Cleaved parp

Exo2, BFA and Secin H3 were obtained from Selleckchem (Houston, TX). Salirasib and β-actin antibody were purchased from Sigma-Aldrich (St Louis, MO). Antibodies that recognize p-AKT, p-ERK1/2, p-STAT3, p-Src, AKT, ERK1/2, STAT3, Src and cleaved (c)-PARP were purchased from Cell Signaling Technology (Beverly, MA). Ki67 and Arf1 antibodies were purchased from Abcam (Cambridge, MA). Cell proliferation was determined by CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) (Promega, Madison, MI) and crystal violet staining. Apoptosis was determined by FITC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend, San Diego, CA). Western blot, Arf1 activation, and Transwell invasion analyses were carried out as described previously [21 (link), 28 (link)–31 (link)].

Cellular protein was extracted from cultured cells and fresh xenograft tumor specimens using lysis buffer (CelLytic MT; Sigma-Aldrich) containing a mixture of protease and phosphatase inhibitors (Sigma-Aldrich). A 20 μg-aliquot of protein extract was analyzed by Western blotting for the proteins of interest^{29 (link)}. The amount of protein in each sample was monitored by expression of β-actin. The following primary antibodies were used at the dilutions shown against both GSK3 isoforms (GSK3α and GSK3β; 1:1,000; Millipore), GSK3β (1:1,000; BD Biosciences) and GSK3β fractions that are phosphorylated at the serine (S) 9 residue (pGSK3β^S9; 1:1,000; Cell Signaling Technology) and the tyrosine (Y) 216 residue (pGSK3β^Y216; 1:1,000; BD Biosciences); glycogen synthase (GS; 1:1,000; Cell Signaling Technology) and its fraction phosphorylated at the S641 residue (pGS^S641; 1:1,000; Cell Signaling Technology); cyclin D1 (1:1,000; MBL); cyclin-dependent kinase (CDK)4 (1:1,000; Abcam), cyclin B1 (1:1,000, Cell Signaling Technology), poly[ADP]-ribose polymerase (PARP; 1:1,000; Cell Signaling Technology), cleaved (c)-PARP (1:1,000; Cell Signaling Technology), and β-actin (1:4,000; Ambion).

Human osteosarcoma cell lines MG-63, Saos-2 and U-2 OS were purchased from the Cell Bank of Chinese Academy of Medical Science (Shanghai, China) and cultured in McCoy’s 5a medium (Gibco, Los Angeles, CA) containing 10% fetal bovine serum (Gibco) and ampicillin and streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO2. All cell lines were used within 20 passages. The antibody against HSP90AA1was obtained from Proteintech. The antibodies against LC3, p62, cleaved PARP, Akt, p-Akt, mTOR, p-mTOR, JNK, p-JNK, p38, p-p38 and actin were obtained from Cell Signaling Technology. Cisplatin, doxorubicin, methotrexate, bafilomycin A1, rapamycin, LY294002 and 3-methyladenine were purchased from Sigma Aldrich (St. Louis, MO, USA).

Proteins were quantified using a modified Bradford protocol (BioRad Laboratories, Hercules, CA, USA) and applied to immunoblotting analysis as described previously (26 (link)). Equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and transferred to nitrocellulose membrane (Whatman, Clifton, NJ, USA). Membrane was blocked with 3% bovine serum albumin (BSA) TBS-T buffer (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20), probed with antibodies targeting to FST (Santa Cruz Biotechnology, Santa Cruz, CA, USA), AUF1 (Millipore Corporation, Billerica, MA, USA), GAPDH (Cell Signaling Technology, Beverly, MA, USA), Lamin (Santa Cruz Biotechnology) or cleaved PARP (Cell Signaling Technology), incubated with horseradish-conjugated secondary antibodies, detected with the SuperSignal West Pico chemiluminescence substrate (Thermo Fisher Scientific), and finally exposed to an X-ray film.