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: 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