Cell culture and reagents
Two human osteosarcoma cell lines (MNNG-HOS,143B) and human osteoblast hFOB1.19 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The American Type Culture Collection (ATCC, Manassas, VA, USA) provided the human osteosarcoma U-2OS cell line. All cell lines were cultured under standard conditions. Human osteosarcoma cells were cultured at 37℃ in a 5% CO2 atmosphere. Meanwhile, the hFOB1.19 cells were cultured in a 5% CO2 atmosphere at 34.5℃. Antibodies against ICT1 (AP20382b; Abgent, San Diego, CA, USA), Cleaved Caspase-3 (Immunoway, YT6161), Cleaved Caspase-9 (Immunoway, YP0598), BCL-2 (ab32124; Abcam, Cambridge, UK) and BAX (ab32503; Abcam, Cambridge, UK) were used.
Establishment of stable ICT1 knockdown cell lines and BCL-2 oversxpression cell lines
The OBiO Technology (Shanghai, China) provided plasmids containing sh-ICT1 or BCL-2 and a negative control plasmid. The shRNA sequences for ICT1 are 5'-GCTGTTAATGCTTGTCTATAACTCGAGTTATAGACAAGCATTAACAGC-3' and 5'-GCAGAATGTGAACAAAGTGAACTCGAGTTCACTTTGTTCACATTCTG C-3'. The overexpression RNA sequence for BCL-2 is : 5′-GTTGTAGCGGGACAC CTACTGAAAGTTCTCTTCAGTAGGTGTCCCGCTACAAAAAAACTTA-3′.HEK 293T cells provided by OBiO Technology (Shanghai, China) were used to package these plasmids into virus particles. Then, the titers of virus were measured. In order to constitute stable ICT1-knockdown cells and BCL-2 overexpression cells , the target cells were infected with 1×108 lentivirus-transducing units with 6 mg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA). After 72 h, infected cells were screened with 2.5 mg/mL of puromycin. Finally, we used western blotting and qRT-PCR to measured the efficiency.
RNA isolation and qRT-PCR analysis
Total RNAs were extracted and reverse transcription were implemented as described before[18]. ICT1, BAX and BCL-2 transcripts were quantified with the SYBR Green qPCR Master Mix (Roche, Switzerland). The primer sequences used for measuring the expression levels of BCL2 , BAX and ICT1 are as follows: BCL-2: forward 5′-GCCCTGTGGATGACTGAGTA-3′, reverse5′-TTCAGAGACA GCCAGGAGAAA-3′; BAX: forward 5′-GCTGGACATTGGACTTCCTC-3′, reverse 5′-GGCGTCCCA AAGTAGGAGAG-3′; ICT1: forward5′-CAGCCTGGACAAGCTC TACC-3′, reverse: 5′-GGAACCTGACTTCTGCCTTG-3′. The reaction conditions were set up in accordance to the instructions. β-actin and 18S were used as internal controls.
Western blotting
Total cellular protein was extracted by making use of a protein extraction buffer (Beyotime, Shanghai, China). Proteins were spilt by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose (NC) membrane (Millipore, USA). After blocking, the membranes were incubated with the primary antibodies at 4 ℃ for one night, Then, the membrane were probed with the secondary antibodies. Finally, the bands were determined using a western electrochemiluminescence substrate (Share-bio, Shanghai, China).
Immunohistochemistry (IHC)
Alena Biotechnology Co., Ltd. (Xi’an, China)provided a microarray containing tissue from 40 OS patients. The IHC assay was carried out as previously described[19]. We employed the corresponding primary antibodies at 1:200 dilutions to detect the Ki67. We took photos of all the sections using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). The final IHC score was counted by multiplying the intensity score with the quantity score as previously described[20]. These scores were judged independently by two veteran pathologists in a blinded manner.
Cell counting kit-8 (CCK-8) assay and Colony formation assay
The CCK-8 assay was performed according to the vendor’s instructions (Dojindo Molecular Technologies, Japan). Briefly, 3×103 cells were seeded into 96-well plates. The absorbance at a wavelength of 450 nm was measured at 0, 24, 48, 72, 96, and 120h using a tablet reader (Thermo Fisher Scientific, Waltham, MA, USA).
The infected OS cells were cultured in a 6-well plate at an initial cell density of 1×103 cells/well. After two weeks, the ice-cold PBS was used to wash the colonies. Then, the cell pellet was fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Eventually, we took photos and counted cell numbers.
Cell apoptosis
In oreder to cell apoptosis analysis, the adherent cells were separated after culturing for 24 h in serum-free medium. Cells were stained with the Annexin V/FITC Kit (BD Biosciences, San Jose, CA, USA) following the manufacturer’s instructions. Finally, the flow cytometry was use to analyze results.
Xenograft Tumor in Nude Mice
The Balb/c nude mice (no sex limitation; 20-25 g) were fed and all animal experiments were taken place in the East China Normal University and were carried out according to the animal experimental protocols authorized by the Animal Care and Use Committee of the Animal Care and Use Committee of the Affiliated Hospital of Nanjing Medical University, Changzhou No.2 People's Hospital. The mice were assigned into several groups randomly. In brief, the Balb/c nude mice were anaesthetised with 1.5% pentobarbital sodium. Then, U-2OS cells (1.5×106) were subcutaneously inoculated into mice. The data of the tumor size was collected every 5 days. All of mice were euthanized by CO2 After 20 days. All Tumors were removed and the weight and size data of which were recorded.
Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay
The percentage of apoptotic cells in the xenograft tumors was quantified using the TUNEL kit (Roche, Basel, Switzerland). This assay was carried out as previously reported[20].
Statistical analyses
The Graphpad software was used for statistical analyses. The expression of all data was represented as mean ± SD. The comparison between different groups was performed by Student's t-test. The p< 0.05 was considered to show a statistically remarkable difference.