The human OVTC cell line PA1 and the human embryonic kidney cell line HEK293T were cultured in ((D)MEM) (Gibco, USA) with 10% FCS and 1% penicillin/streptomycin (Gibco, USA). PA1 cells were provided courtesy of >Dr. Min-Chie Hung (MD Anderson, Houston, TX, USA) and HEK293T cells were obtained from Dr. Yuh-Pyng Sher (Center of Molecular Medicine, China Medical University Hospital, Taichung, Taiwan). The cell lines were maintained at 37°C in a humidified atmosphere of 5% CO2.
Western blotting assay
Protein extraction and the immunoblot assay were performed as previously described . Briefly, cells were washed with 1 x PBS and resolved in RIPA buffer (100 mM Tris, 5 mM ethylenediaminetetraacetic acid (EDTA), 5% NP40; pH8.0) with protease inhibitors (1 mM phenylmethyl sulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin). Proteins were resolved by SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. Blocking of non-specific binding was accomplished by adding 5% non-fat milk. After application of primary antibodies, secondary antibodies were applied (1:3,000, horseradish peroxidase (HRP)-goat-anti-mouse and HRP-goat-anti-rabbit) for one hour at room temperature. Signals were enhanced using an enhanced chemiluminescence kit (Millipore, Billerica, Massachusetts 01821, USA) and detected by ChemiDoc ™ XRS + (BioRad, Hercules, CA 94547).
Transfection and infection procedure
Basically, the lentiviral production and infection procedures were carried out as reported previously . Briefly, cells were transfected with the following lentivirus plasmids: psPAX2 packaging plasmid, pMD2G envelope plasmid (Addgene, Cambridge MA 02139) [37, 38], miRZip-21  (anti-miR-21 microRNA construct; System Biosciences, SBI, Johnstown, PA 15901, USA), and hsa-miR21  (Human Lentiviral Primary microRNA Over-Expression Construct in pMIRNA1; System Biosciences, SBI). Both lentiviral plasmids carried the GFP gene and were co-transfected with psPAX2 and pMD2G into HEK293T cells at a ratio of 3:2:4 by lipofectamine2000 (Invitrogen, USA) as per the manufacturer’s instructions. After six hours, the medium was replaced with fresh (D)MEM/10% fetal bovine serum (FBS) medium and cells were maintained at 37°C in a humidified incubator in an atmosphere of 5% CO2 for 48 hours. Medium containing virus was collected by centrifugation and filtered through a 0.45 μm filter. Medium containing 0.8 mg/ml polybrene (Sigma-Aldrich, USA) was then added to culture dishes containing 106 PA1 cells. After 16 hours of infection, the medium containing virus was replaced with fresh (D)MEM/10% FBS medium and cells were maintained at 37°C in a humidified incubator in an atmosphere of 5% CO2 for 48 hours. Infected cells were then collected and analyzed. The GFP+ cells were measured by flow cytometry (BD LSR II Flow Cytometry) to determine infection efficiencies. GFP + cells with infection efficiencies greater than 85% were subjected to the following experiments (data not shown).
Total RNA isolation and cDNA synthesis
RNA was extracted from PA1 cells as reported previously . Briefly, cells that had reached approximately 80% to 90% confluence in 100-mm dishes were lysed with 1 ml Trizol (Invitrogen). Phenol/chloroform was then added and RNA-rich layers were separated by centrifugation. Soluble RNA was precipitated with 2-propanol. RNA was then rinsed with 75% ethanol and dissolved in RNase-free water. For first-strand cDNA synthesis, 5 μg of total RNA was used to perform reverse transcription PCR by the PrimeScript™ RT reagent kit (Takara Bio Inc., Tokyo, Japan). MiRNA cDNA was synthesized using the Mir-X™ miRNA First-Strand synthesis Kit (Clontech, Mountain View, CA 94043, USA). cDNA and miRNA were synthesized according to the manufacturer’s instructions.
Quantitative real-time PCR analysis
A real-time detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the KAPA™ SYBR FAST One-Step qRT-PCR Kit (Kapa Biosystems, Woburn, MA, USA) were used according to the manufacturers’ instructions. Relative gene expression was determined by normalizing the expression level of the target gene to the expression level of housekeeping genes (U6 or actin). Threshold value (Ct) dynamics were used (2-ΔΔCt) for quantitation of gene expression. The qRT-PCR primer sequences were as follows: CD133 forward 5′- TCT CTA TGT GGT ACA GCC G-3′, reverse 5′- TGA TCC GGG TTC TTA CCT G-3′; CD24 forward 5′- TTT ACA ACT GCC TCG ACA CAC ATA A-3′, reverse 5′- CCC ATG TAG TTT TCT AAA GAT GGA A-3′; CD44 forward 5′- GAC CTC TGC AAG GCT TTC AA-3′, reverse 5′- TCC GAT GCT CAG AGC TTT CTC-3′; CD117 forward 5′- CAA GGA AGG TTT CCG AAT GC-3′, reverse 5′- CCA GCA GGT CTT CAT GAT GT-3′; Nanog forward 5′- GGG CCT GAA GAA AAC TAT CCA TCC-3′, reverse 5′- TGC TAT TCT TCG GCC AGT TGT TTT-3′; OCT-4 forward 5′- GGC CCG AAA GAG AAA GCG AAC C-3′, reverse 5′- ACC CAG CAG CCT CAA AAT CCT CTC-3′; SCF forward 5′- ACT GAC TCT GGA ATC TTT CTC AGG-3′, reverse 5′- GAT GTT TTG CCA AGT CAT TGT TGG-3′; miR-21 5′- TAGCTTATCAGACTGATGTTGA-3′.
Cell growth analysis
The WST-1 assay (Roche, Indianapolis, Indiana 46250, USA)  was used to assess cell growth. Briefly, 103 cells/100 μl/well were seeded in 96-well plates with (D)MEM in 10% FBS and were incubated for the designated time periods (one, two, four, six and eight days). Then 10 μl of WST-1 solution was added to each well and cells were allowed to incubate at 37°C in an incubator for one hour. Cell viability was quantified by colorimetric detection in an ELISA plate reader (Beckman Coulter Paradigm™ Detection Platform, USA) at an absorbance of 450 nm and 690 nm to generate an optical density (OD) proportional to the relative abundance of live cells in the given wells.
Verification and sorting of CD133+ cells
Cells were detached with 1% trypsin/EDTA and cell membrane non-specific binding to antibodies was blocked by 5% BSA for 30 minutes at room temperature. Cells were then incubated with antibody CD133- allophycocyanin (APC) (magnetic cell separation (MACS), Miltenyi Biotec, Auburn, CA 95602) for 30 minutes and then subjected to flow cytometry (fluorescence-activated cell sorting (FACS), BD FACSAria, USA) analysis. The CD133+/− cells were collected by a cell sorter (BD FACSAria).
Sorting of CD133+ and CD133– populations
A CD133 MicroBead kit (Miltenyi Biotec) was used to sort PA1 CD133+ cells according to the manufacturer’s instructions. Briefly, 108 cells were washed, resuspended in 300 μl of buffer (1 X PBS/ pH 7.2, 0.5% BSA/2 mM EDTA), with 100 μl FcR Blocking Reagent and 100 μl of CD133 MicroBeads, mixed well, and then incubated for 30 minutes at 2°C to 8°C. The labeled cells were then washed, resuspended in 500 μl buffer and applied on a MACS Column of MiniMACS separation kit to collect the unlabeled negative fraction (that is, CD133– cells). Finally, we moved the column onto collection tubes to collect labeled cells (that is, CD133+ cells).
Sphere formation assay
Cells were collected and washed to remove serum and then suspended in serum-free (D)MEM supplemented with 20 ng/ml human recombinant epidermal growth factor (hrEGF), 10 ng/ml human recombinant basic fibroblast growth factor (hrbFGF), 5 μg/mL insulin and 0.4% BSA (Sigma). The cells were subsequently cultured in ultra low attachment 6-well plates (Corning Inc., Corning, NY, USA) at a density of less than 5,000 cells/well for 14 days. Spheres were observed under a microscope and images were photographed under a phase contrast fluorescence microscope (Nikon, ECLIPSE 80i).
Statistical analyses were performed using the Student’s t-test. All experiments were repeated at least three times, and P-values less than 0.05 were considered to indicate statistical significance.