Pistritto et al.
Supplemental Materials and Methods
Cell cultures and treatments
Human ovarian cancer 2008 cell line carrying endogenous wtp53 (kindly provided by Dr. S.B.
Howell, University of San Diego, La Jolla, CA, USA) and H1299 (p53 null) lung carcinoma cell
line were cultured in RPMI-1640 (GIBCO-BRL, Life Technology, Grand Island, NY)
supplemented with 10 % heat-inactivated fetal bovine serum (FBS, GIBCO-BRL) plus glutamine
and antibiotics in humidified atmosphere with 5 % CO2 at 370C.
For drug treatment, subconfluent cells were treated for 8 and 24 h with adriamycin (ADR) diluted
into the medium to a final concentration of 2 g/ml.
Irreversible caspase inhibitors zVADfmk (for all caspases) and zIETDfmk (for caspase-8)
(Calbiochem) were diluted in DMSO, stored at –200, and used at a final concentration of 40 M for
16 h.
TUNEL assay
For TUNEL assay, 4x104 cells were spun on a slide by cytocentrifugation and subsequently fixed in
4 % paraformaldehyde for 30 min at room temperature. After rinsing with PBS the samples were
permeabilized in a solution of 0.1 % Triton X-100 in sodium citrate for 2 min. Samples, washed
with PBS, were then incubated in the TUNEL reaction mix for 1 h at 370C, according to the
manufacturer’s instructions (Roche, Germany). Cells were counter-stained with Hoechst 33342
before analysis with a fluorescent microscope (Zeiss). Standard deviations of three independent
experiments were indicated.
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Caspase activity
After treatments, both adherent and floating cells were collected by centrifugation at 1100 rpm for
10 min. After washing with PBS, the cell pellets were lysed in ice-cold lysis buffer provided by the
caspase assay kits (R&D Systems, Minneapolis, MN, USA). The lysates were centrifuged for 20
min at 14,000 x g at 40C. The resulting supernatants were analyzed for protein concentration by
Lowry method for normalization of assay results on protein basis. The caspase fluorometric
enzymatic activity assays were carried out following manufacturer’s instructions. The fluorogenic
substrates were DEVD-amino-4-trifluromethyl coumarin (AFC) (caspase-3) and IETD-AFC
(caspase-8) (Valter Occhiena, Torino, Italy).
Data are presented as representative of three independent experiments. Standard deviations were
indicated.
Western blot, antibodies
Cells were washed with ice-cold phosphate-buffered saline (PBS), collected by scraping, spun
down, rinsed with PBS, and lysed for 20 min on ice in Chaps Cell Extract Buffer (50 mM
Pipes/NaOH pH 6.5; 2 mM EDTA; 0.1 % Chaps; 5 mM DTT) plus protease inhibitors.
Resuspended cells in the buffer were subjected to freeze and thaw for three times before centrifuge
them at 14.000 rpm to pellet cell debris. Equal amount of proteins were mixed with SDS sample
buffer (62.5 mM Tris-HCl pH 6.8; 2 % w/v SDS; 10 % glycerol; 50 mM DTT; 0.01 % w/v
bromophenol blue) resolved in 12 % sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and
transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA). Western blot analysis was
performed with an enhanced chemiluminescence detection system (Amersham Corp., Arlington
Heights, IL) in accordance with the manufacturer’s instructions.
The antibodies used were: rabbit polyclonal anti-caspase-3, mouse monoclonal anti-caspase-8,
and mouse monoclonal anti-PARP antibodies (all form BD Pharmingen), following the
manufacturer’s instructions.
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Transfection and plasmids
Transient transfection were performed using the modified calcium phosphate BES methods as
described (D’Orazi et al., 2002). The amount of plasmid DNA in each sample was equalized by
supplementing with empty plasmid. Expression vectors used in this study were: pSUPER and
pSUPER-HIPK2 (Di Stefano et al., 2004), HIPK2-Flag and K221R-Flag (D’Orazi et al., 2002),
pCAG3.1wtp53, p53S46A (kindly provided by DR. E. Appella, NIH, Bethesda, MD, USA), and
DN-caspase-8 (kindly provided by Dr. Valerie Castle, University of Michigan, MI, USA).
siRNA interference and Reverse Transcriptase-PCR (RT-PCR)
For siRNA transfection, 2008 cells were plated at semiconfluence in 35 mm dishes the day before
transfection. si-Control, si-DR5 (Dharmacon) (Wang and El-Deiry, 2003), and si-Noxa
(Dharmacon) were transfected overnight using Lifectamine Plus method (Invitrogen) following the
manufacturer’s instructions and 24 h later cells were transfected with HIPK2 and K221R expression
vectors. RNA was isolated 48 after siRNA transfection by using the RNeasy mini kit (Qiagen
S.P.A., Milano, Italy). cDNA and PCR were performed essentially as previously described (Di
Stefano et al., 2005). Total RNA (2 g) were reverse transcribed using MuLV reverse transcriptase
and the reverse transcribed material was used in PCR reactions with the AmpliTaq DNA
polymerase (Gene Amp RNA PCR kit, Perkin Elmer, Roche mMlecular System, Brachburg, NJ,
USA). The GAPDH and p21Waf1 primers were described elsewhere (Di Stefano et al., 2004, 2005).
The following primers were used to amplify:
KILLER/DR5
Forw 5’-GATTGTACACCCTGGAGTGACATCG-3’
Rev 5’-CCACAGTAAAGACTTGCAAACAAACAC-3’;
Noxa
Forw 5’-AGGACTGTTCGTGTTCAGCTC-3’
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Rev 5’-GTCCACCTCCTGAGAAAACTC-3’;
Fas/Apo1
Forw 5’-ATTTCTGCCACTGCAGCCCTCAGG-3’
Rev 5’-TCCAGTTCGCTGGGCAGACTTCTC-3’;
p53AIP1
Forw 5’-CCAAGTTCTCTGCTTTC-3’
Rev 5’-AGCTGAGCTCAAATGCTGAC-3’.
PCR products were separated on 1.5 % agarose gels and visualized by ethidium bromide staining.
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