Materials and methods
Drugs and chemicals
Sources of chemicals are as in (Krämer et al., 2008); CAPE was from Axxora (Lausen,
Switzerland); TNF- was purchased from ReliaTech (Wolfenbüttel, Germany) or Promokine
(Heidelberg, Germany) respectively. Human cells were treated with human TNF-and
murine cells with mouse TNF-.
Generation of primary murine PDAC cell lines
Low passage cell lines from primary pancreatic tumors of PTF1a/p48ex1Cre/+;LSLKRASG12D/+;LSL-p53R172H+/+;LSL-R26Tva-lacZ/- (5436 cell line) or PTF1a/p48ex1Cre/+;LSLKRASG12D/+;TP53lox/lox;LSL-R26Tva-lacZ/- (W22 and 6554 cell lines) mice were generated as
described (Seidler et al., 2008; von Burstin et al., 2009). Expression of tumor virus A (TVA)
receptor enables retroviral transduction of 6554 cells with RCASBP(A) (replication competent
avian sarcoma-leucosis virus long terminal repeat with splice acceptor, Bryan RSV
polymerase and subgroup A envelope) vectors (Seidler et al., 2008).
siRNA transfection
Double-stranded siRNAs (50 nM) were transfected with oligofectamine (Invitrogen,
Karlsruhe, Germany) according to the manufacturer. siRNAs were purchased from Eurofins
(Ebersberg, Germany). Sequences of the siRNAs were described or are available upon
request (Schneider et al., 2006). The shRNA vector pSUPER-shp53 and the control shRNA
vector have been described (Brummelkamp et al., 2002; Krämer et al., 2009) and were
transfected according to the Amaxa protocol for MCF7 cells.
RCASBP(A) vectors
The coding sequence of murine p53 was amplified by RT-PCR using PfuUltra (Stratagene,
La Jolla, CA, USA) and ligated upstream of the internal ribosome entry site (IRES) of pEntrIRES-Puro. This plasmid contains an IRES-puromycin (Puro) cassette from pIRES-Puro3
(Clontech, Mountain View, CA, USA) ligated into pEntr-D-TOPO (Invitrogen). To generate
the R172H mutation, we used the Quick Change Mutagenesis Kit (Stratagene), with the
following primers: forward 5´-CGGAGGTCG-TGAGACACTGCCCCCACCATGA; reverse 5´-
TCATGGTGGGGGCAGTGTCTCACGACCTCCG. As a control we used pEntr-IRES-Puro.
The p53R172H-IRES-Puro and the control IRES-Puro cassettes were transferred into Gateway
compatible RCASBP(A) vectors (Seidler et al., 2008). Integrity of cloned sequences was
confirmed by sequencing (GATC, Konstanz, Germany).
Virus Preparation and Infection
RCAS viruses were generated, DF1 cells were transfected and virus titer was determined
exactly as recently described (Seidler et al., 2008). The TVA receptor-positive pancreatic
cancer cell line 6554 were transduced and individual clones were established using
puromycin selection (3 µg/mL).
Preparation of cell lysates, immunoblotting, ABCD assays, EMSA, microscopical
analyses, and ChIP
EMSAs with nuclear extracts, lysate preparations, Western blots, ABCD assays, and coimmunoprecipitations were carried out as described (Krämer et al., 2009; Krämer et al.,
2008; Schneider et al., 2006; Voelcker et al., 2008). Proteins recognized by these antibodies
were detected with Western blots generated with X-ray films (grey backgrounds) or with the
Odyssey Infrared Imaging System (LI-COR, Bad Homburg, Germany) using Alexa680- or
IRDeye800-coupled secondary antibodies (white backgrounds). EMSAs were analyzed with
phosphoimager or X-ray films. NF-B oligonucleotides were for ABCD: Bio 5′GGAATTTCCGGGAATTTCCGGGAATTTCCG-GGAATTTCCC;
5′-
GGGAAATTCCCGGAAATTCCCGGAAATTCCCGGAAATTCC (tandem repeat from the I-B
promotor) or irrelevant biotinylated sequences (mutated sequences from the FASL promoter
(Novac et al., 2006); for EMSA: 5’-GATCCAGAGGGGACTTTCCCACAGGA; 5’-GATCTCCTCTGGGAAAGTCCCCTCTG (from the Ig- chain promoter) (Göttlicher et al., 2001; Voelcker
et al., 2008; Yilmaz et al., 2003).
Antibodies were obtained from Santa Cruz Biotechnology (Heidelberg, Germany; sc-): p65,
p50, Rel B, p53 (sc-263 (α-human)/-6243 (a-human/mouse)), SIRT1, CBP, Caspase-3, p21,
MnSOD2, MCL1, BCL2, p-ATM, GAPDH; Sigma-Aldrich (Munich, Germany): Actin, Tubulin;
BD Pharmingen (Heidelberg, Germany): BCL-XL, HMG1, FASL; Novus Biologicals (Littleton,
CO; USA): Survivin; Upstate/Millipore (Billerica, MA, USA): HDAC1, p52; Merck
Calbiochem/EMD Biosciences (Darmstadt, Germany): p53 (OPO3). The Rel-A antibody for
supershift is described in (Voelcker et al., 2008; Yilmaz et al., 2003). For the p53 supershift 2
µl of a 1:1 mix of p53 antibodies (OPO3 and sc-6243) was used. Microscopical analyses
were carried out as described (Knauer et al., 2007; Krämer et al., 2009).
ChIP assays were performed as recently described (Schneider et al., 2006). An equal
amount of chromatin (50 – 100 µg) was used for each precipitation. Antibodies used were:
p53 (DO-1; sc-126), p65 (c-20; sc-372) and control IgG, all from Santa Cruz Biotechnology
(Heidelberg, Germany). One-twentieth of the precipitated chromatin was used for each PCR
reaction. To ensure linearity, 28 to 38 cycles were performed, and one representative result
is
shown.
Sequences
of
the
promoter
specific
primers
are:
GAPDH-FW
5’-
AGCTCAGGCCTCAAGACCTT, GAPDH-RV 5’-AAGAAGATGCGGCT-GACTGT, FasL-kBFW-396
5’-TACCCCCATGCTGACCTGCTC,
TGTTGCTGACTG, MnSOD-FW
FasL-kB-RV-76
5’-ACGGGACCC-
5’-CGGGGTTATGAAATTTGTTGAGTA, MnSOD-RV 5’-
CCACAA-GTAAAGGACTGAAATTAA.
Cell lines, transfections, luciferase assay, and Caspase-3/7 assays
Cells were cultured and transfected as described recently (Fritsche et al., 2009; Krämer et
al., 2009; Schneider et al., 2006). Luciferase assays were carried out as described
(Göttlicher et al., 2001) with 260-1070 ng NF-B-dependent reporter (Hoffman et al., 2002),
130 ng SV40-lacZ, 1.3 µL LFA per 12-well. The Survivin promoter was described recently
(Retzer-Lidl et al., 2007). Caspase-3/7 activity was assessed with Caspase-Glo 3/7
according to the manufacturer (Promega, Mannheim, Germany). Prof. Wang, Jena, provided
wild-type and p53-p65+ MEFs; Prof. Hoffmann, San Diego, provided wild-type and p53+p65MEFs.
Quantitative RT-PCR
qRT-PCRs were done as described in (Krämer et al., 2009). Primers sequences:
TRAF1_F ACTCTCACCAAATGAGAAGAAAATG;
AAGTCTTCAAATCCAACCC;
TRAF1_R ACTTAA
BCL-XL_F
ATCGCAGCTTGGATGGCCACTT;
BCL-XL_R CGGGTGCTGCATTGT
TCCCATA;
CXCL1_F: GCTGGGATTCACCTCAAGAA; CXCL1_R: TGGGGACACCTTTTAGCATC;
CXCL10_F: AAGTGCTGCCGTCATTTTCT; CXCL10_R: GTGGCAATGATCTCAACACG;
VCAM1_F: CCAAGCCATGCATTCAGACTT; VCAM1_R: CCTCGCGACGGCATAATTT;
-ACTIN_F: TGGCGCTTTTGACTCAGGA; -ACTIN_R: GGGAGGGTGAGGGACTTCC.
References: MATERIALS AND METHODS
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