Saos-2, MG-63, HOS, U20S OS cells and 293T cells were obtained from the American Type Culture Collection and cultured in DMEM medium (Corning, Inc.). All media contained 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), penicillin G (100 U/ml) and streptomycin (100 μg/ml; Sigma-Aldrich; Merck KGaA). All cell cultures were maintained as a monolayer at 37˚C in a humidified atmosphere containing 5% CO2.

The short hairpin (sh)RNA (5'-TTACCTGACCCAGGTTG AA-3') for human CacyBP/SIP gene was inserted into the lentivirus expression plasmid pGCSIL-GFP (Shanghai GeneChem Co., Ltd.) and non-silencing shRNA (5'-TTCTC CGAACGTGTCACGT-3') was used as a negative control. For virus packaging, 0.5 µg CacyBP/SIP-shRNA vector or control (Ctrl) vector together with 0.5 µg pHelper 1.0 and 0.5 µg pHelper 2.0 (Shanghai GeneChem Co., Ltd.), were added to 293T cells with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Saos-2 cells were transduced with either the CacyBP/SIP-shRNA lentivirus or Ctrl lentivirus at a multiplicity of infection of 10 for 72 h. The transduced cells expressing GFP protein were observed by using fluorescence microscopy to determine the transduction efficiency. CacyBP/SIP expression was confirmed by reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis after lentivirus transduction.

After 3 days of transduction, total RNA was isolated from Saos-2, MG-63, HOS and U20S OS cells (2x10⁵ cells/well) using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Total isolated RNA (2 µg) was used to synthesize cDNA using reverse transcription system composed by M-MLV reverse transcriptase (Promega Corporation), M-MLV reverse transcriptase 5X reaction buffer (Promega Corporation), dNTPs (Promega Corporation) and random primers (Invitrogen; Thermo Fisher Scientific, Inc.) for 60 min at 37˚C according to the manufacturer's protocol. CacyBP/SIP mRNA expression was evaluated by RT-qPCR on Roche LightCycler 480 platform with SYBR Master Mixture (Takara Bio, Inc.). The following thermocycling conditions were used for qPCR: Initial denaturation at 95˚C for 30 sec; followed by 40 cycles of denaturation at 95˚C for 5 sec and extension at 60˚C for 30 sec. The absorbance value was obtained at the extension stage. GAPDH was used as the internal reference control. The following PCR primers were used: CacyBP/SIP forward, 5'-CTCCCATTACAACGGGCTATAC-3' and reverse, 5'-GAACTGCCTTCCACAGAGATG-3'; GAPDH forward, 5'-TGACTTCAACAGCGACACCCA-3' and reverse, 5'-CACCCTGTTGCTGTAGCCAAA-3'. Data were analyzed using the 2^-∆∆Cq method (18). Results were presented as CT values, which were defined as the threshold PCR cycle number at which an amplified product was first detected. The average CT was calculated for both CacyBP/SIP and GAPDH, and ΔCT was determined as the ratio of the mean of the triplicate CT values for CacyBP/SIP to the mean of the triplicate CT values for GAPDH. Each experiment was performed in triplicate and repeated three times.

Cells were washed with ice cold PBS and then lysed in 2X lysis buffer (100 mM Tris-HCl, pH 6.8; 2% mercaptoethanol; 20% glycerinum; 4% SDS) on ice for 15 min. The lysates were clarified by centrifugation at 12,000 x g for 15 min at 4˚C and the supernatants were employed for further analysis. The total protein concentration was estimated using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Protein samples (20 µg) were loaded and electrophoresed in an SDS-PAGE (10% gel) at 120 mA for 1 h and subsequently transferred to PVDF membranes (EMD Millipore) at 300 mA for 120 min. After being blocked with TBS with Tween-20 (TBST) containing 5% (w/v) non-fat dried skim milk powder for 24 h at 4˚C, membranes were incubated with a CacyBP/SIP antibody (cat. no. 3354; 1:1,000) or a GAPDH antibody (cat. no. 3683; 1:1,000; both from Cell Signaling Technology, Inc.) overnight at 4˚C. p21 antibody (ab188224; 1:1,000), cyclin-dependent kinase (CDK) 2 antibody (ab32147; 1:1,000), CDK4 antibody (ab199728; 1:2,000), Cyclin D1 antibody (ab226977; 1:2,000), Cyclin E1 antibody (ab33911; 1:2,000), Bax antibody (ab32503; 1:2,000) and Bcl-2 antibody (ab692; 1:500) were purchased from Abcam. After washing with TBST, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (cat. no. 7074; 1:2,000; Cell Signaling Technology, Inc.) at room temperature for 2 h. The membranes were analyzed and visualized by Pierce ECL Western Blotting Substrate (cat. no. 32109; Pierce; Thermo Fisher Scientific, Inc.). Each experiment was repeated three times. The quantification of proteins was performed in ImageJ software (version 1.7.9; National Institutes of Health).

Cell growth was measured using a Celigo Imaging Cytometer (Nexcelom Bioscience LLC). Briefly, 3 days after Saos-2 cells were transfected with the negative Ctrl lentivirus or CacyBP/SIP-shRNA lentivirus, cells in logarithmic phase were digested, resuspended, counted and inoculated in 96-well plates for 5 days (2,000 cells/well). Cell growth rate was defined as cell count on day n / cell count on day 1, where n=2, 3, 4 and 5. Plates were analyzed with using the Celigo image cytometer (4.1.3.0; Nexcelom Bioscience LLC).

Following a previous report (19), Saos-2 cells from different groups (shCacyBP/SIP, shCtrl) were seeded in 96-well plates at a density of 3,000 cells/well after 72 h of lentivirus transduction. On days 1, 3 and 5, MTT was added into each well at a final concentration of 5 mg/ml for 4 h. Acidic isopropanol [10% SDS, 5% (v/v) isopropanol and 0.01 mol/l HCl] was subsequently added to stop the reaction. Absorbance was measured with an ELISA reader (Bio-Rad Laboratories, Inc.) at a wavelength of 595 nm. Viability of cells was calculated from absorbance. Each experiment was performed in triplicate and repeated three times.

In order to assay monolayer colony formation, stably transduced Saos-2 cells from shCacyBP/SIP and shCtrl groups after a 72-h transfection were seeded into 6-well plates at a density of 600 cells/well. After culture for 16 days, cells were fixed with 4% paraformaldehyde (1 ml/well) for 30 min at room temperature. The cells were washed with PBS and then stained with 500 µl Giemsa staining solution at room temperature for 15 min. Colonies (>50 cells/colony) were photographed by using a fluorescence microscope under x100 magnification (IX71; Olympus Corporation) and counted by using the ImageJ software (version 1.7.9; National Institutes of Health). Each experiment was performed in triplicate and repeated three times.

A total of 3 days after transfection, the cells were seeded in 60-mm-diameter plates for 72 h and were collected and fixed and permeabilized in 70% ethanol on ice for 1 h. After washing with PBS twice, the cells were incubated with staining buffer (including 10 µg/ml RNase, propidium iodide) at room temperature for 10 min and were subsequently subject to cell cycle analysis by flow cytometry (Guava easyCyte HT; EMD Millipore). The data were analyzed using the ModFit LT™ 3.3 software (Verity Software House, Inc.).

For the apoptosis assay, after 3 days of CacyBP/SIP lentiviral transfection, the cells were seeded in a 6-well plate for 72 h. Subsequently, the cells were harvested and washed with binding buffer, resuspended in staining buffer and then incubated with Annexin V-APC (cat. no. 88-8007; eBioscience; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions and detected by a flow cytometer (Guava easyCyte HT; EMD Millipore). The data were analyzed by CytoSoft 5.3.1 software (EMD Millipore).

The expression profiles of CacyBP/SIP as well as clinical information of sarcoma (SARC) samples were obtained from TCGA database (https://cancergenome.nih.gov/), including level 2 RNA-seq data from 262 SARC patients (20,21). The K-means clustering analysis with K=2 was performed using R 3.6.1 (https://www.r-project.org/) to categorize the patients into two groups based on the expression levels of CacyBP/SIP. The K-means clustering is an unsupervised clustering, mainly used to group unlabeled data, where K represents the number of packets. Packet represents the grouping condition of unsupervised clustering. In the present study, K=2 means clustering into two groups after unsupervised clustering. The K-means algorithm iteratively assigns each data point to K packets, so that the data points are aggregated based on feature similarity (22).

All statistical analyses were performed using SPSS 13.0 software (SPSS, Inc.). The differences between groups were compared using Student's t-test and data are presented as the mean ± standard deviation of three independent experiments. The log-rank test and Kaplan-Meier curve were employed to evaluate the association between CacyBP/SIP expression and prognosis. P<0.05 was considered to indicate a statistically significant difference.

The levels of CacyBP/SIP expression in a variety of OS cell lines was first evaluated and CacyBP/SIP mRNA expression was detected in all four OS cell lines including Saos-2, MG-63, HOS and U20S (Fig. 1A). After 3 days of CacyBP/SIP lentiviral transfection, the transfection efficiency was found to be ~80% for the CacyBP/SIP-shRNA lentivirus and the Ctrl shRNA lentivirus (Fig. 1B). The fluorescence level of GFP protein was observed to determine the transfection efficiency. CacyBP/SIP mRNA level was confirmed by RT-qPCR. The result indicted that CacyBP/SIP-shRNA lentivirus transfection significantly reduced CacyBP/SIP expression compared the Ctrl lentivirus-transfected cells (Fig. 1C). Decreased CacyBP/SIP protein level was detected in CacyBP/SIP-shRNA lentivirus-transfected Saos-2 cells and the knockdown efficiency was quantitatively evaluated (Fig. 1D).

In order to explore the function of CacyBP/SIP on cell growth, Saos-2 cells expressing either CacyBP/SIP-shRNA lentivirus or Ctrl lentivirus were seeded in 96-well plates and cell growth was monitored with a Celigo Imaging Cytometer daily for 5 days. The results demonstrated that following the downregulation of CacyBP/SIP expression, the total number of cells remained almost unchanged from day 2 to day 5. The cell growth rate was stalled compared with that in the shCtrl group (Fig. 2A and B). There was a consistent result from the MTT assay (Fig. 2C).

The colony formation was assayed to determine CacyBP/SIP knockdown in Saos-2 and U20S cells tumorigenesis in vitro. The results demonstrated that CacyBP/SIP knockdown in Saos-2 cells caused a substantial reduction in colony formation compared with the Ctrl cells (Fig. 2D). Similarly, CacyBP/SIP knockdown in U20S cells also caused a substantial reduction in colony formation (Fig. S1). The number of colonies in the CacyBP/SIP-shRNA lentivirus-transduced cells was significantly decreased compared with the Ctrl group, with shCtrl 112±6 vs. shCacyBP/SIP 45±8 in Saos-2 cells (P<0.01; Fig. 2E).

Alterations in cell proliferation is frequently caused by cell apoptosis (23). To further explore this mechanism, cell apoptosis was examined by Annexin V-APC. As illustrated in Fig. 3A and B, 6 days after cell seeding, the percentage of apoptotic Saos-2 cells was significantly increased in the shCacyBP/SIP group compared with that in the shCtrl group (shCtrl, 4.93±0.06%; shCacyBP/SIP 10.3±0.31%; P<0.01). This result indicated that CacyBP/SIP may be associated with the apoptosis of Saos-2 cells.

To further explore the mechanism by which CacyBP/SIP promoted Saos-2 cell growth, the effects of silencing CacyBP/SIP expression on the cell cycle were investigated by flow cytometry. A total of 67.54±0.76% of shCacyBP/SIP cells were in the G₁ phase, compared with 61.32±0.69% of Ctrl cells (Fig. 3C and D; P<0.01). In addition, S phase distribution of Ctrl cells and shCacyBP/SIP cells were 17.18±0.16 and 10.82±1.01%, respectively (Fig. 3C and D; P<0.01). The results indicated that downregulation of CacyBP/SIP induced G₁/S phase cell cycle arrest.

Next, the levels of key proteins involved in the intracellular mechanisms of the cell cycle and apoptosis were evaluated. The data demonstrated that knockdown of CacyBP/SIP protein was associated with an increase in p21 levels and a decrease in CDK2, CDK4, cyclin D and cyclin E levels (P<0.01; Fig. 4), indicating that CacyBP/SIP arrested the process of cell cycle G₁ to S phase transition. Knockdown of CacyBP/SIP resulted in an increase of Bax (P<0.01; Fig. 4), and decrease of Bcl-2 (P<0.05; Fig. 4), which are two key regulators in the apoptosis process. The results demonstrated that CDK/cyclin/P21 axis and Bcl-2/Bax axis were activated by CacyBP/SIP.

Patients with high CacyBP/SIP mRNA levels demonstrated worse prognosis compared with patients with low CacyBP/SIP mRNA levels, from both the disease free survival (Fig. 5A) and overall survival (Fig. 5B) data. The results demonstrated that CacyBP/SIP had a tumor-driving role in TCGA samples.