Cell cultures and reagents
Human osteosarcoma cell lines MG63 and U2OS were purchased from Cell Resource Center of Shanghai Institutes for Biological Sciences (Shanghai, China) and cultured in Minimal Essential Medium (Gibco, Waltham, Massachusetts, USA) with 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), 1% non-essential amino acid (Gibco), and penicillin/streptomycin (Gibco) in a humidified incubator under 95% air and 5% CO2 at 37 °C. All other cell culture materials were obtained from Gibco; all chemicals were obtained from Sigma-Aldrich (St. Louis, Missouri, USA).
Virus packaging and infection
pQCXIH empty vector and pQCXIH-YAP constructs were gifts from Bin Zhao (Zhejiang University, China) [18]. pLKO empty vector and pLKO-YAP-knockdown expressing lentivirus were also constructed to obtain YAP knockdown cell lines. MG63 cells were infected with retrovirus that expresses empty vector and wild-type (WT) YAP separately to generate control and YAP-overexpressing stable cell lines. pLKO empty vector and pLKO-YAP-knockdown expressing lentivirus were used to treat MG63 cells to generate control and YAP-knockdown stable cell lines. Hygromycin and blasticidin screening was performed 48 h after infection.
RNA extraction and quantitative real-time polymerase chain reaction (RT-PCR) analysis
Total RNA was isolated from cells using TRIzol reagent (Invitrogen-Life Technologies, Waltham, Massachusetts, USA). The reverse transcription products were used for RT-PCR with specific primers: MST1 (forward: 5′-AGACCTCCAGGAGATAATCAAAGA-3′; reverse: 5′-AGATACAGAACCAGCCCCACA-3′), Beta-Actin (forward: 5′-GTCTGCCTTGGTAGTGGATAATG-3′; reverse: 5′-TCGAGGACGCCCTATCATGG-3′).
Immunofluorescence staining
MG63 and U2OS cells were fixed using 4% paraformaldehyde in phosphate buffered saline (PBS) for 15 min. After permeabilization, using 0.1% Triton X-100 in PBS and blocking in 3% bovine serum albumin in PBS, the cells were incubated in primary antibodies overnight at 4 °C. Alexa Fluor 546-conjugated secondary antibodies (Invitrogen-Life Technologies; 1:1000 dilution) were used. The samples were mounted using ProLong Gold Antifade Reagent with DAPI (Invitrogen-Life Technologies), and immunofluorescence was detected using an Olympus confocal microscope.
Co-immunoprecipitation
Cells were collected, and proteins were solubilized in immunoprecipitation buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP40, 1% protease inhibitor cocktail) at 4 °C. Then, 1 mg of lysed protein was incubated with YAP antibody (ABclonal Biotech, A1002, College Park, Maryland, USA) and precipitated with protein A or G agarose (Upstate Biotechnology, Lake Placid, New York, USA) at 4 °C overnight. The immune complex was collected, washed three to five times, and probed with 14-3-3β antibody (Cell Signaling Technology, #9636, Danvers, Massachusetts, USA) and YAP antibody (ABclonal Biotech).
Cell counting
MG63 cells were cultured in 96-well flat plates for 6 days. Before seeding, cell numbers were calculated using a countess automated cell counter (Invitrogen-Life Technologies) to keep the initial cell numbers equal. Culture media were rejuvenated every 48 h, and total cell numbers of cells were counted every 24 h. In this study, three independent experiments were performed.
Cell viability assay
Cell Titer-Glo Luminescent Cell Viability Assay (Promega, Madison, Wisconsin, USA) was used to monitor cell total adenosine triphosphate (ATP). MG63 cells were seeded in a 96-well flat plate for 24 h and exposed to methotrexate (20 mM) or doxorubicin (10 μM) for another 24 h. Then, the Cell Titer-Glo reagent was added to the cells for 10 min. ATP was measured using a reporter luminometer. Relative cell viability was calculated according to the manufacturer’s instructions. This experiment was repeated three times.
Cell apoptosis assay
Cell apoptosis was examined by flow cytometry analysis using the Annexin V-FITC and propidium iodide (PI) double-staining technique. MG63 cells were seeded in a 24-well culture plate at greater than 80% confluence and subjected to methotrexate (20 mM) or doxorubicin (10 μM) treatments for 24 h. Cells were stained following the Annexin V-fluorescein isothiocyanate (FITC) cell apoptosis detection kit’s instructions (Beyotime Biotechnology, C1062, Shanghai, China). To confirm our results, three independent experiments were conducted.
Western blotting
Cells were lysed in a RIPA buffer (Beyotime Biotechnology), and total protein concentration was measured using a BIO-RAD Quick Start Bradford Dye Reagent (#500-0205; Bio-Rad Laboratories, Hercules, California, USA) according to the manufacturer’s instructions. Western blotting procedures were performed as reported previously [22]. Grayscale analysis was conducted using Image J software (National Institute of Health, Bethesda, Maryland, USA), and results were calculated from three independent experiments. The primary antibodies used in our experiments were as follows: YAP (ABclonal Biotech, A1002), Phospho-YAP (Cell Signaling Technology, #13008), LATS2 (Sigma-Aldrich, WH0007004M1), LATS1 (Bethyl laboratory, A300-477A; Montgomery, Texas, USA), MST1 (Cell Signaling Technology, #3682), 14-3-3β (Cell Signaling Technology, #9636), and GAPDH (Cell Signaling Technology, #5174).
Statistical analysis
Results are presented as mean ± standard deviation. Comparisons between two groups were assessed using the unpaired Student’s t test. Cyclohexamide grayscale comparison was made using the paired t test. P values less than 0.05 were considered statistically significant. All statistical analyses were conducted using GraphPad Prism software (GraphPad Software, San Diego, California, USA).
