All reagents, prepared or manufactured plasmids, cell types and competent cells that were utilised in this study are listed below:
Table
2.1 List of cell lines, their description and source
Table 2.2 List of viruses
Virus Description Source and references
Wt300 Wild type Ad5 (Jones and Shenk, 1978)
(Jones and Shenk, 1978) E4orf3 Implanting of E4 frame-shift
(300 backbone)
(Huang and Hearing, 1989)
Table 2.3 Restriction enzymes
Cell type Description Source and suppliers
HEK293 Embryonic Kidney immortalized with adenovirus type 5- express E1A and E1B proteins
Supplied by Dr. Keith Leppard, University of Warwick
(Graham et al., 1977)
HeLa Human cervical cancer cells provided by Prof. M.A. McCrae, University of Warwick
MRC5 Normal diploid human lung fibroblasts ATCC No.
HB-8117TM
Hybridoma cells produce anti-Ad5 hexon antibodies
Provided by Dr. Hans- Gerhard Burgert.
293T cells HEK293 cells; transformed with SV40 to increase protein production
Restriction enzymes Description
AgeI Promega
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Table 2.4 Bacterial cells
Table 2.5 Plasmids
Table 2.6 PLKO. Sequencing primers
Primers Sequence Source
PLKO.1 A GACTATCATATGCTTACCGT Addgene
PLKO.1 B CTTTCCCCTGCACTGTACC This study
PLKO.1 C GCTATTATGTCTACTATTC This study
Competent cells Description
DH5α supE44, lacU169(ϕlacZ M15), hsdR17, recA1,
gyrA96, thi-1, relA1
Plasmid Description and sources
PLKO.1 Shuttling plasmid with ampicillin bacterial resistance and puromycin as selective marker for production of recombinant lentivirus, (with cloning site 5 AgeI and 3 EcoRI and a stuffer insert of 1900bp.(Addgene)
psPAX2 Packaging plasmids, contains a robust CAG promoter to express packaging proteins for PLKO.1(Addgene) pMD2 Envelope plasmid for PLKO.1 packaging (Addgene) PGEM-T PGEM R-T Easy Vector Promega
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Table 2.7 ShRNA constructs shRNA
functional products
Sense 5 Antisense 3 Description
PML-II CATCCTGCCCAGCTGCAAATT TTTGCAGCTGGGCAGGATGTT Specific sequence targets PML-II within exon 7b (Wright, 2010)
Exon3 GAGCTCAAGTGCGACATCATT TGATGTCGCACTTGAGCTCTT Targets all PML isoforms (Wright, 2010) Scrambled
DNA
GAGCCGGACGCCAAAGAAATT TTTCTTTGGCGTCCGGCTCTT Control shRNA with no target effect of any human gene (Wright, 2010)
GFP GCAAGCTGACCCTGAAGTTCAT ATGAACTTCAGGGTCAGCTTGC Dr. Kirsten Bentley
Table 2.8 SiRNA
Target Sense 5 Antisense 3 Description
HSP70 -- -- Targets HSPA1 and HSPB1 (Ambion)
IRF3 UGGAUGAGUUACUGGGUAATT UUACCCAGUAACUCAUCCAGA Targets IRF3, gene ID = 3661 (QIAGEN) STAT1 GAAAGAGCUUGACAGUAAATT UUUACUGUCAAGCUCUUUCTG Targets STAT1 gene ID = 6772 (QIAGEN) PML-II CAUCCUGCCCAGCUGCAAAUU UUUGCAGCUGGGCAGGAUGUU Targets exon 7b (Wright, 2010)
Scramble siRNA “A” GAGCCCGGACGCCAAAGAAAUU UUUCUUUGGCGUCCGGCUCUU Control siRNA with no target effect of any human gene (Wright, 2010)
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Table 2.9 PCR and qPCR primers
Primer Forward Reverse Description
genomic GAPDH
CCCCACACACATGCACTTACC CCTAGTCCCAGGGCTTTGATT (Krog et al., 2007)
PML-II AGGCAGAGGAACGCGTTGT GGCTCCATGCACGAGTTTTC (Chen, 2014)
PML Exon3 ACTGCCGAGGATGTTCCAAG TCCTGTCGCTGCTGGATCTC This study
IL-6 AAAGAGGCACTGGCAGAAAA TTTCACCAGGCAAGTCTCCT (Chen, 2014)
ISG56 GCCATTTTCTTTGCTTCCCCTA TGCCCTTTTGTAGCCTCCTTG (Chen, 2014)
GAPDH GGTCGGAGTCAACGGATTT CCAGCATCGCCCCACTTG (Vestergaard et al., 2011) Β-Actin AAAGACCTGTACGCCAACAC GTCATACTCCTGCTTGCTGAT (Shi et al., 2010)
18S ACGCTGAGCCAGTCAGTGTA CTTAGAGGGACAAGTGGCG (Menager et al., 2009) HSP70 AGGCCAACAAGATCACCATC TCGTCCTCCGCTTGTACTT (Tanaka et al., 2007) HSPA1A GCCGAGAAGGACGAGTTTGA TCCGCTGATGATGGGGTTAC www.rtprimerdb.org HSPA1B AGGCCAACAAGATCACCATC TCGTCCTCCGCTTTGTACTT www.rtprimerdb.org Hsp705 GGCCGCACGTGGAATG ACCTCCAATATCAACTTGAATGTATGG www.rtprimerdb.org HSP706 GCCCTTCAGAGATGAACTTT TGTTAAAGTGCCACACAAGT www.rtprimerdb.org HSP27 AAGCTAGCCACGCAGTCCAA CGACTCGAAGGTGACTGGGA (Wang et al., 2013) Daxx TCTACAACTTTGGCTGTCACCTC GTCTCTTCTGTCTCTCGCCCT (Pan et al., 2013) hexon CGCTGGACATGACTTTTGAG GAACGGTGTGCGCAGGTA (Schreiner et al., 2013) E1A GTGCCCCATTAAACCAGTTG GGCGTTTACAGCTCAAGTCC (Schreiner et al., 2013)
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Table 2.10 primary antibodies
Antibody Description Dilution Sources
and suppliers
Western blotting Immunofluorescence FACS
Viral proteins Rat-anti E4Orf3 monoclonal Detects E4Orf3 expression 1/500 -- -- (Nevels et al., 1999) Mouse anti- E1B55kmonoclonal Detects E1B55K expression 1/250 -- -- (Sarnow et al., 1982b) Mouse anti DBP Detects Ad5 DNA
binding protein 1/10000 1/5 (Reich et al., 1983) Mouse anti-Hexon monoclonal antibodies 2Hx-2 IgG2a Adenovirus group specific antigen
-- No dilution No dilution Provided by
Dr. Hans- Gerhard Burgert Rabbit poly clonal anti-
late (AdJLB1)
Detects all Ad5 late gene expression
1/10000 -- 1/500 (Farley et
al., 2004)
Cellular proteins
Anti-Hsp70 Rabbit polyclonal 1/50000 1/300 -- StressMarq
(SPC- 103C/D)
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(SC-9082X) Anti PML Detect total PML
(Rabbit polyclonal)
1/50000 1/500 -- (Xu et al.,
2003) Anti- PML-II Detect PML-II(Rabbit
anti-peptide)
1/50000 1/500 -- (Xu et al.,
2003) Anti- PML-V Detect PML-V(Rabbit
anti-peptide)
1/50000 -- -- (Xu et al.,
2003) Mouse anti- GAPDH Mouse GAPDH(
monoclonal Detect)
1/50000 -- -- MA5-15738
Anti-STAT1 Detects STAT1 (Rabbit polyclonal)
1/1000 -- -- Santa Cruz
(SC-346)
Anti-Sp1 polyclonal Ads 1/1000 -- -- Santa Cruz
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Table 2.11 Secondary antibodies
Antibody Description Dilution Supplier
Western blotting Immunofluorescence FACS
Goat anti-Mouse IgG Horse Radish
peroxidase conjugate
1/10000 -- -- Sigma
Goat anti- Rabbit IgG Horse Radish
peroxidase conjugate
1/20000 - 100000 -- -- Santa Cruz (SC-2054)
Goat anti Rat IgG Horse Radish
peroxidase conjugate
1/5000 -- -- Chemicon
International Goat anti- Rabbit
IgG(H+L)
AlexaFluor 488(conjugate)
-- 1/500 1/50 Life Technology
Goat anti- Rabbit IgG(H+L)
AlexaFluor 594 (conjugate)
-- 1/500 -- Life Technology
Goat anti- Mouse IgG (H+L)
AlexaFluor 488(conjugate)
-- 1/500 1/50 Life Technology
Donkey anti-Goat IgG Horse Radish
peroxidase conjugate
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2.12 A list of reagents
Reagent Supplier
Dulbecco's modified Eagle's medium(DMEM 1X) Gibco
Fetal bovine serum (FBS) Sigma Roswell Park Memorial Institute (RPMI) Gibco
Minimum Essential Medium(MEM) Gibco
Puromycin dihydrochloride Sigma
QNZ, 6-Amino-4(-4-Phenoxyphenylethylamino)quinazolin Santa Cruz Recombinant Human Tumor necrosis factor (TNF-α)
Lyophilised
Invitrogen
Interferon-α (IFN-α) PbI interferon
source
Lipofectamine 2000 (LF2000) Invitogen
TransITR LT1 transfection reagent (LT1) Mirus
Dimethyl Sulfoxide(DMSO) Sigma
GenEluteTM Mammalian Total RNA Miniprep kit Sigma
SV Total RNA Isolation System Promega
GenEluteTM Mammalian Total DNA Miniprep Kit Sigma GoScriptTM Reverse Transcription System Promega
QIAquickR PCR Ourification kit QIAGEN
QIAprep Spin Miniprep Kit Using a Microcentrifuge QIAGEN
GeneJET Gel Extraction Kit Thermo Scientific
Gene detecting primers Sigma
Brilliant III Ultra-Fast SYBR Green qPCR Master Mix with Low Rox
Agilent
DNase treatment kit Promega
100bp DNA Ladder Promega
1kb DNA ladder Promega
PageRuler Plus Prestained Protein Ladder Thermo Scientific Super Signal R West Femto Maximum Sensitivity Substrate Thermo Scientific Nitrocellulose Blotting Membrane GE Health
Care Amersham
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