(Supplementary Methods online)

Transcription

1 (Supplementary Methods online) Production and purification of either LC-antisense or control molecules Recombinant phagemids and the phagemid vector were transformed into XL-1 Blue competent bacterial cells that were previously infected with a helper bacteriophage, M13K07 (New England Biolabs, Beverly, MA). The orientation of the cloned cdna in the phagemid vector determines the identity as sense or antisense. After overnight culture of helper phage-infected cells in 2 YT (10 g NaCl, 10 g yeast extract, and 16 g bactotrypton/1000 ml), 20% polyethylene glycol (PEG 8000) was added to culture supernatant to precipitate bacteriophages. Bacteriophage precipitate was resuspended in TE (ph 8.0), and phage genomic DNA was extracted with phenol and precipitated with ethanol. Phage genomic antisense and control molecules were purified with gel filtration column chromatography (1 50 cm) for large-scale purification. The column resin used for gel filtration was superfine Sephacryl S-1000 (molecular cutoff: 20,000 bp) (Amersham Biosciences, Uppsala, Sweden), and was packaged and equilibrated with 50 mm Tris-HCl (ph 8.3) buffer containing 0.2 M NaCl. The starting volume of the molecules was adjusted to 5% of the gel void volume, and DNA elution was carried out with the same buffer used for resin equilibration (flow rate: 0.3 ml/min). Samples were UV scanned at 260/280 nm with a dual UV detection system and were collected every 5-10 min during elution. Sample fractions were washed and precipitated with 70% cold ethanol and were resuspended in TE buffered for subsequent experiments. The

2 purified molecules were tested for quantity and purity on a 1% agarose gel. Sequence integrity for singlestranded sense or LC-antisense molecules was confirmed by employing the universal primers for sequencing. Detection of polypeptides with ELISA or Western Blotting Quantification of target proteins after LC-antisense treatment was performed with either ELISA or Western blotting method. For the ELISA assay, cell culture supernatant was diluted 50 fold and added to an ELISA plate coated with anti-tnf- antibody. Biotinylated secondary antibody to anti-tnf-α was added to each well of the ELISA plate and incubated at room temperature for 90 min. The plate was then washed three times and incubated with streptavidin-peroxidase for 45 min. The plate was washed four times to remove unbound streptavidin-peroxidase, which was followed by addition of chromogen and 20 min incubation for color development. Optical density was measured at 450 nm. Western blotting was performed to examine the presence of CDK2 and CDK4. The amount of total proteins was determined with the BCA protein assay kit (Pierce, Milwaukee, WI). Twenty g protein samples were loaded onto a 12% polyacrylamide gel and electrophoresed. The proteins were then transferred onto PVDF membrane (Bio-Rad, Hercules, CA). The membrane was blocked by incubating with PBS buffer containing 3% non-fat milk and 0.05% Tween 20. The membrane was then incubated with rabbit polyclonal IgG antibody to either human CDK2 or CDK4 (Santa Cruz Biotechnology, Santa Cruz, CA). The secondary antibody of horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Biosciences, Uppsala, Sweden) was added, and protein bands were visualized

3 with an ECL kit (Amersham Biosciences). Construction of a unidirectional subtracted liver cdna library To clone differentially expressed genes in hepatoblastomas, a cdna library was constructed by employing a subtractive hybridization method 37. The procedures were performed with the PCR-Select cdna Subtraction Kit (Clontech, Palo Alto, CA) using cancer and normal tissues as tester and driver, respectively. The tissue samples were washed with phosphate-buffered saline (PBS) and sliced into smaller pieces. Total RNA was extracted with Tri reagent (MRC, Cincinnati, OH). Poly(A) + mrna was isolated with poly(a) Quick mrna Isolation Kit (Stratagene, La Jolla, CA) and used as template for cdna synthesis. To clone the cdna inserts unidirectionally, cdna synthesis primer with the HindIII recognition sequence supplied in the kit was replaced with a primer with an XhoI recognition sequence (5'- TTTTGTACCTCGAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3'). First-strand cdna was synthesized with the modified primer followed by second-strand cdna synthesis using T4 DNA polymerase. The tester cdna and driver cdna, prepared according to the protocol, were digested with RsaI, a four-base cutting restriction enzyme that yields blunt ends. The tester cdnas were then subdivided into two groups, and cdnas from each group were separately ligated to group-specific cdna adaptors (Adaptor 1: 5'- ACCTGCCCGG-3', complementary to a portion of 5'-CTAATACGACTCACT ATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3'; Adaptor 2: 5'-ACCTCGGCCG-3', complementary to

4 a portion of 5'-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT-3'). Two hybridization reactions were performed, which were followed by suppression PCR to amplify differentially expressed sequences using a pair of primers (forward 5'-TCGAGCGGCCGCCCGGG CAGGT-3' and reverse 5'-AGCGTGGTCGCGGCCGAGGT-3') and the Advantage cdna polymerase mix (Clontech). The subtracted cdna pool was cloned into NotI and XhoI restriction sites of the multiple cloning site of pbluescript (pbs) SK(-) vector (Stratagene) in the same direction as the LacZ gene. The cdna plasmid library was transformed into Epicurian Coli XL-10 Gold competent cells (Stratagene) by the calciumchloride method. To determine the total number of primary transformants, 1 or 10 l per 1 ml pilot transformation was plated onto LB ampicillin agar plates. After overnight incubation at 37ºC, the number of ampicillin-resistant colonies was counted and multiplied by a factor of 100 or To determine the percentage of vectors with inserts and the average insert size, recombinant plasmids of 40 randomly selected individual clones were purified by the alkaline lysis method and digested by the same restriction enzymes used in cloning. Finally, to determine if unidirectional cloning was successfully achieved, cdna regions of purified plasmids derived from 100 randomly selected individual transformants were sequenced from the 5' end of the (+) strand by employing the T3 primer in an automated DNA sequencer. Treatment of phosphorothioate (PS) end capped AS-oligos

5 Five different AS-oligos derived from WGSL11 were synthesized with phosphorothioate end capping at both ends, one residue at the 5' terminus and two at the 3' terminus to improve stability. Sense or mismatched oligonucleotides were employed as controls (see Supplementary Table 4 online). HepG2 cells were fed with fresh medium without antibiotics 1 day prior to the addition of PS end capped AS-oligos and washed twice with Opti-MEM just before an experiment. For MTT assay, the cell density was adjusted to cells/ml and divided into 100 µl aliquots for each well of a 96-well plate. Cells were transfected with 0.16 M of the AS-oligos complexed with Lipofectin according to the manufacturer s instructions. Media was replaced with fresh EMEM 24 h after the AS-oligos treatment and incubated for 3 days. The assay was performed in triplicate and scored as mean SD. For RT-PCR and Southern hybridization, the cell density was adjusted to cells/ml and divided into 300 µl aliquot in each well of a 48-well plate. Cells were transfected with 1.1 M of the AS-oligos complexed with Lipofectin. Transfection cocktail was replaced with fresh media 24 h after the AS-oligo treatment and incubated 48 h further before RNA extraction.