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Supplementary Material and Methods
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Cell lines and cell culture
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The human melanoma cell line A375N was kindly provided by Dr. Medrano (Houston,
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USA). 293 cells were purchased from Microbix (Toronto, Canada), while 911 cells were
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courtesy of Dr van der Eb (University of Leiden, The Netherlands). Ovarian
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adenocarcinoma cell lines OVCAR-3 (HTB-161), PA-1 (CRL-1572) and lung carcinoma
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A549 (CCL-185) were obtained from the ATCC (Manassas, VA). Ovarian adenocarcinoma
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cell lines SKOV3.ip1, and OV-4 cells were obtained from Dr. Price, Dr. Wolf (both MD
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Anderson Cancer Center, Houston, TX, USA) and DrEberlein (Harvard Medical School,
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Boston, MA). Firefly luciferase-expressing ovarian adenocarcinoma cell line SKOV3-luc
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was kindly provided by Dr. Negrin (Stanford Medical School, Stanford, CA). All the cell
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lines were grown in the recommended medium supplemented with 10% of fetal bovine
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serum (Natocor, Cordoba, Argentina), 2 mM glutamine, 100 U/ml penicillin and 100 g/ml
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streptomycin and maintained in a 37 °C atmosphere containing 5% CO2.
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Construction and production of adenoviruses
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In order to introduce the chimeric fiber 5/3, the entire cassette I-F512-E1Awt was extracted
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from pAd-I-F512-E1Awt 1 with SpeI / SalI and subcloned in pShuttle-1 vector (Stratagene,
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CA) in XbaI / SalI sites to obtain pShuttle-I-F512-E1Awt. The latter was digested with
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PmeI enzyme and used for homologous DNA recombination with pVK500C 5/3
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Escherichia coli BJ5183 by electroporation 3. One positive clone (pVK500-I-F512-E1Awt
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in
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5/3) was selected, sequenced and amplified by transforming DH5eletromax cells
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(Invitrogen, Carlsbad, CA) followed by DNA-maxiprep (Qiagen, Hilden, Germany). The
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resulting plasmid pVK500-I-F512-E1Awt 5/3 was linearized with PacI, purified by ethanol
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precipitation and transfected in 911 cells using LTX lipofectamine (Invitrogen, Carlsbad,
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CA). The rescued adenovirus (AdF512wt) was used to infect 293 cells and produce the
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stocks 1.
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E1A mutants were obtained by PCR-based techniques using as template pGEM-T-E1Awt
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(499-1632). The E1ARbmutant (1130bp) carrying the CR2 mutation 123-127
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(corresponding to aminoacid residues TCHEA) was generated by a two-step amplification
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procedure. First, a 430bp fragment was amplified using primers PRE5´ and DelE1Ar (Table
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S4). A second fragment (700bp) downstream to the mutation region was amplified with
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primers DelE1Afand PRE3´ (Table S4). Both products were mixed and used as templates
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for a second PCR round with PRE5´/PRE3´ primers to obtain the E1ARb mutant.
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To obtain the E1Ap300 mutation (amino acids 4-25, IICHGGVITEEMAASLLDQLIE)
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the first round of PCR was carried out with primers delE1Ap300 5’/ PRE3’ Nhe (about 900
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bp) and the second with primers delE1Ap300 Nco 5’/PRE 3´. Finally, the double mutant
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E1ARbp300 was constructed using as templateE1Ap300 mutant and PRE5´/DelE1Ar
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andDelE1Af/PRE3´primers set (Table S4) for the first round of PCR; then, both products
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were mixed and used as templates for a second PCR round with primersdelE1Ap300 Nco
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5’ / PRE 3´(Table S4). PCR products E1ARb, E1Ap300 and E1ARbp300 were
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cloned into pGEM-T vector (Promega Corp., Madison, WI), to obtain pGEM-E1ARb,
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pGEM-E1Ap300 and pGEM-E1ARbp300, respectively. Each E1A mutant was
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released from pGEMs vectors with NcoI/NheI enzymes and subcloned into NcoI / XbaI
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digested pGL3-Basic vector (Promega Corp., Madison, WI). The E1A mutants were
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extracted with HindIII / SalI from pGL3-E1ARb, pGL3-E1Ap300 or pGL3-
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E1ARbp300 and cloned into HindIII / SalI digested pShuttle-I- F512 vector. pShuttle-I-
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F512-E1ARb, pShuttle-I-F512-E1Ap300 or pShuttle-I-F512-E1ARbp300 were
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digested with PmeI enzyme and utilized for homologous DNA recombination with
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pVK500C 5/3 to obtain AdF512v1, AdF512v2 and AdF512v3, respectively, each one
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containing the different E1A mutants. Viral constructs were confirmed by restriction
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pattern and automatic DNA sequencing (ABI PRISM 377 DNA Sequencer, Applied
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Biosystems, Foster City, CA). Ad-SV40(Luc 5) and Ad-wt 5/3 were already described
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1, 4
.
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Ad-SV40(Luc 5/3) and Ad-F512(Luc 5/3) were prepared using pAd-SV40-luc
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pAd(I)-F512-luc 1. The fragments SV40-luc and (I)-F512-luc were extracted from the
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plasmid by digestion with SpeI / SalI enzymes and cloned into pShutlle-1 vector digested
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with XbaI / SalI enzymes. The resulting plasmids pShuttle-SV40-luc and pShuttle-(I)-F512-
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luc were used to obtain the corresponding viruses as described above.
and
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Immunoprecipitation
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Ten g of antibody agarose conjugated rabbit anti-adenovirus-2 E1A protein 13 S-5 (Santa
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Cruz Biotechnology Inc, CA), was added to 500 g of total cell proteins from cells infected
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during 14 hours with Ad-wt 5/3 or AdF512v1, and incubated at 4°C overnight. The pellet
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was collected by centrifugation, washed with RIPA, and subjected to 7% SDS
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polyacrylamide gel electrophoresis followed by immunoblottingwith rabbit anti-human Rb
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C-15 antibody (Santa Cruz Biotechnology Inc, CA).
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Western blot analysis
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Twenty four hours after adenoviral infection at 1000 MOI, 1 x105 cells were scraped into
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200 l lysis buffer (150 mmol/L NaCl, 50 mmol/L tris (pH 7.5), 0.05% SDS, 1% Triton X-
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100) and sonicated on ice. Protein (about 10 g) was electrophoresed on SDS-
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polyacrylamide gels and transferred onto a nitrocellulose filter. Antibody binding (mouse
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Anti-E1A sc-430, Santa Cruz Biotechnology Inc, CA) was visualized using enhanced
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chemiluminescence (Amersham Pharmacia, Buckinghamshire, United Kingdom). We used
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-tubulin III as housekeeping (Anti-β-Tubulin III, Sigma, St. Louis, MI, USA).
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E1A RT-PCR
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Seven thousand A549 cells were infected at 1000 MOI and after 24 hours total RNA was
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isolated by using TRizol (Invitrogen, Carlsbad, CA). E1A transcripts were assesed by
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semiquantitative RT-PCR using primers FE1A560 and RE1A1632 (Table S4). The
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expression was normalized with actin (primers ACTBas and ACTBAse, Table S4).
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Histology
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For histology studies, samples were fixed in 10% neutral-bufferedformalin before paraffin
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embedding and cutting of 8-µmsections. For immunohistochemical studies of SPARC
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expression eight m-thick tumor sections were deparafinized in xylene and rehydrated in
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graded ethanol. For antigen unmasking, sections were immersed in citrate buffer (pH=6)
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and boiled twice in a microwave oven. Endogenous peroxidase activity was blocked by
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soaking the sections in 3 % hydrogen peroxide in methanol for 15 minutes. Non-specific
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binding sites were blocked by incubating the sections in normal goat serum (10 % in PBS).
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Excess serum was then removed and the tissue sections were incubated overnight with anti-
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hSPARC antibody (R&D Systems, Minneapolis, MN, USA) at 1/50 dilution. After washing
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the slides twice in PBS for 10 min the sections were incubated with biotinylatedgoat anti-
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mouse antibody (Vector Laboratories, Burlingame, CA) at 1/400 dilution for 45 min,
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followed by a PBS washing. The sections were subsequently incubated with Vectastain
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ABC reagent (Vector Laboratories, Burlingame, CA) for 45 min. Color was developed
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incubating the sections with liquid DiaminoBenzydine (substrate chromogen system,
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DakoCytomation, Carpinteria, CA). Finally the sections were counterstaining with
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hematoxylin, dehydrated and mounted.
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Flow cytometry
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Cells (1 x 105/well MW6) were arrested in G0/G1 by serum starvation for 48 hours and
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then maintain in 1 ml of D/F12 2% during two hours. After that, cells were infected at 500
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of MOI in the same medium (non-infected cells were hold in D/F12 2% during this period
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of time). After 4 hours the culture medium was added in a 2 ml volume (6.5% SFB, final
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serum concentration of 5%). At 4 days cells were harvested, washed with PBS, and fixed in
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70% ethanol at 4 °C overnight. They were then washed with PBS, resuspended in PBS-
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triton and treated with RNase (Sigma, San Luis, MO) and stained with propidium iodide.
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Cell cycle status was analysed using FACSCalibur flow cytometer (Becton Dickinson,
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Oxford, United Kinngdom). In all the cases 10000 events were measured in each analysis.
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References
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Lopez MVn, et al.(2009). Tumor Associated Stromal Cells Play a Critical Role on
the Outcome of the Oncolytic Efficacy of Conditionally Replicative Adenoviruses.
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receptor-independent cell entry mechanism. J Virol72: 9706-9713.
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Chartier C, Degryse E, Gantzer M, Dieterle A, Pavirani A, Mehtali M (1996).
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