Rat C6 glioma cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, as described previously (6). Cells were cultured at 37°C in an incubator containing 5% CO₂ and 100% humidity. Cells in the mid-log phase were used for experiments.

C6 cells were grown on coverslips in 24-well culture plates for 24 h. After fixation for 10 min in 4% formalin or paraformaldehyde, cells were permeabilized with methanol for 2 min at room temperature. After fixation/permeabilization, the slides were rinsed with phosphate-buffered saline (PBS), blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature or overnight at 4°C and incubated with primary antibody (1:500; anti-Nogo-66 receptor, Santa Cruz Biotechnology). The cells were washed again with PBS and incubated for 1 h at room temperature with FITC-conjugated secondary antibody (1:200 dilution). Slides were washed and rinsed as described above and then mounted in anti-quenching medium (Sigma, St. Louis, MO, USA). The stained and mounted slides were stored in the dark at 4°C and fluorescence was visualized with a laser confocal microscope (FV500; Olympus, Tokyo, Japan).

To knock down NgR expression, a vector-based short hairpin RNA (shRNA) expression system was used. The shRNA vector (pGenesil1.1) was purchased from Wuhan Genesil Biotechnology Co., Ltd. The designation of 21 nucleotide target sequences was based on a computer algorithm and 5′-AATCTCACCATCCTGTGGCTG-3′ was selected as the target sequence. The negative control was pGenesil1.1 containing a non-effective scrambled shRNA. For transfection, C6 cells were seeded in 6-well plates to 80% confluency and transfected with 4 μg of plasmid DNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). RNA and proteins from scrambled shRNA- or shRNA-transfected cells were analyzed 72 h after transfection.

Total RNA extraction and cDNA amplification were performed as described previously (7). The following oligonucleotides were used for RT-PCR analysis: NgR: 5′-AATGAGCCCAAGGTCACAA-3′ (sense), 5′-CCATGCAGAAAGAGATGCGT-3′ (antisense); β-actin: 5′-GAGAGGGAAATCGTGCGTGAC-3′ (sense), 5′-CAT CTGCTGGAAGGTGGACA-3′ (antisense). The PCR conditions were as follows: predenaturation at 94°C for 10 min; denaturation at 94°C for 50 sec, annealing at 59°C for 50 sec and extension at 72°C for 1 min; and a final incubation at 72°C for 7 min. The amplified products were resolved on 1% agarose by performing homeothermic gel electrophoresis at 80 V for 30 min. The bands were excised and eluted from the gel, purified, precipitated overnight with ethanol and sequenced. The electrophoresis results were observed under an ultraviolet lamp and a density scan of the positive bands was performed. Then, the refractive index (RI) of NgR mRNA was calculated using the formula RI = NgR mRNA density/β-actin density × 100%.

Cells were lysed with RIPA buffer, electrophoresed on a 10% SDS-PAGE gel at 100 V for 2 h and electroblotted onto a polyvinylidene fluoride membrane at 275 mA for 2 h. The membrane was incubated with 5% fat-free milk in PBS for 2 h at room temperature. Then, the membrane was incubated in rabbit anti-NgR primary antibody (1:200; Santa Cruz Biotechnology), rabbit anti-cleaved caspase-3 mAb (1:300; Cell Signaling Technology), or mouse anti-β-actin primary antibody (1:1,000; Abcam) overnight at 4°C. After the membranes were washed with PBS three times, they were incubated in horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (1:5,000; Pierce Chemical Co.) for 1 h at room temperature. The samples were washed three times with PBS and developed with an enhanced chemiluminescence reagent (Thermo).

The proliferation rates of sh-NgR cells and control cells were measured by an MTT assay. Briefly, NgR-targeted cells or control cells were plated at an initial density of 5×10³/well in 96-well plates and incubated for 1, 2, 3, 4 or 5 days in complete culture medium. Twenty microliters of MTT (5 mg/ml) (Sigma) was added and the cells were incubated for 4 h. After the entire medium was discarded, 150 μl of dimethyl sulfoxide was added into each well. The optical density was determined in a microplate reader at 490 nm with subtraction of the baseline reading. Each time-point was repeated six times.

The adhesion assay was done as described previously (8). Briefly, a 96-well microtiter plate was coated with Matrigel (50 μl/well) (Vigorous Biotechnology, Beijing, China) for 1 h at 37°C and then blocked with BSA (10 mg/ml). After being treated with Nogo-66 (15 μg/ml) (Biosynthesis Biotechnology, Beijing, China) or IgG (10 μg/ml) for 2 h at 37°C, C6 cancer cells were then seeded onto these components. The cells were allowed to adhere to each well for 1 h at 37°C and were gently washed three times with PBS. The adhesion of C6 cancer cells to the extracellular components was counted at five fields per well at random under a microscope. All experiments were performed in triplicate.

The invasion activity of C6 cancer cells was measured by the previously reported method with some modifications (6). Briefly, C6 cells (1.25×10⁵ per well) were seeded in the upper chamber separated with an 8-μm membrane filter coated with 15 μg/ml Nogo-66 or IgG (10 μg/ml), which was diluted into extracellular matrix (ECM, Sigma). Medium in the upper chamber was supplemented with 10% fetal bovine ferum (FBS). In the lower chamber, the FBS concentration was 20%. After incubation for 72 h at 37°C, C6 cells invading the lower chamber were manually counted under a microscope. Six randomly selected fields were counted for each assay. Mean values from six fields were calculated as sample values. For each group, the cell culture was performed in triplicate.

C6 cells were grown to confluency in 35-mm wells (3×10⁵ cells/well). Wounds were scratched using a 200 μl pipette tip and cells were washed three times with PBS. Serum-free DMEM was then added. Triplicate wells were used per condition and three fields per well were captured at each time-point over a period of 24 h. Scratch wound assays were performed for cells from three independent infections. For experiments in which Nogo-66 was used, confluent monolayers were treated with 15 μg/ml Nogo-66 before wound initiation.

Wounds were visualized using a Nikon Eclipse TS100 microscope (Nikon, Kingston Upon Thames, UK), images were captured using a Coolpix 4500 camera (Nikon) and cells migrating through a central strip were counted in each group.

Spontaneous apoptosis of C6 cells was examined by an Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit (BestBio, China). After shRNA vector was transfected for 48 h, cells were washed with ice-cold PBS and resuspended in 400 μl of 1X binding buffer and the cells were stained with 5 μl of Annexin V and 5 μl of PI for 15 min at 4°C in the dark. Then, the stained cells were analyzed by fluorescence-activated cell sorting (BD Biosciences, USA) using CellQuest Research Software (BD Biosciences).

To observe the extent of spontaneous apoptosis, cells were cultured on coverslips in 6-well culture plates. Cells were fixed with 4% formalin for 10 min at 4°C and then permeabilized with 0.1% Triton-X 100 for 5 min, incubated with reagents from a fluorescein-conjugated TUNEL kit (Roche, Canada) for 1 h at 37°C and stained with Hoechst 33342 dye (5 μg/ml) for 15 min at room temperature. After washing with PBS, the number of apoptotic cells was visualized by using a laser scanning confocal microscope (FV500; Olympus, Tokyo, Japan) from five random fields.

Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS; version 11.5; Bizinsight, Beijing, China). P<0.05 was considered statistically significant. Intergroup data were compared using analysis of variance (ANOVA). The quantities of NgR mRNA were consistent with normal distributions and were analyzed using the Student-Newman-Keul’s (SNK) test.

Immunofluorescence was used to identify the presence of NgR in C6 cells. The immunofluorescence experiment was performed with anti-rabbit NgR antibody. As expected, strongly positive cytoplasmic NgR staining was evident in C6 tumor cell lines (Fig. 1).

RT-PCR analysis was used to detect the changes of NgR mRNA expression after transfection. A 450-bp band was observed in the blank control, the scrambled shRNA group and the NgR shRNA group; and the relative densities of the NgR band compared with the β-actin band were 0.591±0.038, 0.539±0.028 and 0.105±0.009, respectively. There was no significant difference in the density of the NgR mRNA band between the blank control and the scrambled shRNA groups; whereas compared with the blank control, the density of the band in the shRNA group was dramatically less (Fig. 2A and C). Similar to the RT-PCR results, expression of the NgR protein in each group was also observed by western blotting and the relative densities of the NgR band compared with the β-actin band were 0.446±0.059, 0.389±0.085 and 0.162±0.048, respectively (Fig. 2B and D). These data indicate that NgR shRNA can inhibit NgR mRNA and protein expression after transfection into C6 cells.

Cell proliferation after NgR silencing was assessed by an MTT assay. As shown in Fig. 3, the sh-NgR cells had a lower proliferation rate than the sh-scramble cells. Knockdown of NgR led to a decrease in C6 cell growth at 3 (23.0%), 4 (27.7%) and 5 days (26.1%) (P<0.05), compared with the scramble control. These results demonstrate that NgR silencing inhibits the proliferation of C6 cells.

In the Nogo-66-coated experiment, 1 h after seeding, the number of adherent living cells was significantly increased in the NgR knockdown group compared to the C6 scramble control group (Fig. 4). As a result, the total number of cells increased in the NgR knockdown group. In the IgG-coated experiment, 1 h after seeding, the number of adherent living cells in the NgR knockdown group was not significantly different than that in the C6 control group. These results suggest that NgR knockdown in C6 cells enhances cell adhesion to Nogo-66-coated Matrigel but does not affect cell adhesion to IgG-coated Matrigel.

We tested whether NgR knockdown affected the invasion capability of C6 cells by using a Boyden chamber assay. Cells were seeded in the upper part of a Nogo-66/ECM-coated invasion chamber containing a low (10%) FCS concentration. After 72 h, cells that migrated in the lower chamber containing a higher (20%) FCS concentration were stained and counted. In NgR-shRNA C6 cell lines, the number of cells in the membrane was significantly greater than that of the scramble control (Fig. 5). In the IgG/ECM-coated invasion chamber, no further decrease in invasion was observed between NgR-shRNA cells and control cells. These results indicate that invasion through the Nogo-66-coated membrane was significantly enhanced by NgR knockdown in C6 cells but did not affect cell adhesion to the IgG/ECM-coated membrane.

To further test whether NgR knockdown affected the invasion capability of C6 cells, a scratch wound migration assay was used. A wound was scratched in a confluent monolayer of NgR-shRNA cells and scramble control cells coated with Nogo-66 (Fig. 6). The edge of the wound was monitored over 24 h and images of the same fields were taken 0 and 24 h later. The width of the wound in NgR-shRNA C6 cells was significantly narrower than that in the control group. This result indicates that the invasion ability of C6 cells to Nogo-66-coated coverslips was significantly enhanced by NgR knockdown.

C6 cells stained with the double stain Annexin V/PI were analyzed by flow cytometry (Fig. 7A and D). The apoptotic rate of C6 cells was 3.21±1.29% in the scramble control group. After NgR knockdown for 48 h, the rate of apoptosis increased to 16.63±0.58%. Thus, the rate of apoptosis in NgR knockdown cells showed a notable increase compared to controls. NgR knockdown in C6 cells significantly increased the expression of caspase-3 (active form) when compared with the scramble control group (Fig. 7B and F). The increased apoptosis rate in NgR knockdown-C6 cells was also evident from the TUNEL-positive staining results (Fig. 6C and E). The rate of TUNEL-positive cells in sh-NgR-C6 cells was 10.21±1.04%, whereas that in the control group was 1.43±0.69%. These results suggest that knockdown of NgR increased the apoptotic rate of C6 cells.