The Saos-2 cell line was purchased from the Institute of Biochemistry and Cell Biology (Shanghai, China). KLF8 and GAPDH primers were synthesized by Applied Biosystems (Carlsbad, CA, USA). All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) unless stated otherwise.

3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-di-phenytetrazoliumromide (MTT) was purchased from Shanghai Dingguo Biological Technology Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). TRIzol^® Reagent and Lipofectamine^® 2000 were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). M-MLV Reverse Transcriptase was purchased from Promega Corporation (Madison, WI, USA) and SYBR^®-Green PCR Master mix was purchased from Takara Bio Inc. (Shiga, Japan). A Cell cycle analysis kit and an apoptosis kit were purchased from Nanjing KeyGen Biotech., Co., Ltd. (Nanjing, China). An ECL-PLUS™ kit was purchased from GE Healthcare (Piscataway, NJ, USA).

Saos-2 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were incubated in a humidified atmosphere containing 5% CO₂ at 3°C.

Small interfering (si)RNA targeting the KLF8 gene (CAGCACTGTTTAATGACAT) and negative control siRNA (TTCTCCGAACGTGTCACGT) were cloned into a pGCSIL-green fluorescent protein (GFP) vector (Shanghai GeneChem Co., Ltd., Shanghai, China). The siRNA plasmids were transfected into 293T cells, together with two lentiviral packaging plasmids (pHelper1.0 and pHelper2.0; Shanghai Gene ChemCo., Ltd.) to generate a lentivirus. After three days of incubation, the culture medium containing the recombinant virus was collected and concentrated using Centricon^®-plus-20 (Millipore, Billerica, CA, USA). For lentiviral infection, 5×10⁴/well Saos-2 cells were incubated with the KLF8 siRNA-expressing lentivirus and non-silencing control lentivirus [multiplicity of infection (MOI)=20] for 24 h. The culture medium was then replaced.

Lentiviral transduction efficiency was validated using qPCR analysis after transduction for 72 h. Total RNA was extracted using TRIzol reagent and reverse transcribed using M-MLV Reverse Transcriptase according to the manufacturer’s instructions. The resulting complementary (c)DNA was used for qPCR analysis using SYBR-Green PCR Master mix. qPCR analysis was performed in triplicate using the TP800 qPCR System (Takara Bio Inc.). Target gene expression was normalized to that of the endogenous control GAPDH. The relative quantitative expression of the target gene compared with GAPDH was expressed as 2^−(Ct-Cc) (Ct and Cc represent the mean threshold cycle differences following normalization to GAPDH). The qPCR primer sequences were as follows: Forward: 5′-TTCAGAAGGTGGCTCAATGC-3′ and reverse: 5′-GGAGTGTTGGAGAAGTCATATTAC-3′ for KLF8; and forward: 5′-TGACTTCAACAGCGACACCCA-3′ and reverse: 5′-GGAGTGTTGGAGAAGTCATATTAC-3′ for GAPDH.

Lentiviral transduction efficiency was validated using western blot analysis after transduction for four days. In brief, cells were harvested following four days of infection and treated with buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 25 mM β-glycerophosphate, 50 mM NaF, 1 mM Na₃VO₄, 1% Triton X-100, 10% glycerol and protease inhibitors (1 mM phenylmethylsufonyl fluoride and 1 mg/ml aprotinin, pepstatin A, and leupeptin). Cell lysates were separated using 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore). Subsequent to blocking, membranes were incubated in milk containing mouse anti-KLF8 monoclonal antibodies (dilution, 1:1,000; Abcam, Cambridge, MA, USA). Western blots were developed using horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (dilution 1:5,000) and the immunoreactive bands were detected using an enhanced chemiluminescence reagent (Millipore). Endogenous GAPDH was used as an internal control.

Following confirmation of the transduction efficiency, cells were seeded onto 96-well plates at a density of 2,000 cells/well on day zero for the MTT assay. Cell growth was measured daily until day five. A volume of 20 μl MTT solution (5 mg/ml) was added into each well. Following incubation for 4 h at 3°C, 150 μl dimethylsulfoxide was added to dissolve the crystals. After incubation for 10 min at room temperature, the absorbance was read at 490 nm on the Shimadzu UV-1603 spectrophotometer (Shimadzu Corp., Kyoto, Japan).

To determine the long-term inhibitory effect of the lentivirus, Saos-2 cells were cultured in six-well plates at a density of 200 cells/well and were treated with KLF8 siRNA lentivirus (RNAi⁺) or non-silencing siRNA lentivirus (RNAi⁻). Cells were incubated at 37°C in air with 5% CO₂ and the medium was replaced every three days. After ten days, colonies were stained with Giemsa and the number of colonies containing >50 cells were counted in each well.

Saos-2 cells infected with the KLF8 siRNA-expressing lentivirus and negative control lentivirus were collected three days following infection. For cell cycle analysis, cells were collected and cultured in 6-cm dishes until they reached 80% confluency. A total of 1×10⁶ cells were harvested and fixed in 70% ethanol for 1 h. Subsequent to three washes, cells were treated with 50 μl/ml propidium iodide (PI) solution (Sigma-Aldrich, St. Louis, MO, USA) and 100 μl/ml RNase in phosphate-buffered saline for 15 min at room temperature in the dark. Flow cytometric analysis was then performed using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

An in vitro cell invasion assay was performed using a Transwell unit (8-μm pore size) with polyvinylpyrrolidone-free polycarbonate filters coated with 500 μg/ml BD Matrigel™ Basement Membrane Matrix (BD Biosciences) placed in 24-well Transwell chambers. Saos-2 cells were placed in the upper compartment of the chamber and cells were allowed to attach for 8 h. Cells were then incubated in FBS-free medium for 36 h at 37°C in 5% CO₂. DMEM containing 10% FBS was placed in the lower compartment of the chamber. Following incubation for 24 h, the filter inserts were removed from the wells and the cells on the upper side of the filter were removed using cotton swabs. The cells that had invaded the lower surface of the membrane were fixed using methanol and stained with 0.5% crystal violet for 10 min. Cells that had migrated to the lower side of the filter were scored visually in five random fields using a light microscope (10× objective lens; Nikon, Tokyo, Japan). The number of cells from three filters was then averaged. In addition, the invaded cells were lysed and quantified at 570 nm using the Shimadzu UV-1603 spectrophotometer. The experiments were repeated three times with three wells for each treatment.

Statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). Continuous variables were compared using Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

To determine the effect of KLF8 expression on osteosarcoma cell growth, lentiviruses expressing KLF8-specific siRNA were generated. GFP expression was observed in >90% of Saos-2 cells 72 h after lentivirus infection at an MOI=40 (Fig. 1A). qPCR analysis revealed that KLF8 mRNA expression in Saos-2 cells infected with KLF8 lentiviral siRNA was significantly decreased. (P<0.05; Fig. 1B). Western blot analysis of cell lysates extracted four days after lentiviral infection revealed that KLF8 protein expression was also decreased (Fig. 1C). These findings demonstrate that the lentivirus transduction system successfully downregulated KLF8 expression at the mRNA and protein levels compared with the Saos-2 cells infected with nonsense lentiviral siRNA.

To assess the effect of KLF8 knockdown on osteosarcoma cell proliferation, Saos-2 cells were infected with KLF8 siRNA-expressing lentiviral vectors and viable cells were counted using an MTT assay five days post-infection. As shown in Fig. 2A, KLF8 knockdown was found to decreased the number of Saos-2 cells compared with the control siRNA-infected cells (P<0.05). Lentiviral KLF8 siRNA-infected Saos-2 cells were analyzed using a colony forming assay. Downregulation of KLF8 was found to reduce the number of viable Saos-2 cell colonies (Fig. 2B), suggesting that the KLF8 siRNA-treated cells had a lower colony formation ability compared with the control siRNA-infected cells (P<0.05; Fig. 2C). In summary, KLF8 knockdown was observed to inhibit cell growth and colony formation in Saos-2 cells.

Cell cycle distribution was assessed in KLF8-knockdown cells using PI staining and FACS analysis five days after lentiviral infection. Lentivirus-mediated KLF8-siRNA infection affected cell cycle distribution in Saos-2 cells, as shown in Fig. 3A. Statistical analysis revealed that KLF8 siRNA treatment arrested Saos-2 cells in G₀/G₁-phase of interphase of the cell cycle. Furthermore, the number of Saos-2 cells in G₂/M phase was observed to be significantly reduced (P<0.05; Fig. 3B), indicating that DNA replication was impaired following KLF8 knockdown.

To investigate the effect of KLF8 knockdown on osteosarcoma cell invasion, Saos-2 cells were analyzed using a Transwell assay and crystal violet staining. As shown in Fig. 4, the invasive potential of Saos-2 cells was significantly reduced with lentivirus-mediated KLF8 siRNA treatment compared with the control group (P<0.05), indicating that KLF8 may have a role in promoting osteosarcoma cell invasion.