Cell culture and reagents.
The human chondrosarcoma cell line, SW1353 cell line, was obtained from American Type Culture Collection. The cell was cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, Invitrogen Corp., USA). Niclosamide was supplied by Sigma Chemical Corporation (St. Louis, MO, USA),
Cell viability and clonogenic assays.
SW1353 cells (5 x 103/well) were incubated in the 96-well plates. After being treated with niclosamide or DMSO for 24 hours, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazoliumbromide (MTT) solution (5 mg/ml) was added to the culture medium. Viable cells were detected with an MRX II absorbance reader (DYNEX Technologies, Chantilly, Virginia, USA).
To evaluate cell colony-forming ability, SW1353 cells were incubated in 6-well plates for 10 days before being fixed with methanol and stained with Gentian Violet.
Apoptotic cells were measured with Annexin V/FITC kit (BD Biosciences, USA) according to the instructions. Samples were analyzed by a flow cytometer (Becton Dickinson, USA).
Cell cycle analysis
SW1353 cells were seeded in 6-well plates at 2× 105 per well. After being treated with niclosamide for 24 hours, cells were harvested and washed with cold phosphate-buffered saline (PBS). And then cells were stained with PI for 30 min. Samples were analyzed by flow cytometer (Becton Dickinson, USA).
Cells apoptosis was identified after staining with 40,6-diamidino-2-pheny-lindole (DAPI). Condensed chromatin as well as nuclear fragmentation indicate apoptotic nuclei.
Cells were washed and resuspended in lysis buffer (50 mM Tris-HCl,150 mM NaCl,, 1% NP-40, 2mM EDTA, pH 7.4,) containing protease inhibitor (Amresco, USA). Protein (40 µg per lane) was separated by electrophoresis and transferred to a PVDF membrane (Millipore, USA). Following primary antibodies were used in this study: PDGFRβ (Cell Signaling Technology), phospho-PDGFRβ (Thr751, Cell Signaling Technology), STAT3 ( Bioworld Technology Inc.) phospho-STAT3(Ser 727, Bioworld Technology Inc.), phospho-ERK (Thr202/Tyr204, Cell Signaling Technology), phospho-MEK (Ser217/221, Cell Signaling Technology), ERK (Cell Signaling Technology), MEK (Cell Signaling Technology), cyclin D1 (Cell Signaling Technology), Rb (BS1310, Bioworld Technology Inc.), Bcl-2 (sc-783, Santa Cruz), Bax (sc-493, Santa Cruz), Bcl-xl (BS1032, Bioworld Technology Inc.,), Mcl-1 (BS1220, Bioworld Technology Inc.,), β-actin (sc-1616-R, Santa Cruz), and the appropriate peroxidase-conjugated secondary antibodies were then used, followed by detection by enhanced chemiluminescence (Amersham, USA).