Minimal essential medium with Earle's salt (EMEM), glutamine, trypsin-versine, anti-mycotic antibody mixture (penicillin-streptomycin-fungizone) and phosphate-buffer saline (PBS), pH 7.2 and 7.4 were from BioSource (Camarillo, CA). Fetal bovine serum (defined) was from HyClone, Logan, UT. Dimethylsulfoxide, nystatin, and ethidium bromide were obtained from Sigma Aldrich, St. Louis, MO. Texas-Red conjugated WGA was purchased from EY Laboratory, San Mateo, CA. iScript™ cDNA synthesis kit, Tween-20, biotinylated protein molecular weight markers, DNA markers and all electrophoresis reagents were obtained from BioRad Laboratories, Hercules, CA. Lipofectamine™ Reagent, Plus™ Reagent, G418 and TRIzol were from Invitrogen, Life Technologies, Carlsbad, CA. XhoI, BamHI, HindIII, T4 DNA ligase were from New England BioLab, Ipswich, MA. dNTPs and Taq polymerase were obtained from Promega, Madison, WI. Rabbit polyclonal anti-DPMS antibody was developed in-house. Horseradish peroxidase conjugated (HRP) goat anti-rabbit IgG, ECL chemiluminescence detection kit and GDP-[U-14C]-mannose was from GE Healthcare, Piscataway, NJ. Mouse monoclonal antibody to actin was obtained from BD Biosciences, San Jose, CA. PCR grade water and DNA decontamination kit were from Applied Biosystems, Austin,TX. All other chemicals and solvents were of the highest purity grade and obtained from commercial suppliers.
Culturing of endothelial cells and stable transfectants
Wild type cells were maintained in EMEM containing 10% fetal bovine serum (heat-inactivated), glutamine (2mM), penicillin (50 units/ml), streptomycin (50μg/ml), nystatin (1,000 units/ml) (21
). Overexpressing DPMS stable cell clone and the cell clone carrying the vector alone were cultured in the same media but containing G418 (0.55μg/ml) at 37°C in an humidified incubator (5% CO2
Construction of DPMS overexpression plasmid
Cellular RNA was isolated with TRIzol reagent and treated with DNase to remove any possible genomic DNA contamination. Total RNA was quantified in a NanoDrop Bioanalyzer. First strand cDNA was synthesized using the iScript™ cDNA synthesis kit according to the manufacturer's instructions. The primers for amplifying full DPMS gene were: 5′-ccgctcgagATGGCTGCCGAGGAAGCAAGTC-3′ (forward) and 5′-ccgggatccCTCCAGAAAACAGACTTGCCTTACCT-3′ (reverse), which provided with the XhoI and BamHI site. PCR reactions were carried out as follows: initial denaturation at 94°C for 4 min, 30 cycles at 94°C for 50s, 64°C for 50s, 72°C for 50s, and a final extension for 10 min at 72°C in 50μl containing 2μl each cDNA, 0.2μM each primer, 0.2mM dNTP, and 2 units of Taq DNA polymerase. 5 μl each reaction mixture was used for product identification and detected by 1% agarose gel electrophoresis followed by ethidium bromide staining. PCR products were digested with XhoI and BamHI, purified, and cloned into XhoI and BamHI sites of pEGFP-N1 vector to obtain pEGFP-N1-DPMS overexpression plasmid. The plasmids were confirmed by XhoI/BamHI double digestion and DNA sequencing.
Sequencing of DPMS overexpression plasmids
For sequencing, plasmids were isolated and 20μl sequencing reaction was designed according to the BigDye terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) containing 3.2 pmol primer [(DPMS overexpression plasmid sequencing primers: forward Primer: 5′-TTTCCAAAATGTCGTAACAACTCCG-3′; reverse Primer: 5′-TGAACTTGTGGCCGTTTACGT-3′)] and 300 ng plasmid. The reaction was carried out as follows: initial denaturation at 96°C for 1 min, 25 cycles at 96°C for 10s, 50°C for 50s, 60°C for 4 min, and rapid cooling to 4°C. This was followed using BigDye XTerminator™ Purification Kit (Applied Biosystems), and then electrophoresis on the ABI PRISM 310 Genetic Analyzer.
Transfection of capillary endothelial cells with constructed plasmids
Transfection of capillary endothelial cells with pEGFP-N1-DPMS overexpression plasmid, and pEGFP-N1 vector were performed using Lipofectamine™ Reagent and Plus™ Reagent according to the manufacturer's instruction. After transfection, the cells were cultured in regular EMEM containing 0.55 mg/ml of G418 for 4 weeks, and cells overexpressing DPMS and the vector pEGFP-N1 alone (carrying a non-transcribable gene; control) were collected.
Mannosylphospho dolichol synthase assay
Enzymatic transfer of mannose from GDP-mannose was assayed using a procedure described earlier (22
) with the following modification. Total cell lysates from overexpressing DPMS clone and the clone carrying vector only were incubated at 37°C for 5 min in 50 mM Tris-HCl (pH 7.0), 0.125M sucrose, 0.5mM EDTA, 2.5mM 5′-AMP, 10mM MnCl2
, 10μg Dol-P and 2.5μM GDP–[U-14
C]mannose in a total volume of 150μl. The reaction was stopped with 3ml of chloroform/methanol (2:1, v/v). Chloroform/methanol extracts containing Dol-P-Man were washed with 0.5 volume of 0.9% sodium chloride and twice with chloroform/methanol/water (3:47:48, v/v/v) to remove free GDP-[U-14
C]mannose. A lower organic phase containing the Dol-P-Man was quantified in a liquid scintillation spectrometer.
Quantitative RT-PCR of DPMS
Total cellular RNA was extracted and the cDNA was synthesized using the iScript™ cDNA synthesis kit as mentioned above. Primers used for QRT-PCR were: DPMS gene forward primer 5′-GCTGAGCAGTTGGAGAAG-3′, reverse primer 5′-TGGATGGTGTGAGAGGTC-3′ (PCR product size: 153 bp); GAPDH gene forward primer 5′-TGACCCCTTCATTGACCTTC -3′, reverse primer 5′- GATCTCGCTCCTGGAAGATG -3′ (PCR product size: 143 bp) (XXIDT integrated DNA Technologies). QRT-PCR was performed in an iCycler (BioRad Laboratories) using iQ™ SYBR® Green Supermix (BioRad Laboratories) as a fluorescence dye for the double-stranded DNA. After optimizing the PCR conditions, reactions with SYBR® Green PCR master mix, 10μM forward/reverse primer and 100ng cDNA were performed following the instruction of manufacturer.
Quantitative PCR was accomplished under the following conditions: 95°C for 3 min, and 40 cycles of 95°C for 10s and 50°C for 60s. The relative gene expression for each sample was determined using the formula 2ΔΔ = 2ΔCt (GAPDH – target), which reflected the target gene expression normalized to GAPDH levels. Each sample was run three times. After amplification, 5 μl each reaction mixture was subjected to 1.5% agarose gel electrophoresis, and the bands were visualized by ethidium bromide staining. GAPDH expression was detected as the internal control.
To prepare whole cell extracts, stable cells were washed in PBS and resuspended in cell lysis buffer (10% SDS, 1M Tris.HCl, pH7.5, 100mM Na3
). After sonication and heating for 5 min, cell lysates were clarified by centrifugation at 9000 ×g for 10 min, and the supernatants were collected. Protein concentration was determined with BioRad DC Protein Assay using bovine serum albumin as a standard. Total protein (50μg) from the whole cell lysate was separated by 10% SDS-PAGE in a mini-gel. Proteins separated in the gel were transferred electrophoretically onto nitrocellulose membranes. After blocking with TTBS [50 mM Tris-HCl (pH 7.5), 0.15M NaCl, 0.1% Tween-20] containing 5% fat-free dry milk for 1 h, the membrane was incubated overnight with a specific polyclonal antibody directed against DPMS (6:1000 dilution) at 4°C. After washing with TTBS three times, 5 min/each, the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit antibody (1:1000) for 1 h at 4°C. The membrane was developed with an enhanced chemiluminescence (ECL) detection system. The same blot was stripped and reprobed with anti-actin antibody (BD Biosciences) to confirm the equal loading (23
The stable clones were subcultured on cover glasses (15mm diameter, Warner Instruments, Hamden, CT) for 24 h under standard conditions. The cells were washed three times with PBS, pH 7.4 and fixed for 5 min in ice-cold methanol. The cells were washed three times with PBS, pH 7.4 and incubated with anti-DPMS antibody (rabbit polyclonal 1:50 dilution) for 1 h at room temperature. After washing three times with PBS, pH 7.4, the cells were incubated with rhodamine-conjugated goat anti-rabbit IgG (1:300 dilution) and Hoechst 33342 (1:10,000 dilution) for 30 min at room temperature. After washing three times with PBS, pH 7.4, the cover glasses were mounted and examined in a Zeiss AxiocamMRc (Carl Zeiss, Germany) fluorescent microscope at 40X magnification (24
For glycoprotein detection, cells were seeded on cover glasses as mentioned above, and washed three times with buffer (50mM Tris-HCl, pH, 0.15M NaCl, 4mM CaCl2). Texas-Red conjugated WGA (100ug/ml in Tris-CaCl2 buffer) was then added and the cells were incubated for 30 min at room temperature. After washing three times, the cover glasses were mounted and the images were captured in a Zeiss AxiocamMRc (Carl Zeiss, Germany) fluorescent microscope at 40X magnification.
Cell proliferation and migration assay
The vector (carrying no transcribable gene) and DPMS overexpressing clones were seeded in 24-well plates (2×104
cells/well) in 1 ml of normal medium containing 10% serum and 0.55 mg/ml G418. After 24 h the medium was removed, cells were washed three-times with PBS (pH 7.4) and incubated in a serum-free medium for 24 h. At the end of incubation, the medium was replaced with normal medium containing 10% serum and 0.55 mg/ml G418. The cell numbers were counted after every 24 h for seven days. The experiment was conducted in triplicate wells and averaged after counting the cells in four quadrants for each well (25
For migration assay cells (1×106) were plated into 6-well cell culture plate in normal media containing 0.55 μg/ml G418. After 24h the media was removed, cells were washed with PBS, pH7.4 and changed to a serum-free medium. The cells were continued to incubate for another 24h when the media were removed and replaced with the normal media. After 48h, a 1-mm wide scratch (reminiscent of a wound) was made across the cell monolayer using a pipette tip. The monolayer was washed with PBS, pH 7.4; normal media were added and photographed. After incubating at 37°C for 6 h the plates were again photographed. Quantitative analyses of the scratches were performed by counting the cells migrated towards the scratch. Results are presented as mean ± S.D. averaged from three low power fields (magnification 10X).
The results were analyzed statistically using the student t-test.