PC tissue specimens
A total of 82 clinical PC tissue samples were obtained from patients who underwent radical prostatectomy at Chiba University Hospital between 2006 and 2014. The present study was conducted in accordance with ethical standards that promote and ensure respect and integrity for all human subjects and the Declaration of Helsinki. The study was approved by the Review Board (approval number 408), and the patients signed a written, informed consent form.
Reagents and antibodies
In this study, siRNAs 4F2 cell-surface antigen heavy chain (si4F2hc) (Stealth siRNAs: HSS109825 and HSS109826), siRNAs S-Phase Kinase Associated Protein 2 (siSKP2) (Stealth siRNAs: HSS185711 and HSS109780), and siRNA Negative Control (Stealth RNAi™, Thermo Fisher Scientific, MA, USA) were used. The antibodies used in the study were: anti-4F2hc (CD98, Santa Cruz Biotechnology, TX, USA); anti-4F2hc (CD98, Trans Genic,Tokyo, Japan); anti-LAT1 (Trans Genic); anti-SKP2, anti-MAPK, anti-phosphorylated MAPK, anti-AKT, anti-phosphorylated AKT, anti-p21cip1, and anti-p27kip1 (Cell Signaling Technology, Danvers, MA, USA); Anti-GAPDH (Ambion, Waltham, CA, USA).
Cell culture and transfection
DU145 and C4-2 cell lines were obtained from the RIKEN Cell Bank (Tsukuba, Japan). PC cell lines were supplemented with 10% foetal bovine serum (FBS) in RPMI 1640 culture medium and maintained in a humidified atmosphere incubator (95% air, 5% CO2, 37 °C). Lipofectamine RNAiMAX reagent (Invitrogen) was used, and siRNA was transfected with PC cells. Detailed experimental methods have been previously reported.35
mRNA expression evaluation
Total RNA was isolated using the RNeasy Mini Kit. cDNA was synthesized with the ImProm-II™ Reverse (Promega, Madison, WI, USA). mRNA expression was carried out as described previously35 with ABI 7300 (Applied Biosystems, Foster, CA, USA) and SYBR Green PCR Master Mix (QPS-201, Toyobo, Japan). GAPDH (internal control) and PCR primers used in this study are listed in Table S1.
Western blot analysis
Protein expression levels were measured using GAPDH as the control. Protein samples (24 mg) were subjected to SDS-PAGE and transferred to Hybond-C membranes (GE Healthcare, Chicago, IL, USA). The membranes were then blocked (5% skim milk, 30 min, 37 °C). The primary antibody was incubated overnight at 4 °C. Detailed experimental methods have been previously reported.35
Immunohistochemistry procedures were performed according to a previously described method.35 The slides were treated with endogenous peroxidase (30% hydrogen peroxide solution, 100% methanol, 10 min), and then incubated with anti-4F2hc and anti-SKP2 (4 °C overnight). Finally, the slides were lightly counterstained with haematoxylin, dehydrated with ethanol, cleared with xylene, and mounted.
IHC scores were as follows: 3 intense staining; 2 moderate; 1 very weak; and 0 no staining. IHC scores were calculated as follows: IHC score = 3× (mean percentage of intensely stained cells in the field) +2× (mean percentage of moderately stained cells in the field) +1× (mean percentage of weakly stained cells in the field). Two independent investigators blinded to patient clinical status scored each specimen.
Growth assay, cell migration, and invasion assay
Detailed experimental methods have been previously reported.35 Following the manufacturers’ instructions, the migration assay was performed using the Cell Counting Kit-8 (343-07623, Dojindo, Japan) and the Falcon Permeable Support Plate (353097, Corning, NY, USA), and the invasion assay used the Matrigel invasion chamber (354480, Corning).
Flow cytometric analysis
For cell cycle analysis, cells were stained with propidium iodide using the FITC BrdU Flow Kit (51-2359KC, BD Biosciences, Bedford, MA, USA) according to the manufacturer’s instructions and examined using the FACS Celesta Analyzer (660344, BD Biosciences). Flow cytometry data were analysed using FlowJo using the previously reported flow cytometric method.35
In order to examine the downstream signal of 4F2hc, RNA sequencing was performed based on previous reports,35 using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech, Palo Alto, CA, USA), NEBNext Ultra DNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA), HiSeq 1500 system (Illumina, Santiago, CA, USA), and Cuffdiff (Cufflinks version 2.2.1).
The Kaplan–Meier method and univariate and multivariate Cox proportional models were used for statistical analyses. Mathematical calculations were performed using JMP Pro 15 (SAS Institute, Cary, NC, USA).