Construction of Dp71 short hairpin RNA plasmid
According to the open reading frame of the human Dp71 gene (NM_004015), one siRNA sequence (5′-gcactttaattatgacatc-3′) was selected. The scrambled sequence (5′-ttctccgaacgtgtcacgt-3′) which has no significant homology with human gene sequences was included as a negative control. Two complementary oligonucleotides for Dp71 (5′-gatcccgtctttagctgacctgaataactcgagttattcaggtcagctaaagactttttggat-3′ and 5′-agctatccaaaaagtctttagctgacctgaataactcgagttattcaggtcagctaaagacgg-3′), and for the negative control (5′-gatcccttctccgaacgtgtcacgtctcgagacgtgacacgttcggagaatttttggat-3′ and 5′-agctatccaaaaattctccgaacgtgtcacgtctcgagacgtgacacgttcggagaagg-3′), were synthesized by Invitrogen. Sense or antisense strands are in bold letters and stem loop sequences are in italics. They were annealed to generate double-stranded DNAs and ligated into the linearized shRNA (short hairpin RNA) eukaryotic expression vectors purchased from Genechem (Shanghai, China, containing hU6-MCS-CMV-GFP-SV40-Neomycin elements) to construct Dp71 shRNA or control empty shRNA vectors, which were termed Dp71AS and Dp71 empty shRNA vector (E) respectively. The nucleotide sequences of the plasmids were verified by automated DNA sequencing.
Cell culture and generation of stable transfectants
HBE was obtained from the Culture Center, Chinese Academy of Medical Sciences (Shanghai, China). HBE cells were cultured in the same condition as described previously . For stable transfectants, 5 μg of Dp71shRNA plasmid or 5 μg of control empty shRNA plasmid was mixed with 15 μl of Lipofectamine in serum- and antibiotics-free 1640, and the DNA/Lipofectamine mixture was added to the cell culture medium and incubated in the incubator for 4 h. The transfection mixture was removed and cells were maintained in 1640 supplemented with sera. Selection of stable transfectants was initiated with 600 μg/ml of G418 (Invitrogen) 48 h after transfection, a neomycin analog. The stable transfected HBE cells were named HBE-Dp71AS and HBE-Dp71E respectively.
Isolation of cell extracts and western blot analysis
Cultured cells were collected by centrifugation at 1200 rpm for 5 min, and washed twice with PBS. Protein extraction, concentration determination, 10% SDS-PAGE electrophoresis, and membrane incubation with the corresponding primary antibody (rabbit anti-dystrophin, rabbit anti-RAD51 polyclonal antibody purchased from Abcam; rabbit anti-FAK polyclonal antibody, p-FAK polyclonal antibody; rabbit anti-Akt polyclonal antibody, p-Akt polyclonal antibody; rabbit anti-phospho-histone H2AX (γH2AX; Ser 139) antibody (Bioworld Technology, Inc) was performed as described previously. After three washes with TBS-T, horseradish peroxidase-conjugated anti-rabbit IgG was used as the secondary antibody and developed using the ECL Western blotting analysis system (Amersham-Pharmacia).
Quantitative real-time polymerase chain reaction (QRT-PCR) and RT-PCR
The following primers were used and they produced a 157 bp PCR product for Dp71: 173 bp PCR product for FAK, 146 bp PCR product for lamin B1, 160 bp PCR products for RAD51 and 181 bp PCR product for 18 s. The primers are: LMNB1 (Human Accession NM_005573) F:5′tccaggagaaggaggagctg3′, R:5′ggtctcgtagagcgccttg3′; Dp71 (Human Accession NM_004017.2) F:5′ttggcagtcaaacttcggactc3′,R:5′gtgtcctctctcattggctttccag3′; FAK (Human Accession L13616.1) F: 5′ tccccagagctcctcaagaa 3′,R: 5′ tactcgctccattgcaccag3′; RAD51 (Human Accession D14134.1) F: 5′gggaagacccagatctgtca 3′, R: 5′catcactgccagagagacca 3′; Human 18S (NM_022551) F: 5′ aaatagcctttgccatcactgcc3′,R: 5′ gttcaagaaccagtctgggatc3′.
Cell viability assay
The cell viability was assessed by conducting the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT assay and results interpretations were performed as described previously .
Plate colony formation assay
Clone formation assay was performed as described previously. Clone formation efficiency was calculated according to the formula: (clone number/plated cell number) × 100% .
Apoptosis of HBE, HBE-Dp71AS and HBE-Dp71E cells in the log growth phase were induced by 0.2 mM H2O2 (Sigma, St. Louis, USA) for 16 h. The cells then were harvested by trypsinization for flow cytometry. Apoptosis was quantified using the PE Annexin V apoptosis detection kit (BD Pharmingen, San Diego, USA) according to the manufacturer’s protocol. Cell analyses were made using a FACSCalibur flow cytometer (Becton-Dickinson, Mountain View, CA) and CellQuest software (BD Biosciences). Each assay was repeated 3 times .
Measurement of caspase 3, 8, 9 activities
The caspase fluorescent assay kits specific for caspase 3, caspase 8 and caspase 9 (BioVision, San Francisco, USA) were used to detect caspase activation by measuring the cleavage of a synthetic fluorescent substrate. Cell treatment and fold increases in caspase 3, caspase 8 and caspase 9 activities were determined as described previously .
Alkaline comet assay for DNA damage
To perform the comet assay, the cell suspension of each cell group was mixed with low melting-point agarose at 37 °C, to a final concentration of 0.7%. The mixture (15 μl) was pipetted onto slides pretreated with 0.5% normal-melting-point agarose, to retain the agarose cell suspension. The drop containing the cells was covered with a glass cover slip (24 mm × 24 mm) and left at 4 °C for 5 min. The cover slips were gently removed and the slides were then ready for processing. The alkaline comet assay was performed using the basic rationale of Singh et al. The slides were then incubated in the dark for 30 min in cold electrophoresis buffer (300 mM NaOH, 1 mM EDTA, 1% (v/v) DMSO, pH 13) to allow the DNA to unwind before electrophoresis at 25 V for 25 min. After neutralization with 0.5 M Tris–HCl (pH 8.0), the slides were stained with 50 μl ethidium bromide (30 μg/mL, Absin Bioscience Inc., China). Finally, the images were taken by fluorescence microscope and at least 120 randomly selected cells (30 cells from each of the three replicate slides) were analyzed per sample and analyzed using the Comet Assay Software Pect (CASP 1.2.3 beta 1) (http://casplab.com/download). Parameters of tail moment (% DNA in tail × tail length), tail length, and percent of DNA in tail, the most frequently used parameters in the comet assay, were used in this study.
Immunofluorescence and confocal microscopy analysis
The immunofluorescence and confocal microscopy analysis of Dp71, Rad51 and γ-H2AX in HBE was as follows: After the three HBE cells were cultured on glass coverslips for 24 h, cells were treated with 200 μM H2O2 for 30 min as described previously, treated cells and untreated cells were incubated overnight at 4 °C with the primary anti-dystrophin, anti-RAD51 and anti-γ-H2AX antibody. Cells were incubated for 10 min at 37 °C with 1 mg/ml 49,6-diamidino-2-phenylindole (DAPI) for counterstaining, After washing, coverslips were mounted on microscope slides with VectaShield (Vector Laboratories, Inc., Burlingame, CA, USA) and analyzed in a confocal and multiphoton microscope (TCS-SP5, Leica Microsystems, Heidelberg, Germany), using an oil immersion 636 objective. Co-localization of FITC, TRITC, and DAPI staining was analyzed in single optical sections obtained for two channels throughout the Z axis.
Total protein extracts in a final volume of 250 ml were incubated overnight at 4 °C with 5 μg of rabbit anti-lamin B1, 5 μg of rabbit anti-Dp71 antibody, 5 μg of rabbit anti-FAK, and 5 μg of rabbit anti-RAD51 antibody, previously bound to protein G magnetic beads (Millipore). An irrelevant rabbit polyclonal antibody bound to protein G magnetic beads was performed as a negative control. The immune complexes were precipitated by placing the tube into the magnetic stand (Millipore) and washing 3 times with 500 μl of PBS containing 0.1% Tween 20. Precipitated proteins were separated by SDS-PAGE and analyzed by Western blotting with mouse anti-lamin B1, mouse anti-Dp71 antibody, mouse anti-RAD51 and mouse anti-FAK antibody.
All assays were repeated 3 times to ensure reproducibility. Results were displayed as mean ± SE. One-way ANOVA and LSD were used to analyze all experimental data. All statistical analyses were performed with SPSS software (version 17.0; SPSS Inc., Chicago, IL, USA). P < 0.05 was considered as indicating a statistically significant difference.