Active mmp 2

The culture supernatants were collected from vehicle, 1.0 μM trabodenoson and 10 μM trabodenoson treatments at the 2- and 8-day time points. Samples were mixed with tris-glycine SDS-PAGE sample buffer (1:1; Life Technologies, Carlsbad, CA, USA) without a reducing agent, and were subjected to electrophoretic analysis using tris-glycine-buffered SDS-polyacrylamide gel containing 0.1% gelatin (Thermo Fischer Scientific). The gel was developed according to manufacturer's instructions and stained with SimplyBlue Safestain (Life Technologies) before imaging. Active MMP-2 (Abcam) was loaded on the gel to help identify the MMP-2 bands for each sample. Densitometry was performed (using ImageJ) subtracting background and normalizing to vehicle-treated samples.

Cultured cells were lysed by use of the RIPA lysis buffering system (Santa Cruz Biotechnology) supplemented with 1% PMSF. Total protein was extracted by Protein Extraction Reagents (Pierce) according to the manufacturer’s instructions. The protein concentration was detected by BCA method with a BCA protein assay kit (Beyotime). Protein samples were separated by SDS-PAGE. Then, the separated proteins were transferred to PVDF membranes electrically. Specific antibodies against Shh (Cell Signaling Tech, 1: 500), Ptch1 (Abcam, 1: 500), Smo (Abcam, 1: 500), active MMP2 (Abcam, 1: 1000), active MMP9 (Abcam, 1: 500), caspase3 (Abcam, 1: 500), cleaved caspase3 (Abcam, 1: 250), Bcl2 (Sigma-Aldrich, 1: 500), and GAPDH (Sigma-Aldrich, 1: 500) were used to incubate the membranes at 4°C for 12 h. Corresponding second antibodies conjugated to HRP (Cell Signaling Technology) were used to incubate the membranes at room temperature for 2 h. The immunoblots were visualized on X-ray films with Super Signal West Pico chemiluminescence reagent (Pierce).