magnification. Area of MyHC⁺ myotubes and number of nuclei in MyHC⁺ myotubes were quantified in six randomly selected fields per group. Fusion index (%) was calculated as follows: 100 × number of nuclei in myotubes with two or more nuclei/total number of nuclei.

The following compounds were tested for toxicity with the CellTiterGlo assay after a 4-day treatment in A375 melanoma cells and used at the maximal nontoxic dose in the transwell assay: Integrin inhibitor (Echistatin, 100 nM, R&D Systems), FAK inhibitor (PF562271 besylate, 500 nM, Cayman Chemicals), SRC inhibitor (Dasatinib, 10 nM, LC Laboratories), RAC1/CDC42 inhibitor (MBQ-167, 50 nM, Selleckchem), Wnt inhibitor (XAV939, 1 μM, Cayman Chemicals), JNK inhibitor (SP600125, 1 μM, LC Laboratories), ROCK inhibitor (RKI-1447, 500 nM, Cayman Chemicals), PKC inhibitor (Staurosporine, 1 nM, LC Laboratories), AMPKa inhibitor (Dorsomorphin, 1 μM, LC Laboratories), integrin inhibitor (SB273005, 100 nM, Selleckchem), FAK inhibitor (GSK2256098, 1 μM, Cayman Chemicals) and SRC inhibitor (Bosutinib, 100 nM, LC Laboratories). Echistatin was resuspended in water. All other compounds were dissolved in DMSO.
In addition, CD49f (Integrin alpha 6) monoclonal antibody (GoH3) (14-0495-85, eBioscience) was used at 40 μg ml⁻¹ to block integrin α6β4.

Rat mucosal mast cell line (RBL-2H3; CRL-2256^™, ATCC, Manassas, VA, USA) was maintained in ATCC-formulated Eagle’s minimum essential medium (30-2003, ATCC) supplemented with 15% fetal calf serum and 1% penicillin/streptomycin (Gibco). Normal human dermal fibroblast (NHDF; PCS-201-012^™, ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (Welgene, Daegu, Korea) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin (Gibco). Cells were maintained at 37 °C, under 5% CO₂.
For treatment with LIFE, echistatin (3202, R&D system), and ellagic acid (5070, ChromaDex^™), RBL-2H3 cells (passage 3–7) at 90% confluence were treated with substances for 24 h.