Materials and reagents.
Rabbit polyclonal anti-Cx43 antibody (Cat. No 71-0700) was purchased from Invitrogen, N-terminal monoclonal anti-Cx43 antibody and monoclonal anti-NEDD4 antibody were obtained from BD Bioscience Inc. Polyclonal anti-GAPDH, anti-beta Tubulin were acquired from Sigma Aldrich, anti-Cx37 antibody, SB431542, monoclonal antibodies for TGF beta1 and TGF beta2, Latrunculin A and Nocodazole were obtained from Invitrogen, Tocris, BD Bioscience Inc., and Sigma-Aldrich, respectively.
HUVECs were purchases from American Tissue and Cell Culture (ATCC) and cultured in a 75 ml flask in F-12K cell culture medium containing 10% FBS, 1% penicillin and 0.03 mg/ml endothelial cell growth supplement (ECGS) according to the instructions from the manufacturer. After the cells reached about 90% confluency, they were transferred to three collagen I coated Bio flex 6-well plates (Flex cell International Corporation, Burlington, NC, USA) in F-12 K medium containing serum, after culturing the HUVECs for three days and the cells con-fluency reached about 80%, the cell culture medium changed to serum free. After five hours stabilization, the cells were undergoing 12 or 24 hours of mechanical stretch. For mRNA studies, samples were collected from both 12 and 24 hours of mechanical stretch condition, for protein studies, samples were collected only from 24 hours of mechanical stretch condition. Control cells plated the same condition in collagen I coated Bio flex 6-well plates without being subjected to mechanical stretch.
For mechanical stretch, after cultured cells reaching around 80% confluence, HUVEC cells were switched to serum-free DMEM for five hours, followed by cyclic mechanical stretch for 24 hours with the following parameters: (10% stretching, 1 cycles/second) using the FX-5000 Tension System controlled by the computer (Flexcell International Corporation).
For administration of actin and microtubule inhibitors, the cells were incubated with 50uM of actin cytoskeleton inhibitor Latrunculin A or microtubule inhibitor Nocodazole or Latrunculin A and Nocodazole combined together (all dissolved in dimthylsulfoxide, DMSO), which has been demonstrated previously by other groups that using the same dosage of Nocodazole or Latrunculin A can significantly block actin and microtubule cytoskeleton. Moreover, administration of DMSO was used as a control, then undergoing mechanical stretch treatment. All experiments were performed in duplicate and repeated up to three times.
After 24 hours mechanical stretch, the cell culture serum free medium from control and stretched plates were collected and were subjected to measurement of TGF beta1 and TGF beta2 concentration using Elisa kits with standard protocol provided by the manufacture (R & D systems).
Total RNA extraction, RT-PCR and real time PCR.
The procedure for RNA extraction, RT-PCR and real time PCR were used and described previously and complied with the manufacturer’s protocol . Briefly, cells were collected after 12 or 24 h mechanical stretch, total RNA was extracted using trizol reagents (Invitrogen) and RNA concentrations were determined using a Nanodrop machine. Reverse transcription was conducted using SuperScript® III reverse transcriptase (RT) and oligo (dT)20 primer. The PCR conditions for annealing is dependent on the primers. Single band PCR product were verified by electrophoresis in a 2% agarose gel stained with ethidium bromide. Real time PCR was performed using RT² SYBR Green Fluor qPCR Master mix and the standard protocol.
The following primers were used in this study.
GAPDH Sense: 5’-AATCCCATCACCATCTTCCAGGAG-3’
GAPDH antisense: 5’-CACCCTGTTGCTGTAGCCAAATTC-3’
Cx37 Sense: 5’-TCAGCACACCCACCCTGGTCT-3’
Cx37 antisense: 5’-GGATGCGCAGGCGACCATCTT-3’
Cx43 sense: 5’- GGTCTGAGTGCCTGAACTTGCCT-3
Cx43 antisense: 5’-AGCCACACCTTCCCTCCAGCA-3’
TGF-beta2 sense primer: 5′- GAAGACCCCACATCTCCTGCTA-3’
TGF-beta2 antisense primer: 5′- AGCAATAGGCCGCATCCAA-3’
Human Cx43 expression vector construction:
The procedure for constructing human Cx43 expression vector was conducted as previously described18. Oligonucleotide primers were obtained from Integrated DNA technologies (IDT) Inc. (Coralville, Iowa), transfection reagents were purchased from Life Technologies Inc. Primers chosen for RT-PCR were based on the human Cx43 sequence (NCBI Reference Sequence: NM_000165.5). These were: full length Cx43 sense primer: 5’-ATGGGTGACTGGAGCGCCTTAG-3’; full length Cx43 antisense primer: 5’-CTAGATCTCCAGGTCATCAGG-3’.
For amplification of human Cx43 coding cDNA, PCR was conducted in 20 µL solution containing 2 µL 10 × PCR buffer; 0.8 µL 50 mM MgCl2; 200 µM dNTP; 100 ng sense and antisense primers;1 unit Taq DNA polymerase; and 1 µL template cDNA. For PCR, DNA was denatured at 95°C for 3 min, and then subjected to 30 cycles at 95°C for 60 s, 55°C for 60 s and 72°C for 90 s; this was followed by a final extension at 72°C for 10 min for T-A cloning. PCR products were separated by electrophoresis in 1% agarose gel, stained with ethidium bromide and purified using a gel purification kit (Qiagen Inc, Mississauga, ON, Canada). PCR products were subcloned into PCR 2.1 vector, digested with BamHI and ApaI, and ligated into pcDNA3.1 expression vector using T4 DNA ligase according to the manufacturer’s instructions. Recombinant plasmids were extracted, orientation was verified with KpnI and BstX1 digestion, and at least two recombinant plasmids were sequenced using T7 universal primer and specific Cx43 primer for confirmation of sequence.
HeLa cells (American Type Culture Collection, Rockville, MD, USA) were grown in Dulbecco’s modified Eagle’s medium with low glucose and supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. For transient transfection, HeLa cells with 80% confluence were transfected for 48 h with pcDNA3.1 vector, full length Cx43-pcDNA3 or truncated Cx43-pcDNA3.1 plasmids using LipofectAMINE 3000 reagent as described previously18,19, and the cells were harvested followed by Western blotting assay for validating anti-Cx43 antibody specificty.
The procedure for immunofluorescence labeling was used as we previously described18,20. Briefly, mechanical stretched and non-stretched HU-VECs grown in 6 well Bio flex plates were fixed with 2% cold paraformaldehyde for 10 min, and then were washed with PBS. For immunolabelling, slides were incubated in 50 mM Tris-HCl, pH 7.4, containing 1.5% sodium chloride (TBS) and 0.3% Triton X-100 (TBSTr) and 5% NGS for 24 h at 4 ℃ with Cx43 polyclonal antibody at 1:1000, they were then washed for 1 h in TBSTr and incubated for 1.5 h at room temperature simultaneously with Alexa Fluor 488-conjugated goat anti-rabbit IgG at 1:1500 dilution (Molecular Probes Eugene, Oregon). Following incubation with secondary antibodies, slides were sequentially washed in TBSTr for 20 min, in 50 mM Tris-HCl buffer, pH 7.4 for 30 min, and then coverslipped using anti-fade medium. To test for inappropriate cross-reactions between primary and secondary antibodies or between different secondary antibodies, control procedures included omission of one of the primary antibodies with inclusion of each of the secondary antibodies. Confocal immunofluorescence images were gathered on an Olympus confocal microscope using the Fluoview program. Some confocal images are presented as z-stacks of six to ten scans at z scanning intervals of 0.5 µm.
Western blotting. The procedure for western blotting was described previously18,21. Briefly, Cells in mechanical stretch and non-stretch condition were harvested in an IP buffer (20 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EGTA, 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml each of leupeptin, pep-statin A, and aprotinin) and sonicated. Homogenates were centrifuged at 20,000 x g for 20 min at 4°C, and the supernatants were taken for protein determination using Bradford reagent (Bio-Rad Laboratories). Proteins containing 5% β-mercaptoethanol were boiled for 5 min and were separated by SDS-PAGE (5 µg of protein per lane) using 10% or 12.5% gel followed by transblotting to polyvinylidene difluoride membranes (Bio-Rad Laboratories) in standard Tris-glycine transfer buffer, pH 8.3, containing 0.5% SDS. Membranes were blocked for 2 h at room temperature in TBSTw (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.2% Tween 20) containing 5% nonfat milk powder, rinsed briefly in TBSTw, and incubated overnight at 4°C with polyclonal anti-Cx43 primary antibody diluted at 1: 1000 in PBS containing 1% nonfat milk or polyclonal anti-alpha-GAPDH antibody diluted at 1:1000 (Novus Biotechnological Inc) in TBSTw containing 1% nonfat milk powder. Membranes were washed four times in TBSTw for 40 min, incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG or anti-mouse IgG diluted 1:3000 to 1:5000 (Sigma-Aldrich) in TBSTw containing 1% nonfat milk powder, washed four times in TBSTw for 40 min, and re-solved using an Odyssey imagers. The optical density (OD) of specific band was also acquired.
Statistical analysis. All data were expressed as mean ± SEM. The one-way ANOVA test was employed to examine the statistically significant differences between groups. Significant differences were set at * (P<0.05), ** (P<0.01) and *** (P<0.001).