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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 37 0C, according to the
manufacturers 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.
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 manufacturers 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.
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.
References
Di Stefano V, Rinaldo C, Sacchi A, Soddu S, DOrazi G. (2004). Homeodomain-interacting protein
kinase-2 activity and p53 phosphorylation are critical events for cisplatin-mediated apoptosis.
Exp. Cell. Res., 293, 311-320
DOrazi G, Cecchinelli B, Bruno T, Manni I, Higashimoto Y, Saito S, Gostissa M, Coen S,
Marchetti A, Del Sal G, Piaggio G, Fanciulli M, and Soddu S. (2002). Homeodomaininteracting protein kinase-2 phosphorylates p53 at Ser46 and mediates apoptosis. Nature Cell
Biol., 4, 11-19
Di Stefano V, Soddu S, Sacchi A, DOrazi G. (2005a). HIPK2 contributes to PCAF-mediated p53
acetylation and selective transactivation of p21Waf1 after non-apoptotic DNA damage.
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Di Stefano V, Mattiussi M, Sacchi A, DOrazi G. (2005b). HIPK2 inhibits both MDM2 gene and
protein by, respectively, p53-dependent and -independent regulations. FEBS Letters, 579: 547380.
Wang S and El-Deiry W (2003). Requirement of p53 targets in chemosemsitization of colonic
carcinoma to death ligand therapy. P.N.A.S., 100: 15095-150100.