Tissue preparation and patient follow up
BCa tissue samples (N = 57) were obtained from the Department of Urology, at the First Affiliated Hospital of Xi’an Jiaotong University (32 males; age range 39–78 years-of-age; mean 63.7 ± 7.5 years). Pathological grading was monitored by three independent hospital pathologists and 15, 18, and 24 samples of grade I–III were noted, and all were transitional cell carcinomas. Samples were fixed in 4% formalin and paraffin-embedded.
To assess any TN-C expression and tumor grade correlation, TN-C and survival time was assessed and patients who gave the tumor samples were contacted by telephone. Survival was a normal distribution as demonstrated by Shapiro-Wilk test. This study was approved by the Ethics Committee of Xi’an Jiaotong University.
Immunohistochemical (IHC) staining of TN-C in BCa tissues
IHC was performed with a Dako Autostainer Plus system (Dako Corporation, Carpinteria, CA). Tissues were de-paraffinized, rehydrated and subjected to 5-min pressure-cooking antigen retrieval, 15-min endogenous enzyme block, 60-min primary antibody (1:300) incubation and 30-min DakoCytomation EnVision-HRP reagent incubation with rabbit antibodies (1:200). Signals were measured according to substrate hydrogen peroxide using DAB as a chromogen followed by hematoxylin counterstaining. Negative controls were prepared by omitting primary antibody. Stained (brown) cells were quantified by counting the positive cells X 100/total cells in 10 random microscopic (400X) fields in each slice.
Human BCa cell lines 5637, T24, RT4, J82, 253J, UM-UC-3 were obtained from ATCC (Manassas, VA), RPMI-1640 (for 5637) and DMEM (for the other cell lines) were from Invitrogen, (Carlsbad, CA), and medium was supplemented by 10% FBS (Invitrogen). Cells were cultured under 5% CO2 at 37 °C. (Incubators: Thermo-scientific, location, Germany).
Total RNA was isolated from frozen tissues and cell lines using Trizol reagent (Invitrogen) and quantified by reading the absorbance at 260 nm. RNA (2 μg) was reverse transcribed using Revert AidTM First Strand cDNA Synthesis Kit (MBI Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s protocol.
For real-time PCR, we used the SYBRR Premix Ex TaqTM II system (TaKaRa Biotechnology Co., Ltd, Dalian, China) and a Bio-Rad CFX96TM Real-time system (Bio-Rad, city, CA). Then, 12.5 μl SYBRR Premix Ex TaqTM II, 1 μl primer (10 μM, primers; Table 1), 200 ng cDNA and 9.5 μl double de-ionized water were mixed. Then, pre-degeneration was conducted at 95 °C, 30 sec, for one repeat, and PCR was carried out at 95 °C for 5 sec followed by a 60 °C incubation for 30 sec, and 35 repeats. Next, dissociation was carried out at 95 °C for 15 sec followed by a 60 °C incubation for 30 sec, and another 95 °C incubation for 15 sec and data collection. GAPDH was a loading control.
Cells are harvested when 80% confluent and washed with cold PBS three times. Total cellular protein lysates were prepared with RIPA buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% NP40 and 0.5% sodium deoxycholate] containing proteinase inhibitors (1% Cocktail and 1 mM PMSF, Sigma, St Louis, MO). Protein (30 μg) was separated with 6% (for TN-C) or 10% (for others) SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked at room temperature for 1 h with 5% skim milk in Tris-buffered saline without Tween 20 (pH 7.6, TBS). Polyclonal antibody TN-C was applied (1:300 dilution; Table 2) with 5% skim milk in TBS at 4 °C overnight, followed by washing with TBST (with Tween 20, pH 7.6), and membranes were incubated with secondary antibodies (Licor, Rockford, IL) coupled to the first antibody at room temperature in the dark for 1 h. Then, membranes were washed in the dark room, dried with neutral absorbent paper, and scanned using an Odyssey detection system (Licor). GAPDH was a loading control.
Boyden chamber assay and wound healing assay
Migration and invasion were tested with a Boyden chamber assay (Millipore, city, Switzerland). For the migration assay, 0.2 ml FBS-free DMEM medium suspension with 1 x 104 cells was added to the upper chamber and then 0.8 ml FBS-free DMEM was added to the lower chamber. After 24 h incubation, chambers are washed with PBS (pH 7.4) three times to remove attached cells in the upper chamber. Prior to staining (25 min) with crystal violet (0.01% in ethanol), cells were fixed with 4% formalin for 15 min and washed three times. Crystal violet-stained cells were under an inverted microscope and five cell count observations were randomly taken under a 200× objective, and cell counts were averaged. For the invasion assay, suspension in the upper chamber contained 0.2 ml FBS-free DMEM/Matrigel=8/1 (Matrigel, Sigma) and 1 × 104 cells were incubated for 36 h. The cells were treated as in the migration assay.
Wound healing was assessed by scratching 6-well dishes with a 10 μl pipette tip when cells were 80% confluent. Scratch widths were compared at 0, 12, and 36 h.
Preparing stable clone cell lines
PsiCHECK-2TNC plasmids (Addgene plasmid 26995, http://www.addgene.org) and a vector were transfected into 5637 and 253J (TN-C negative) BCa cell lines, respectively. LipofectamineTM 2000 (Life Technologies, city, state) was used for transfection according to kit instructions and stable cell clones highly expressing TN-C were selected quantified with Western blot and real time PCR.
Short hairpin RNA (shRNA/Sc) to knock down TN-C expression in T24 and J82 cells (TN-C positive) was measured. pGPU-6-GFPTN-C/Sc was transfected into cell lines as mentioned above, low-TN-C expressing lines were chosen using G418 (600 μg/ml), and Western blot and real time PCR was used to assess shRNA effects. siRNA to knock down syndecan-4 expression was used (Table 1). A protocol for transfection using LipofectamineTM 2000 is the same as mentioned above.
BrdU incorporation assay
BrdU was added to cell media (3 μg/ml) after cells reached 60–70% confluence on coverslips and incubated for 4 h. Then, coverslips were rinsed three times with PBS for 10 min to remove free BrdU and samples were fixed in 4% paraformaldehyde for 45 min, followed by rinsing five times with PBS for 20 min. Then, 0.1% Triton X-100 was added to destroy the cell membrane (15 min) and 2N HCl (25 min) was added to separate DNA into single strands for primary antibody access to incorporated BrdU. Before blocking nonspecific epitopes with 10% BSA for 20 min, cells were rinsed three times with PBS for 10 min to remove HCl and Triton. Then 10% BSA with anti-BrdU antibody (1:200) was added and incubated overnight at 4 °C.
The next day, cells were rinsed five times with PBS to remove free antibody, and cells were incubating with TRTIC-labeled second antibody for 1 h at room temperature and rinsed another three times with PBS to remove free antibody. Fluorescent intensity of TRITC was monitored with a Super Micro Orifice Plate Spectrophotometer (BioTek, city, state) at 547 nm.
TN-C in cell media was measured with ELISA. Briefly, cell line (different groups) media were collected and tested with ELISA analysis according kits instructions (Human TN-C ELISA Kit, Shanghai Westang Biological Technology Co., Ltd. Shanghai, PRC), and optical density was measured at 450 nm. Data are expressed in μg/ml.
Immunofluorescent staining for nuclear translocation of NF-κB
Prepared cells were washed three times in cold PBS (pH = 7.4) and fixed with 4% paraformaldehyde for 15 min, followed by permeabilization in 0.5% Triton X-100 for 10 min and blocking with 1% BSA for 1 h. Rabbit anti-human-P65 in 1% BSA was added to media and incubated overnight at 4 °C. Mouse anti-rabbit TRITC (Red) IgG antibody (Santa Cruz, city, CA) diluted 1:100 in blocking buffer was added to media and incubated 1 h. Then cells were washed with cold PBS three times and cell nuclei were stained with DAPI (10 μg/ml, Sigma) for 3 min. Cells were observed under an Image Pro Plus System mounted on a fluorescent microscope (Olympus, Japan).
Other reagent and experiments
TN-C peptide (Millipore, location) was an exogenous TN-C added to media (1 μg/ml), and TN-C neutralizing antibody (1 μg/ml) (MAB2138, Novus, location) was used to neutralize TN-C in the media.
Statistical analysis was performed with a SPSS 15.0 statistic package (Chicago, IL). For comparisons among different grades, one-way ANOVA was used, and for comparisons between two different grades, The Student’s t-test was used. A Shapiro-Wilk test was used to confirm distribution of survival time of BCa patients. P-values <0.05 were considered to be statistically significant.