The human embryonic kidney (HEK) 293 and the murine fibroblast cell line NIH 3T3 cells were cultured in DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were obtained from American Type Culture Collection (ATCC) and cultured at 37°C in a humidified atmosphere containing 5% CO₂. The study was conducted under an approved protocol from the Ethics Committee of Pusan National University.

Total RNA was extracted from cells by using the RNeasy mini kit (Qiagen, Hilden, Germany). The total RNA of cDNA was synthesized from 1 μg total RNA by using M-MLV reverse transcriptase (Promega, Fitchburg, WI, USA). Primers used were NY-SAR-35-forward (5′-CTTGGTGCGA TCAGCCTTAT-3′) and NY-SAR-35-reverse (5′-TTGATGCA TGAAAACAGAAC-3′). PCR amplification was carried out as follows: denaturation for 5 min at 94°C, then 35 cycles consisting of 30 sec at 94°C, 30 sec at 55°C, and 1 min at 72°C and then 10 min at 72°C for final extension. PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide.

Cells were lysed in lysis buffer and cellular debris was removed by centrifugation at 15,000 rpm for 10 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes (Hybond-ECL, GE Healthcare, UK), blocked with 5% skim milk and incubated with anti-V5 or anti-His (Life Technologies, Carlsbad, CA, USA) and anti-β actin antibodies (Sigma-Aldrich). Membranes were then incubated with horseradish peroxidase-conjugated goat anti-mouse-IgG secondary antibodies for 1 h at room temperature and detected by enhanced chemiluminescence (Perkin-Elmer Life Science, MD, USA).

The open reading frame (ORF) cDNA inserts of NY-SAR-35 (AY211917) were selected to generate His-tagged NY-SAR-35 protein. The primers for the partial protein were GGAATTCCATATGCT GCCCAACAGT (forward) and CCGCTCGAGCATTTGCA TCATCT (reverse) containing NdeI and XhoI restriction enzyme sites. PCR products were subcloned into the pET21a expression plasmid containing a polyhistidine-tag. The recombinant NY-SAR-35 fusion protein was induced by isopropyl β-D-thiogalactoside (IPTG) with a final concentration of 0.4 mM at 37°C. Affinity chromatography using Ni-NTA agarose (Qiagen) was performed to purify NY-SAR-35 recombinant protein. The purified recombinant protein was determined by SDS-PAGE and Coomassie Blue staining.

Antibodies against NY-SAR-35 were raised in a naïve New Zealand white rabbit immunized subcutaneously with 200 μg of recombinant NY-SAR-35 protein. Three booster injections were given using the same antigen, the first booster three weeks after the first immunization and then each week. The rabbit was then sacrificed and the crude antiserum was collected. Especially for the isolation of NY-SAR-35 purified antibodies, affinity purification was performed using cyanogen bromide (CNBr)-activated sepharose beads (GE Healthcare). Normally, coupling to beads was achieved at pH 8.3, while elution occurred at pH 2.8. Neutralization by adding Tris buffer with pH 8.8 was performed as soon as possible after elution to prevent damage of the antibody.

To generate stably transfected cell lines, ORF of NY-SAR-35 including 5′-untranslated region was cloned into pcDNA3.1/V5-HisA vector which has C-terminal fusion tag (V5 and 6X His epitopes) containing EcoRI and XhoI sites. Then, HEK293 cells (1×10⁶ cells in 60-mm dishes) were transfected with 4 μg of cloned pcDNA3.1/V5-HisA-NY-SAR-35 using Lipofectamine LTX (Life Technologies). Transfected cells were selected in medium containing G418 (1 mg/ml). The selected clones were maintained in medium containing G418 (0.3 mg/ml).

For transient transfection, the cDNA of complete coding region of NY-SAR-35 was generated by PCR amplification using primers containing XhoI and PstI restriction sites. The PCR products were then inserted in-frame within the pEGFP-N1 vector. The NIH 3T3 cell was transfected with cloned pEGFP-N1-NY-SAR-35.

The small interefing RNA (siRNA) used for the targeted silencing of NY-SAR-35 (5′-GCCUUAUCCUGGU AUGUCUTT-3′) was purchased from Qiagen. In brief, cells (2×10⁵ cells/ml) were seeded on 6-well plates and transfected with 200 nM of siRNA using oligofectamine reagent, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Cells were then incubated at 37°C for 4 h in serum-free media. The transfected cells were collected after 48 h and NY-SAR-35 expression levels in the cells were determined using RT-PCR.

Staining was conducted with the peroxidase-based EnVision Detection kit (DakoCytomation, USA) by following the user manual. Briefly, tissue sections were deparaffinized and hydrated in xylene and graded alcohol series. Then, the sections were incubated with the purified NY-SAR-35 antibody for 1 h at room temperature. After incubation with EnVision solution, the 3, 3′-diaminobenzidine (DAB) substrate was added followed by hematoxylin counterstaining and mounting.

Cell numbers were determined as follows: cells (5×10⁵/90-mm culture plate) were cultured for 4 days in standard culture medium supplemented with 1 or 10% FBS. After incubation for 4 days, adherent and vital cells were counted in a trypan blue exclusion assay.

Cell proliferation was measured by 5-bromo-2′-deoxyuridine (BrdU) incorporation using the Cell Proliferation ELISA, BrdU kit (Roche Diagnostics, Germany) according to the manufacturer's instructions. In brief, cells were grown in 96-well plates and labeled with BrdU for 2 h. Subsequently, anti-BrdU-POD Fab fragments and substrates were added to the medium. The optical density (OD) was determined at 450 nm using an ELISA reader (Bio-Tek Instruments, VT, USA).

Cells grown on coverglass-bottom confocal dish were fixed with 4% paraformaldehyde. Then, cells were blocked in 3% FBS/PBS and incubated with anti-PCNA (proliferating cell nuclear antigen) or Ki-67 antibody. The cells were washed three times with PBS, incubated with FITC-coupled secondary antibodies. Images were captured using confocal microscopy (Olympus).

To measure the cell migration activity, transwell chamber assays were performed using a transwell plate (Becton-Dickinson, MA, USA). The lower surface of the filters was coated with 1% gelatin. Cells were resuspended in serum-free medium and then added to the upper chamber at a density of 5×10⁴ cells/insert. The medium containing 10% FBS was added to the lower chamber. After the cells were incubated at 37°C for 18 h, the number of cells that migrated to the lower side of the upper chamber was counted by staining with crystal violet. The invasive potentials of the cells were analyzed using a Matrigel-coated modified Boyden chamber (Becton-Dickinson). The medium containing 10% FBS was added to the lower chamber as an attractant. The incubations of the cells were continued for 24 h. Inserts were then stained with crystal violet and the number of invaded cells was counted.

To investigate the role of NY-SAR-35 gene, NY-SAR-35-transfected HEK293 cells were established with pcDNA3.1/V5-HisA-NY-SAR-35 vector and named HEK293/NY-SAR-35. Expression of NY-SAR-35 in HEK293/NY-SAR-35 cells was confirmed with RT-PCR (Fig. 1A) and western blot analysis (Fig. 1B). In addition, immunofluorescent staining with EGFP or V5-FITC showed that NY-SAR-35 was predominantly observed at the cytoplasm in NIH3T3/NY-SAR-35 and HEK293/NY-SAR-35 cells, respectively (Fig. 1C).

Using purified antibody, the expression of NY-SAR-35 protein in normal testis tissue was analyzed using immunohistochemical techniques. Expression of NY-SAR-35 was mainly observed in spermatogonia and spermatocytes, as cytoplasmic proteins. In addition, a positive reaction was seen in vascular endothelial cells, but not Leydig cells in interstitium (Fig. 1D).

Because NY-SAR-35 protein seemed to be involved in mitosis during spermatogenesis (Fig. 1D), we tested whether the NY-SAR-35 could regulate cell growth or proliferation. The increase in cell number of HEK293/NY-SAR-35 cells was determined by trypan blue exclusion assay. HEK293/NY-SAR-35 cells grew on average 2.2 times more rapidly than HEK293 cells within the time period of 4 days (Fig. 2A).

Further examination of cell proliferation by BrdU assay showed an increase in DNA synthesis within three days of in vitro growth in HEK293/NY-SAR-35 cells (Fig. 2B). Also, positive staining for Ki-67 (green, Fig. 2C) and PCNA (green, Fig. 2D), markers of cell proliferation, was found in HEK293/NY-SAR-35 cells. Ki-67 staining was restricted to the nucleus. In contrast, PCNA positivity showed staining intensity with both the nuclei and the cytoplasm. It was noted that NY-SAR-35 enhanced cell proliferation.

Furthermore, to confirm these effects of NY-SAR-35, knockdown of NY-SAR-35 was performed using RNAi molecule specific for NY-SAR-35. RT-PCR showed that NY-SAR-35-siRNA effectively silenced NY-SAR-35 expression in HEK293/NY-SAR-35 cells (Fig. 3A). For growth assay, the control scrambled- and NY-SAR-35-siRNA treated cells were incubated for 4 days. The percentage of cell numbers dropped to 20% in NY-SAR-35 knocked-down cells compared to HEK293/NY-SAR-35 cells under media containing 1% FBS (Fig. 3B). Cell proliferation assay showed that DNA synthesis decreased in NY-SAR-35-silenced cells compared with HEK293/NY-SAR-35 cells (Fig. 3C). Immunofluorescent staining also showed that knocking down NY-SAR-35 expression significantly reduced positivity of Ki-67 (Fig. 3D).

To understand the relationship between the expression of NY-SAR-35 and cellular motility, migration and invasion activity, we were examined in transwell assays using a Boyden chamber. HEK293/NY-SAR-35 cells showed that the migratory ability was increased to 2.3-fold and invasive ability was also enhanced to 4.5-fold higher than the control HEK293 cells (Fig. 4A).

Furthermore, to confirm the migratory and invasive effects of NY-SAR-35 expression, NY-SAR-35 was knocked down in the HEK293/NY-SAR-35 cells and migration and invasion assays were performed. NY-SAR-35 silencing reduced migration and invasion of HEK293/NY-SAR-35 cells by ~50% (Fig. 4B). These findings indicated that NY-SAR-35 expression promoted cell migration and invasion in HEK293 cells.