Purecol type 1 collagen

Six well culture plates were coated with 0.05% gelatin type B (Sigma Aldrich, G1393) for 45 min at 37°C. Alternatively, plates were coated with 0.05 mg/mL PureCol Type I collagen (Advanced BioMatrix, #5005) or 1.58 μg/mL fibronectin and were incubated overnight at 37°C. Plates were then rinsed with PBS (pH 7.4) and dried under UV sterilization.

Coverslips were coated with 0.1 mg/mL Purecol Type I collagen (Advanced BioMatrix, #5005) and were incubated overnight at 37°C. Coverslips were then rinsed with PBS (pH 7.4) and allowed to dry. C2C12 myoblasts were seeded onto the coverslips (100,000 cells per well) and transfected the next day with si-RNAs against the control luciferase reporter or si-Snap23 #2 (MSS209236). For myoblasts, the cells were maintained in growth medium for 48h after transfection. For myotubes, the cells were cultured in differentiation medium for four days. Cells were washed briefly with PBS (pH 7.4), fixed in 4% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature, permeabilized in 0.5% Triton X-100 in PBS (pH 7.4) for 20 min at room temperature, and washed briefly with deionized H₂O. As a positive control, cells were incubated with DNase I (Invitrogen, #18068015) for 30 min at room temperature then washed briefly with deionized H₂O. The Click-iT Plus TUNEL Assay kit (Invitrogen, C10617) was used to stain apoptotic cells according to the manufacturer’s protocol. Cells were then stained with 2 μM DAPI (Invitrogen, D1306) for 5 min at room temperature. Cells were washed three times (for 5 min each) with 3% BSA in PBS (pH 7.4) and then imaged by confocal microscopy.