Journal: The Journal of Experimental Medicine

Article Title: Resistance to Granzyme B-mediated Cytochrome c Release in Bak-deficient Cells

doi:

Figure Lengend Snippet: Resistance of Bak-deficient Jurkat cells to GrB-mediated apoptosis. (A) and (B) Flow cytometry analysis of staining by annexin V of Jurkat cells treated with Ad (10 PFU/ml), GrB (1 μg/ml), and a combination of GrB and Ad for 2 h (A) or 24 h (B) at 30°C. The data are means ± SD of results obtained in five independent experiments. The asterisks indicate a statistically significant difference between wild-type and Bak-deficient cells ( P < 0.05, Mann-Whitney U ). (C) GrB-mediated cleavage of PARP and DFF45/ICAD in wild-type, but not in Bak-deficient cells. Wild-type and Bak-deficient cells were treated with Ad, GrB, or a combination of GrB and Ad, as described previously. The cell extracts were resolved on SDS/PAGE and immunoblotted with anti-PARP mAb or anti-DFF45/ICAD Ab. (D) and (E) Lack of mitochondrial apoptotic events in Bak-deficient Jurkat cells treated with GrB. After 2 h of treatment with GrB and Ad, as described previously, the cells were assessed by flow cytometry for mitochondrial staining with CMXRos or NAO. Staining with CMXRos (100 nM) served to assess changes in mitochondria permeability transition; staining with NAO (100 nM) served to assess loss in mitochondrial cardiolipin. (F) and (G) Susceptibility of Bak-deficient Jurkat cells to TRAIL or taxol. Wild-type or Bak-deficient Jurkat cells were treated with TRAIL (100 ng/ml) or taxol (10 μg/ml) for 16 h. The cells were then analyzed by flow cytometry for staining by annexin V or propidium iodide. Percentage of apoptotic cells are indicated for TRAIL.

Article Snippet: We also used anti-Bid Ab from BioVision and from Santa Cruz Biotechnology, Inc.; anticaspase-3 was from BD PharMingen; anti-poly-(ADP-ribose) polymerase (PARP) mAb (C2.10) and Cbz-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) were from Enzyme System; rabbit anti-DNA fragmentation factor (DFF)45/inhibitor of caspase-activate DNase (ICAD) Ab was from ProSci.

Techniques: Flow Cytometry, Staining, MANN-WHITNEY, SDS Page, Permeability

Journal: The Journal of biological chemistry

Article Title: Functional interaction of DFF35 and DFF45 with caspase-activated DNA fragmentation nuclease DFF40.

doi: 10.1074/jbc.274.30.20759

Figure Lengend Snippet: FIG. 2. Both RNA transcripts and proteins of DFF35/45 exist in vivo. A, DFF35 and DFF45 were amplified by human DFF35 (lanes 1 and 3) and DFF45 (lanes 2 and 4) out-encoding region primer sets in human fetal liver cDNA (lanes 1 and 2) or Jurkat cell line (lanes 3 and 4) using PCR. B, expression of DFF45 and DFF35 in Jurkat cells was immunoblotted by polyclonal Ab specific for N-terminal (lane 1), inter- mediate (lane 2), or C-terminal (lane 3), respectively. C, lysate of 293T cells transfected with DFF45 (lane1), DFF35 (lane 2), or an irrelevant protein p53 (lane 3) was immunoblotted by anti-Flag (M5) Ab (top panel), or anti DFF45/35 (bottom panel). Arrows indicate the trans- fected and endogenous DFF45 and DFF35.

Article Snippet: A polyclonal Ab against intermediate peptide of both DFF35 and DFF45 (KQEESKAAFGEEVDAVD) and a polyclonal Ab against C-terminal peptide of DFF45 only (KASPPGDLQNPKRARQDPT) were from Santa Cruz Biotechnology.

Techniques: In Vivo, Amplification, Expressing, Transfection

Journal: The Journal of biological chemistry

Article Title: Functional interaction of DFF35 and DFF45 with caspase-activated DNA fragmentation nuclease DFF40.

doi: 10.1074/jbc.274.30.20759

Figure Lengend Snippet: FIG. 1. Comparison of amino acid sequences between DFF35 and DFF45 (A) or mouse ICAD-S (B). Two putative cleavage sites for caspase-3 are indicated by double underlines. Three peptides (N, I, and C) used to generate polyclonal antibodies are indicated by single under- lines. Identical amino acids between DFF35 and mouse ICAD-S are in bold face.

Article Snippet: A polyclonal Ab against intermediate peptide of both DFF35 and DFF45 (KQEESKAAFGEEVDAVD) and a polyclonal Ab against C-terminal peptide of DFF45 only (KASPPGDLQNPKRARQDPT) were from Santa Cruz Biotechnology.

Techniques: Comparison

Journal: The Journal of biological chemistry

Article Title: Functional interaction of DFF35 and DFF45 with caspase-activated DNA fragmentation nuclease DFF40.

doi: 10.1074/jbc.274.30.20759

Figure Lengend Snippet: FIG. 3. Inhibitory effect of DFF35 in vitro and in vivo. A, Jurkat cells were treated with 1 mg/ml Fas monoclonal Ab 7C11 for 2 h, and 100 ng of cytosolic fraction without (lane 1) or with GST (lanes 2 and 3), GST-DFF35 (lanes 4 and 5), or double mutated GST-DFF35 (lanes 6 and 7) was added to Jurkat nuclei and incubated at 37 °C for 2 h in the presence (lanes 3, 5, and 7) or absence (lanes 1, 2, 4, and 6) of caspase-3, respectively. Jurkat cells were treated with 1 mg/ml of staurosporine for the indicated time, cell lysates were blotted with polyclonal Ab reactive to N-terminal peptide of DFF45 and DFF35 (B), and genomic DNA were extracted and subjected to analysis by 2% agarose gel electrophoresis (C).

Article Snippet: A polyclonal Ab against intermediate peptide of both DFF35 and DFF45 (KQEESKAAFGEEVDAVD) and a polyclonal Ab against C-terminal peptide of DFF45 only (KASPPGDLQNPKRARQDPT) were from Santa Cruz Biotechnology.

Techniques: In Vitro, In Vivo, Incubation, Agarose Gel Electrophoresis

Journal: Cell Death & Disease

Article Title: Induction of necrotic cell death by oxidative stress in retinal pigment epithelial cells

doi: 10.1038/cddis.2013.478

Figure Lengend Snippet: Lack of apoptotic hallmarks in ARPE-19 cells subjected to oxidative stress. ( a ) Light microscopy pictures of MTT crystals in ARPE-19 cells showing decrease in cell number and viability of ARPE-19 cells treated with 300 or 500 μ M of H 2 O 2 , or 150 μ M of tBHP. Test was performed at 24 h after inducing oxidative stress. ( b ) Analyses of DNA fragmentation in ARPE-19 cells treated with 300 or 500 μ M of H 2 O 2 , or 150 μ M of tBHP. DNA from UV-irradiated Hela cells was used as a positive control for apoptosis. DNA was purified from both dead and live cells 24 h after inducing cell death and analyzed by gel electrophoresis. Partial DNA fragmentation in ARPE-19 cells is visible as a smear in the agarose gel. ( c ) Western blotting detection of cleaved caspase-3 and PARP products as a result of cell death. 25 μ g of total cell extract was prepared from both dead and live cells 24 h after subjecting cells to oxidative stress (ARPE-19) or UV irradiation (Hela). GAPDH served as loading control. ( d ) Western blot measurement of DFF45 level in control ARPE-19 cells and cells subjected to oxidative stress. Of note, the DFF antibody recognizes both DFF45 and DFF35. Hela cells were used as reference. GAPDH served as loading control. ( e ) Analyses of ATP levels in ARPE cells subjected to oxidative stress. ATP level was measured by recording luminescence in indicated time points after treating cells with 300 or 500 μ M of H 2 O 2 , or 150 μ M of tBHP. UV-irradiated Hela cells were used as a control for apoptosis. * P <0.05; *** P <0.001

Article Snippet: Antibodies used include: rabbit polyclonal anti-caspase 1 (1 : 1000, Millipore, Billerica, MA, USA), rabbit polyclonal anti-caspase 3 (1 : 1000, Cell Signaling, Danvers, MA, USA), mouse monoclonal anti-cleaved PARP (Asp214; 1 : 1000, Cell Signaling), rabbit polyclonal anti-DFF45/DFF35 (1 : 1000, Cell Signaling), mouse monoclonal anti-GAPDH (1 : 2000, Millipore).

Techniques: Light Microscopy, Irradiation, Positive Control, Purification, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Western Blot, Control