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SUPPLEMENTARY MATERIALS AND METHODS
siRNA battery.
Name
siRNA sequence
Ref.
Source
AIF
(apoptosis-inducing factor)
sense 5’-GAUCCUCCCCGAAUACCUCTT-3’
antisense 5’-GAGGUAUUCGGGGAGGAUCTT-3’
(Vahsen et al.,
2004)
SigmaProligo
ANT1
(adenine nucleotide translocase 1)
sense 5’-ACAGAUCAGUGCUGAGAAGTT-3’
antisense 5’-CUUCUCAGCACUGAUCUGUTT-3’
(Le Bras et al.,
2006)
SigmaProligo
ANT2
(adenine nucleotide translocase 2)
sense 5’-GCAGAUCACUGCAGAUAAGTT-3’
antisense 5’-CUUAUCUGCAGUGAUCUGCTT-3’
(Le Bras et al.,
2006)
SigmaProligo
ANT3
(adenine nucleotide translocase 3)
sense 5’-CUCCCCGACCCCAAGAACATT-3’
antisense 5’-UGUUCUUGGGGUCGGGGAGTT-3’
(de La Motte
Rouge et al., 2007)
SigmaProligo
Apaf-1
(apoptosis-protease activating factor 1)
sense 5’-UUGGUGCACUUUUACGUGATT-3’
antisense 5’-UCACGUAAAAGUGCACCAATT-3’
(Zermati et al.,
2007)
SigmaProligo
ATM
(ataxia telangiectasia mutated)
sense 5’-CAUACUACUCAAAGACAUUTT-3’
antisense 5’-AAUGUCUUUGAGUAGUAUGTT-3’
(Zhou et al., 2003)
SigmaProligo
Bad
sense 5’-GAAGGGACUUCCUCGCCCGTT-3’
antisense 5’-CGGGCGAGGAAGUCCCUUCTT-3’
(Jin et al., 2004)
SigmaProligo
Bak
HP Validated siRNAs
Hs_BAK1_5
Qiagen
Bax
HP Validated siRNAs
Hs_BAX_10
Qiagen
Bcl-2
sense 5’-GCUGCACCUGACGCCCUUCTT-3’
antisense 5’-GAAGGGCGUCAGGUGCAGCTT-3’
(Jiang and Milner,
2003)
SigmaProligo
Bcl-XL
sense 5’-CAGGGACAGCAUAUCAGAGTT-3’
antisense 5’-CUCUGAUAUGCUGUCCCUGTT-3’
(Jiang and Milner,
2003)
SigmaProligo
Bid
sense 5’-GAAGACAUCAUCCGGAAUATT-3’
antisense 5’-UAUUCCGGAUGAUGUCUUATT-3’
(Wagner et al.,
2004)
SigmaProligo
Bim
sense 5’-GACCGAGAAGGUAGACAUUTT-3’
antisense 5’-AAUGUCUACCUUCUCGGUCTT-3’
(Mouhamad et al.,
2004)
SigmaProligo
BLM
(Bloom syndrome protein)
sense 5’-GCUAGGAGUCUGCGUGCGATT-3’
antisense 5’-UCGCACGCAGACUCCUAGCTT-3’
(So et al., 2006)
Personal
gift
BRCA-1
sense 5’-GGGCCUUCACAAUGUCCUUTT-3’
antisense 5’-AAGGACAUUGUGAAGGCCCTT-3’
(Ganesan et al.,
2002)
Personal
gift
Casp-2 (caspase-2)
sense 5’-ACAGCUGUUGUUGAGCGAATT-3’
antisense 5’-UUCGCUCAACAACAGCUGUTT-3
(Lassus et al.,
2002)
SigmaProligo
Casp-3 (caspase-3)
sense 5’-AGUGAAGCAAAUCAGAAACTT-3’
antisense 5’-GUUUCUGAUUUGCUUCACUTT-3
(Dasse et al., 2007)
SigmaProligo
Chk1
(checkpoint kinase 1)
sense 5’-UCGUGAGCGUUUGUUGAACTT-3’
antisense 5’-GUUCAACAAACGCUCACGATT-3’
(Sorensen et al.,
2005)
SigmaProligo
Chk2
(checkpoint kinase 2)
sense 5’-UGUGUGAAUGACAACUACUTT-3’
antisense 5’-AGUAGUUGUCAUUCACACATT-3’
(Vitale et al.,
2007)
SigmaProligo
CypD
(cyclophylin D)
sense 5’-GGCAGAUGUCGUCCCAAAGTT-3’
antisense 5’-CUUUGGGACGACAUCUGCCTT-3’
(Machida et al.,
2006)
SigmaProligo
ERCC1
(excision repair cross-complementing 1)
sense 5’-UAUGCCAUCUCACAGCCUGTT-3’
antisense 5’-CAGGCUGUGAGAUGGCAUATT-3’
(Chang et al.,
2005)
SigmaProligo
siGENOME SMARTpool
Cat. n° 011033
Dharmacon
FANCC (Fanconi anemia group C)
siGENOME SMARTpool
Cat. n° 016376
Dharmacon
FDXR
(ferredoxin reductase)
sense 5’-CGGGAGCCCGGCCCAUUUUTT-3’
antisense 5’-AAAAUGGGCCGGGCUCCCGTT-3’
(Castedo et al.,
2006)
SigmaProligo
GSTA3.3
(glutathione S-transferase A3)
sense 5’-GGUGGAACUUCUCUACUAUTT-3’
antisense 5’-AUAGUAGAGAAGUUCCACCTT-3’
(de La Motte
Rouge et al., 2007)
SigmaProligo
HK1
(hexokinase 1)
sense 5’-GAGAUGAAGAAUGGCCUCUTT-3’
antisense 5’-AGAGGCCAUUCUUCAUCUCTT-3’
(Sui and Wilson,
2004)
SigmaProligo
FANCD2 (Fanconi anemia group D2)
sense 5’-AGGAGAUGGAGAAAGGGCUTT-3’
antisense 5’-AGCCCUUUCUCCAUCUCCUTT-3’
(Sui and Wilson,
2004)
SigmaProligo
sense 5’-GAACGGGGCCUGGAACCAUATT-3’
antisense 5’-UAUGGUUCCAGGCCCCGTTCTT-3’
(Massard et al.,
2006)
SigmaProligo
Ikk1 (I-κB kinase 1)
siGENOME SMARTpool
Cat. n° 003473
Dharmacon
Ikk2 (I-κB kinase 2)
siGENOME SMARTpool
Cat. n° 003503
Dharmacon
IP3R1
(inositol 1,4,5-triphosphate receptor 1)
sense 5’-GCUAUGUCCUAUGAAUCGATT-3’
antisense 5’-UCGAUUCAUAGGACAUAGCTT-3’
(Oakes et al.,
2005)
SigmaProligo
IP3R3
(inositol 1,4,5-triphosphate receptor 3)
sense 5’-UGGCUGUGCCACAUGGGAATT-3’
antisense 5’-UUCCCAUGUGGCACAGCCATT-3’
(Oakes et al.,
2005)
SigmaProligo
Ku80
sense 5’-GAGCUAAUCCUCAAGUCGGTT-3’
antisense 5’-CCGACUUGAGGAUUAGCUCTT-3’
(Waninger et al.,
2004)
Personal
gift
Lck
sense 5’-CUGCAAGACAACCUGGUUAUC-3’
antisense 5’-UAACCAGGUUGUCUUGCAGUG-3’
(Methi et al., 2005)
SigmaProligo
MRE11
(meiotic recombination MRE11)
sense 5’-GAUGCCAUUGAGGAAUUAGTT-3’
antisense 5’-CUAAUUCCUCAAUGGCAUCTT-3’
(Pichierri et al.,
2004)
Personal
gift
mtCK1
(ubiquitous mitochondrial creatine kinase)
sense 5’-CUGGUUGGACGCUAGAUCATT-3’
antisense 5’-UGAUCUAGCGUCCAACCAGTT-3’
(de La Motte
Rouge et al., 2007)
SigmaProligo
NEIL1 (NEI-like mammalian glycosylase 1)
siGENOME SMARTpool
Cat. n° 008327
Dharmacon
Nemo (NF-κB essential modulator)
siGENOME SMARTpool
Cat. n° 003767
Dharmacon
p53
sense 5’-GACUCCAGUGGUAAUCUACTT-3’,
antisense 5’-GUAGAUUACCACUGGAGUCTT-3’
(Gu et al., 2004)
SigmaProligo
p53R2
(p53-inducible ribonucleotide reductase
small subunit 2 homologous)
sense 5’-GCAGAAGAGGUCGACUUAUTT-3’
antisense 5’-AUAAGUCGACCUCUUCUGCTT-3’
(Castedo et al.,
2006)
SigmaProligo
siGENOME SMARTpool
Cat. n° 003533
Dharmacon
PBR
(peripheral-type benzodiazepine receptor)
sense 5’-CACUCAACUACUGCGUAUGTT-3’
antisense 5’-CAUACGCAGUAGUUGAGUGTT-3’
(Gonzalez-Polo et
al., 2005)
SigmaProligo
PIDD
(p53-induced protein with a death domain)
sense 5’-GCCCUCAUUCCAGAAAUGCTT-3’
antisense 5’-GCAUUUCUGGAAUGAGGGCTT-3’
(de La Motte
Rouge et al., 2007)
SigmaProligo
PKCδ
(protein kinase C δ isoform)
sense 5’-GGCUGAGUUCUGGCUGGACTT-3’
antisense 5’-GUCCAGCCAGAACUCAGCCTT-3’
(Yoshida et al.,
2003)
SigmaProligo
Puma
(p53-upregulated modulator of apoptosis)
sense 5’-UCUCAUCAUGGGACUCCUGTT-3’
antisense 5’-UUGAGGUCGUCCGCCAUCCTT-3’
(Gu et al., 2004)
SigmaProligo
Rad17
(Rad17 homolog (S. pombe))
sense 5’-CAGACUGGUUGACCCAUCTT -3’
antisense 5’-GAUGGGUCAACCCAGUCUGTT-3’
(Zou et al., 2002)
SigmaProligo
SMC-1
(structural maintenance of chromosomes 1)
sense 5’-AUCUCAUGGAUGCCAUCAGTT-3’
antisense 5’-CUGAUGGCAUCCAUGAGAUTT-3’
(Musio et al.,
2005)
Personal
gift
TAB1
(TAK1 binding protein 1)
sense 5’-GGCUCAAGUUCAGGAGUGATT-3’
antisense 5’-UCACUCCUGAACUUGAGCCTT-3’
TAB2
(TAK1 binding protein 2)
sense 5’-GGAACGACUUCAAAGAGAATT-3’
antisense 5’-UUCUCUUUGAAGUCGUUCCTT-3’;
Modified from
(Singhirunnusorn
et al., 2005)
Modified from
(Singhirunnusorn
et al., 2005)
TAB3
(TAK1 binding protein 3)
sense 5’-CCACCUCAACAGCCAUCUUTT-3’
antisense 5’-AAGAUGGCUGUUGAGGUGGTT-3’
(Ishitani et al.,
2003)
SigmaProligo
TAK1
(TGF-β-activated kinase)
sense 5’-GUAGAUCCAUCCAAGACUUTT-3’
antisense 5’-AAGUCUUGGAUGGAUCUACTT-3’
(Blonska et al.,
2005)
SigmaProligo
UNR
(“irrelevant”, unrelated siRNA)
sense 5’-GCCGGUAUGCCGGUUAAGUTT-3’
antisense 5’-ACUUAACCGGCAUACCGGCTT-3’
(Criollo et al.,
2007)
SigmaProligo
VDAC1
(voltage-dependent anion channel 1)
sense 5’-GUACGGCCUGACGUUUACATT-3’,
antisense 5’-UGUAAACGUCAGGCCGUACTT-3’
(Criollo et al.,
2007)
SigmaProligo
VDAC2
(voltage-dependent anion channel 2)
sense 5’-CUAGUUGGUUAUCUAGUUATT-3’
antisense 5’-UAACUAGAUAACCAACUAGTT-3’
(de La Motte
Rouge et al., 2007)
SigmaProligo
VDAC3
(voltage-dependent anion channel 3)
sense 5’-GAUUGGACUGGGUUAUACUTT-3’
antisense 5’-AGUAUAACCCAGUCCAAUCTT-3’
(de La Motte
Rouge et al., 2007)
SigmaProligo
HK2
(hexokinase 2)
hTERT
(telomerase reverse transcriptase)
p65 (NF-κB p65 subunit)
SigmaProligo
SigmaProligo
The siRNA downregulating SMC-1 was a present from Dr. Filippo Rosselli; siRNAs targeting
BLM, BRCA-1, Ku80 and MRE11 were kindly gifted by Dr. Bernard Lopez. The remaining
siRNAs of the battery were purchased from the indicated companies: Dharmacon (Lafayette, USA),
Qiagen (Hilden, Germany) or Sigma-Proligo (The Woodlands, USA). The UNR siRNA was
employed as a negative control for transfection.
Reverse transfection in 96-well plates. To screen the battery of siRNAs reported above, NSCLC
A549 cells were reverse transfected in 96-well plates at seeding, following an experimental
approach developed in our lab (de La Motte Rouge et al., 2007). To this aim, 300 pmol of siRNA
dissolved in 2.1 mL of serum-free, antibiotic-free, DMEM:F12 (1:1) with L-glutamine but no
phenol red (Gibco-Invitrogen) were combined with 2.1 mL of the same medium containing 63 μL
of HiPerFect transfection reagent (Qiagen) and let stand at room temperature for 30 min.
Transfection complexes were then mixed to 7.8 mL of complete medium (DMEM/F12 (1:1) with Lglutamine but no phenol red supplemented with 10% FCS and antibiotics) in which approximately
2.35 x 105 cells had been previously resuspended. The resulting cell suspension was seeded into a
96-well plate (approximately 3500 cells in 100 μL of medium per well), and transfected cells were
cultured for 48 h prior to the administration of 50 μM CDDP. Finally (after additional 24 h of
treatment), plates were analyzed for cell proliferation and cytotoxicity.
Mathematical definition of the assay-independent values ΔWST-1 and ΔLDH. To analyze the
influence of siRNAs on CDDP-induced antiproliferative and cytotoxic responses in 96-well plate
assays, we employed the assay-independent indicator Δ, as previously reported (de La Motte Rouge
et al., 2007). Since both residual proliferation and cytotoxicity were assessed, we introduced two
distinct Δ values, namely ΔWST-1 and ΔLDH.
For proliferation assays, first the residual proliferation of CDDP-treated transfected cells was
calculated as the percentage of untreated, but transfected cells (%WST-1siRNA = 100 x WST1CDDP,siRNA / WST-1control,siRNA). Then, for each siRNA, ΔWST-1 was computed as the difference
between its residual proliferation (%WST-1siRNA, as just defined) and the residual proliferation of
cells transfected with a control, irrelevant siRNA (%WST-1siUNR, usually between 50 and 60%).
Accordingly, ΔWST-1 positive values indicated cytoprotective effects of the siRNA against CDDPinduced antiproliferation, whereas negative values were associated with chemosensitization.
For cytotoxicity tests, LDH release was calculated as the percentage of the maximal release induced
by a membrane-permeabilizing agent (%LDHsiRNA), following the manufacturer’s instructions.
Since %LDHsiRNA never exceeded 5 % in untreated cultures (independently from the transfected
siRNAs), the ensuing analysis involved only CDDP-treated wells. In this context, ΔLDH was
defined as the difference between CDDP-induced LDH release in transfected cells (%LDHsiRNA, as
just defined) and the LDH release promoted by CDDP in cells transfected with a control, irrelevant
siRNA (%LDHsiUNR, usually between 30 and 40%). Contrarily to ΔWST-1, ΔLDH positive values
were associated with an increased cytotoxic response following CDDP administration, while
negative values indicated siRNA-mediated cytoprotection. The use of Δs ensured two main
advantages: first, the introduction of assay-independent indicators allowed for normalization among
different assays; second, ΔWST-1 permitted to analyze the influence of siRNAs on CDDP-induced
responses separately from their effects per se on proliferation.
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