Lazebnik for the anti-CARD antibody, and D

Lazebnik for the anti-CARD antibody, and D. how MIHA functions to inhibit it, we analyzed their interactions with each other and with caspases in NT2 cells exposed to apoptotic stimuli and in a yeast system in which the Apaf-1, caspase 9, and caspase 3 pathway had been reconstituted. To inhibit apoptosis, MIHA functioned downstream of mitochondrial release of cytochrome and DIABLO. MIHA was able to inhibit caspase 9 and caspase 3 processing after UV radiation of NT2 cells and was able to bind SLx-2119 (KD025) to both processed caspase 3 and processed caspase 9, indicating it functioned mainly at the level of processed caspase 9. The pattern of caspase activation in mammalian cells and in yeast revealed that caspase 3 and caspase 9 are involved in a feedback amplification loop that can be inhibited by MIHA. When expressed at sufficient levels, DIABLO was able to promote caspase activation by disrupting the interaction between MIHA and processed caspase 9, thereby leading to apoptosis. Materials and Methods Cell Culture and Transfection 293T cells and NT2 cells (a kind gift from D. Nicholson, Merke-Frosst, Quebec, Canada) were maintained in DME supplemented with 10% FCS. Cells were transfected using Effectene (QIAGEN) following the manufacturer’s protocol. For experiments in 6-well plates (Nunc), cells were seeded at a density of 105 cells per well 24 h before transfection. A total of 0.2 g DNA was used for transfections in 6-well plates along with 2.5 l of transfection reagent. For experiments in 100-mm plates, 2C3 106 cells per plate were seeded 24 h before transfection, and a total of 1C2 g of DNA was used along with 10 l (293T cells) or 12 l (NT2 cells) of Effectene. Stable cell lines were SLx-2119 (KD025) made by transfecting NT2 cells with 2 g of DNA linearized by digestion with Fsp1 (New England Biolabs, Inc.). 2 d after transfection, cells were split into four separate GTBP plates and puromycin (8 g/ml) added to the media. Several clones from each individual plate were selected and tested for expression of protein by flow cytometry and by Western blot. Mammalian Cell Constructs NH2-terminally Flag-tagged MIHA was cloned into the pEF vector (Uren et al. 1996; Huang et al. 1997). Bcl-2, LacZ, and caspase 9 were also cloned into the pEF vector but were not Flag-tagged. Untagged DIABLO was expressed in the pEF vector as described previously (Verhagen et al. 2000). Caspase 9 was cloned by PCR from an HeLa cDNA library using the following primers: 5-CGGGGATCCCCATGGACGAAGCGGATCGGCGGC -3 and 5-GCTCTAGATTAGCTAGCTGATGTTTTAAAGAAAAGTTTTTTCC-3. The PCR product was digested with BamH1 and Xba1 and subcloned into the pEF vector. The caspase recruitment domain (CARD) of caspase 9 was PCR amplified with primers 5- CG GGA TCC ACC ATG TCC GGT GCT CTT GAG AGT TTG A-3 and 5-GC TCTAGA TTA GCTAGC TGA TGT TTT AAA GAA AAG TTT TTTCC-3, cut with BamHI and XbaI, and subcloned into the pEF vector. Yeast Constructs Yeast vectors driving expression from either the constitutive ADH promoter or SLx-2119 (KD025) the inducible Gal1/10 promoter based on the pRS31X series of yeast vectors (Sikorski and Hieter 1989) have been described previously (Hawkins et al. 1999, Hawkins et al. 2000; Wang et al. 1999). pGALL-(URA), was generated by swapping the PvuI fragment of pGALL-(TRP1) (Hawkins et al. 1999) containing the TRP-1 gene with that of pRS316 containing the URA selection gene. Various coding sequences were inserted into these vectors as detailed below. Plasmids expressing Apaf-1&1C530, caspase 9, and p35 have SLx-2119 (KD025) been described previously (Hawkins et al. 1999; Wang et al. 1999). The coding region of MIHA was excised from pGALL-(? 2). The times for the doses used were: 10 J/m2 = 11 s, 25 J/m2 = 26 s, 50 J/m2 = 55 s, and 100 J/m2 = 111 s. 6 h after UV radiation, the cells were treated with trypsin, removed from the plate, washed in DME supplemented with 10 mM CaCl2, and incubated with FITC coupled or biotinylated annexin V (a kind gift from D. Huang and A. Strasser, The Walter and Eliza Hall Institute) at a dilution of 1 1:100 in 100 l of DME with CaCl2 and 5% FCS, for 20 min.