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3), and revealed by immunoperoxidase staining. For fluorescent ganglioside studies, cells were incubated for 30 min at 37°C with pyrene-labeled GM3, then stimulated. Gangliosides were then extraded and chromatographed on HPTLC plates. Pyrene-conjugated gangliosides were visualized with an ultraviolet (UV) lamp at 254 nm.
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21
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15144356364
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note
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Cell viability was evaluated by ethidium bromide-acridine orange staining and fluorescence microscopy. Hypodiploid nuclei were evaluated by hypotonic propidium iodide solution staining and flow cytometric analysis, with the use ot a FACScan (Becton Dickinson; San Jose, CA). Internucleosomal DNA fragmentation was analyzed by DNA electrophoresis.
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The GD3 synthase cDNA was cloned in the PUC vector, and a 1152-base pair (bp) Apa I fragment was further cloned in the pEGFP-C3 expression vector (Clontek; Palo Alto, CA). The ΔBH deletion mutant was generated by removing the COOH-terminal portion of the enzyme with Bam HI. The 514-bp Bam HI-Apa I fragment was then cloned in pEGFP-C3. HuT78 cells were electroporated (Gene Pulser, Bio-Rad; Hercules, CA), and viable cells were recovered by lymphoprep density gradient centrifugation. After 24 hours of culture, cells were analyzed by fluorescence microscopy. Transfection efficiency was typically 20 to 25% as measured by fluorescence emission.
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26
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15144354360
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note
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Coupled transcription and translation of PARP, cloned into the pGEM plasmid under the T7 promoter, was performed with the TNT kit (Promega, Madison, WI). Each reaction was carried out for 2 hours at 37°C, then samples were resolved by 10% SDS-polyacrylamide gel electrophoresis and autoradiographed. Caspase inhibitors Ac-YVAD-CMK (Ac-Tyr-Val-Ala-Asp-chlorometylketone), Ac-DEVD-CHO (Ac-Asp-Glu-Val-Asp-CHO), and ZVAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-fluorometyl-ketone) were from Bachern (Bubendorf, Switzerland).
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29
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0029947822
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Eugene, OR
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m were therefore evaluated by the shift in fluorescence emission.
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15144347265
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note
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PDMP was from Matreya (Pleasant Gap, PA). The GD3 synthase antisense oligodeoxynucleotide sequence (5′-CAGTACAGCCATGGCCCCTCT-3′) was selected on the 5′ region of the gene surrounding the second initiator ATG. A scrambled sequence from the same deoxynucleotides (5′-CGACCTACCTATGCGCTACCG-3′) was also used as a control. To increase nuclease resistence, both oligodeoxynucleotide sequences were phosphorothioatemodified on every second deoxynucleotide.
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15144359340
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note
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We thank T. Nishi (Tokyo Research Laboratories, Japan); L. J. Old (Ludwig Institute, New York); R. Beyaert (University of Ghent, Belgium) for kindly providing reagents; and V. Zacchei for expert assistance. Supported by grants to R.T. from Associazione Italiana Ricerca sul Cancro (AIRC), Consiglio Nazionale delle Ricerche (CNR), Ministero dell'Universitá e della Ricerca Scientifica e Tecnologica (MURST), and the European Community Biomed 2 and Human Caprtal and Mobility Programs. R.D.M. is an AIRC fellowship holder.
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