Cell lines
The following human cell lines: DU145 prostate carcinoma, HepG2 hepatocarcinoma, transformed 293 T kidney embryonic, Skmel-25 malignant melanoma, NCI-H1155 lung carcinoma, IM-9 B transformed lymphoblast, SAOS 2 osteosarcoma and HeLa cervix adenocarcinoma were cultured as previously described [13] and recommended by [14].
Reverse transcription and polymerase chain reaction
In order to obtain the coding sequence of the canonical isoform of the CHD7 gene, total RNA samples from diverse tumor cell lines were purified according to the Chirgwin procedure [15]. Poly A+ RNA was isolated from total RNA with the PolyAttract mRNA isolation kit (Promega, Madison, WI) and employed for cDNA synthesis.
cDNA was synthesized using 1 μg of poly A+ RNA, oligodT12-18 and the Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA), according to manufacturer's instruction. PCR reactions were carried out in 25 μl and the reactions mixtures contained 1 μl of a 1/20 dilution of the cDNA preparation, 1× Tuning Buffer® (Eppendorf, Westbury, NY), dNTPs (0.5 mM each), 0.5 μM of each primer and 0.5 U TripleMaster Taq polymerase (Eppendorf, Westbury, NY). The following primers annealing to sequences corresponding to exons 1 and 38 of CHD7 were used:
CHD7F forward: 5'-AAAAAGCAGGCTTGGTCCTCGCCACGCGCTCGTGCTCGGGA-3' and CHD7R reverse: 5'-AGAAAGCTGGGTGGGACATCTCTGCATATCATGGGTCACT-3'.
A "Long distance PCR" cycling protocol was employed, as follows: 3 min at 93°C (initial denaturation); 14 cycles of 20 sec at 93°C and 10 min at 68°C; 21 cycles of 20 sec at 93°C and 11 min at 68°C with additional 20 sec of auto-extension at each cycle; 7 min at 68°C (final extension).
To analyze the expression of the CHD7 novel splicing variant by RT-PCR, its sequence was used to design a set of primers flanking the splicing site at exons 3 (CHD7 E3 forward 5'-AGTGCTGGGATACCAATGGA-3') and 36 (CHD7 E36 reverse 5'-GGAACCCCCATACAGTCAAA-3'), which would yield a PCR band of approximately 2 kbp. Expression of the long transcript from the NOTCH2 gene was evaluated as an internal control using the following primers corresponding to the 5' region of the transcript:
NOTCH2 forward 5'-ACTGTGGCCAACCAGTTCTC-3' and NOTCH2 reverse 5'-CTCTCACAGGTGCTCCCTTC-3', which would yield a PCR band of approximately 300 bp. For this analysis, cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA), according to manufacturer's instruction and 1.5 μg of total RNA from the DU145 prostate carcinoma cell line and from various human tissues (spinal cord, prostate, kidney, lung, placenta, skeletal muscle and liver) obtained from the Human Total RNA Master Panel II (Clontech, Mountain View, CA). Polymerase chain reactions (final volumes 50 μl) containing 1× Phusion HF Buffer (Finnzymes, Finland), 0.5 mM each dNTP, 0.4 uM each primer, 1 ul of the undiluted cDNAs preparations, and 2 U Phusion Hot Start DNA Polymerase (Finnzymes, Finland) were carried out to detect the novel CHD7 alternative isoform and NOTCH2 transcripts in the cDNA samples mentioned above. The cycling protocol employed for the detection of CHD7 CRA_e transcript was as follows: 30 sec at 98°C (initial denaturation); 3 cycles of 10 sec at 98°C, 30 sec at 68°C and 12 min at 72°C; 3 cycles of 10 sec at 98°C, 30 sec at 65°C and 12 min at 72°C; 3 cycles of 10 sec at 98°C, 30 sec at 62°C and 12 min at 72°C; 35 cycles of 10 sec at 98°C, 30 sec at 60°C and 12 min at 72°C; and a 15 min at 72°C (final extension). The cycling protocol employed for the detection of NOTCH2 transcript was as follows: 4 min at 94°C (initial denaturation); 35 cycles of 30 sec at 94°C, 45 sec at 54°C and 45 sec at 72°C; and a 10 min at 72°C (final extension).
A negative control, without cDNA, was run with each reaction. PCR products were fractionated by agarose gel electrophoresis and visualized under UV light and the digital images were acquired using the D-Transilluminator and MiniBIS gel documentation system (DNR Bio-Imaging Systems, Israel).
Sequence analysis of the novel CHD7 transcript
In order to characterize the novel CHD7 transcript, its coding sequence was re-amplified from a cDNA preparation synthesized using the Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA), according to manufacturer's instructions, and 3 μg of total RNA from DU145 prostate carcinoma cell line. PCR reactions were carried out in 50 μl and the reactions mixtures contained 1 μl of a 1/20 dilution of the cDNA preparation, 1× High Fidelity Buffer® (Eppendorf, Westbury, NY), dNTPs (0.4 mM each), 0.4 μM of each primer and 4 U TripleMaster Taq polymerase (Eppendorf, Westbury, NY). The following primers annealing to sequences corresponding to exons 2 and 38 of CHD7 were used: CHD7F forward: 5'-ACCTCAGTGAAGTGAAGCACAGG-3' and CHD7R reverse: 5'-CACACTAGCGTGGAGATTGTCAG-3'. The cycling protocol employed was as follows: 3 min at 94°C (initial denaturation); 1 cycle of 30 sec at 94°C and 12 min at 72°C; 3 cycles of 30 sec at 94°C, 40 sec at 68°C and 12 min at 72°C; 3 cycles of 30 sec at 94°C, 40 sec at 65°C and 12 min at 72°C; 35 cycles of 30 sec at 94°C, 40 sec at 62°C and 12 min at 72°C; and a 15 min at 72°C (final extension).
The approximately 3.3 kpb DNA band was gel purified and subcloned into the pGEM-T Easy vector (Promega, Madison, WI) using the TA cloning system, according to manufacturer's instruction. Three bacterial (Escherichia coli XL1 Blue) clones (pGEM-M1, pGEM-M2 and pGEM-M3) were picked and individually grown in liquid LB medium containing 100 μg/ml of ampicilin overnight at 37°C under agitation (250 rpm). Plasmid DNA was extracted from bacterial cultures using the GFX TM Micro Plasmid Prep (GE HealthCare, Piscataway, NJ), according to the manufacturer's instructions. The three clones containing the novel CHD7 transcript cDNA were subjected to sequencing using the ABI 3700 sequencer and the BigDye 3.1 sequencing kit (Applied Biosystems, Foster City, CA) at the GaTE (Genomic and Transposable Elements) lab, Biological Institute, University of Sao Paulo. Sequencing was carried out using sequencing primers annealing to CHD7 exons 2, 37 and 38. The novel CHD7 transcript cDNA sequences (clones M1, M2 and M3) were individually clustered into contigs using the SeqMan II software (DNASTAR, Inc., Madison, WI). The original sequencing files were evaluated for quality using the Trace Quality Evaluation algorithm within SeqMan II. Poor-quality sequences were trimmed and the trimmed cDNA sequences of each clone were assembled into contigs using the SeqMan II assembly process. The contig cDNA sequences of each clone were individually compared to the canonical CHD7 transcript reference sequence (NM_017780.2) using the BLASTN program [16] at NCBI [17]. Determination of sequence overlap between the contig cDNA sequences described above and the CHD7 canonical transcript reference sequence and CHD7 mRNAs and spliced ESTs sequences from the Genbank was performed using the UCSC Genome Browser [18, 19]. The contig cDNA sequences were aligned to the February 2009 version of the human genome sequence assembly using the BLAT alignment tool provided by UCSC. The contigs cDNA sequences open reading frames (ORFs) were determined using the ORFinder tool at [20]. Translations of the detected ORFs were submitted to alignment to CHD7 reference protein sequence (Genbank: NP_060250.2) using the BLASTP alignment tool [21].
Cloning of the CHD7 CRA_e isoform coding sequence into a bicistronic lentiviral expression vector
The CHD7 CRA_e isoform coding sequence from clones M1, M2 and M3 were amplified by PCR from pGEMT-M1, pGEM-M2 and pGEM-M3 vectors and sub-cloned into the p156RRLsinPPTCMVIRESPRE third generation transfer bicistronic lentiviral vector [22] (heretofore referred to as pLV-EGFP). This pLV-EGFP bicistronic vector was kindly provided by Prof. Inder Verma (The Salk Institute, San Diego, California) and further modified in our lab by Dr. Juan Carlos Bustos Valenzuela to add the 5'-XbaI-EcoRV-MluI-NheI-PstI-XhoI-BamHI-3' multiple cloning site. Polymerase chain reactions (final volumes 50 μl) containing 1× HiFi Buffer (Eppendorf, Westbury, NY), 0.2 mM each dNTP, 0.25 uM each primer, 50 ng of the template plasmids, and 2 U Triple Master DNA Polymerase (Eppendorf, Westbury, NY) were carried out to obtain the CHD7 novel isoform coding sequences from clones M1, M2 and M3. The following primers were used: VL XhoI IM CHD7 forward: 5'-CCCCTCGAGATGGCAGATCCAGGAATG-3' and VL BamHI IM CHD7 reverse: 5'-CCCGGATCCCTTGAACTGGAACTGGTACTGG-3'. The cycling parameters were as follows: 2 min at 94°C (initial denaturation); 24 cycles of 30 sec at 94°C, 30 sec at 58°C and 4 min at 68°C; and 10 min at 68°C (final extension). The purified PCR products were digested by XhoI and BamHI and sub-cloned into the pLV-EGFP vector digested with the same enzymes.
Lentivirus particles production and transduction of DU145 cells
Lentivirus particles containing the M1, M2 and M3 cDNA clones of the CHD7 CRA_e isoform and control EGFP lentiviral expression vectors were produced by transient transfection into 293 T cells. Briefly, 2 × 106 cells were plated in 6-cm diameter Petri dishes 16 h prior to transfection in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), ampicilin (25 μg/ml), streptomycin (100 μg/ml) and 1.2 g/l of sodium bicarbonate in a humidified atmosphere of 2% CO2 in air at 37°C. A sample (total of 5 μg) of plasmid DNA was employed for transfection, as follows: 2.2 μg of the transfer vector plasmid, 1.45 μg of packaging plasmid pMDL (Invitrogen, Carlsbad, CA), 570 ng of the pREV expression vector (Invitrogen, Carlsbad, CA) and 790 ng of the pVSVG envelope plasmid (Invitrogen, Carlsbad, CA). These plasmids were co-transfected into 293 T cells by lipofection using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After 5 h of transfection, the medium was replaced and the conditioned medium containing the pseudo-lentiviral particles were harvested 24, 48 and 72 h after transfection, cleared by low-speed centrifugation and stored at -80°C. For titration of the lentiviral preparations, serial dilutions of the conditioned medium were used to transduce 105 293 T cells as described elsewhere [23]. The transduction efficiency was estimated by counting EGFP-positive cells under a fluorescence microscope (TE300 Nikon, Japan). The viral titer of each preparation was calculated according to the following formula:
where P = % EGFP+ cells, N = number of cells at the time of transduction (105), V = volume of dilution used for transduction and DF = dilution factor.
For transduction of DU145 cells, 2 × 104 cells in suspension were mixed with samples of lentivirus supernatants at a MOI (Multiplicity of Infection) of 10 in the presence of polybrene (10 μg/ml). After mixing, the DU145 cells were plated in 48-well dishes and cultured in the presence of lentivirus for 16 hours. Cells were analyzed for EGFP expression 72 hours after transduction.
Protein extraction
DU145 cells and the transduced DU145 cells generated as described in the item above (DU145 M1, DU145 M2, DU145 M3 and DU145 EGFP) were plated (1.5 × 106 cells) in 6-cm diameter Petri dishes and grown overnight. The culture medium was then removed and the cell monolayers were rinsed twice and then scrapped with cold PBSA. The cell suspensions were transferred to microcentrifuge tubes and pelleted for 5 min at 200 × g at 4°C. The cells were resuspended in RIPA+ buffer (10 mM Tris-HCl pH 7.5, 1% sodium deoxycholate, 1% NP-40, 150 mM NaCl, 0.1% SDS, 1 mM DTT and 1× protease inhibitors cocktail (GE HealthCare, Piscataway, NJ) and incubated on ice for 15 min. Cell lysates were then homogenized by passing through an insulin syringe several times. Finally, cellular debris was removed by centrifugation (20,000 × g for 30 min) at 4°C and the extracts were stored at -70°C. The protein concentration in these extracts was determined using the Bradford assay.
Western blot analysis
Protein samples (30-40 μg) obtained from cell lysates were fractionated in 6% SDS-polyacrylamide gel electrophoresis. The resolved proteins were electro-blotted onto nitrocellulose membranes (Bio-Rad, Hercules, CA), which were blocked with 5% non-fat milk in TBS buffer containing 0.05% Tween 20, overnight at 4°C. After six washes with TBS/0.05% Tween 20, the membranes were incubated with antibodies against CHD7 (1:400 dilution) (ab31824 - Abcam, Cambridge, UK) in the same buffer containing 5% non-fat milk for 1 h at room temperature. The membranes were washed again and then probed with horseradish peroxidase-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA), in the same buffer, for 40 min at room temperature. PACE (Paired basic amino acid cleaving enzyme) detection using the polyclonal antibody against PACE (sc-20801 - Santa Cruz Biotechnology, Santa Cruz, CA) was used (1:500 dilution) as an internal control. The signals were detected using the ECL-Plus detection system (GE HealthCare, Piscataway, NJ) according to the manufacturer's instructions.