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: Molecular Medicine Reports

Article Title: Polyphyllin VII induces apoptotic cell death via inhibition of the PI3K/Akt and NF-κB pathways in A549 human lung cancer cells

doi: 10.3892/mmr.2019.10879

Figure Lengend Snippet: Polyphyllin VII induces apoptosis of A549 cells via the PI3K/Akt and NF-κB pathways. A549 cells were treated with 0.41 µM Polyphyllin VII in the presence or absence of wortmannin and PDTC for 24 h. Protein expression levels were then evaluated by western blotting. GAPDH was used as a loading control for whole cell extract and lamin B was used as the loading control for nuclear extract. Densitometry results were expressed as average density relative to GAPDH or lamin B, or as a ratio of phosphorylated to total levels. Data are presented as mean ± standard deviation of 3 independent experiments. *P<0.05 and **P<0.01 vs. control group; # P<0.05 and ## P<0.01 vs. Polyphyllin VII-treated group. PDTC, pyrrolidine dithiocarbamate; p-, phosphorylated; PARP, poly-(ADP-ribose) polymerase; ICAD, inhibitor of caspase-activated DNase.

Article Snippet: Polyclonal antibodies against PI3K (cat. no. 20584-1-AP), Akt (cat. no. 10176-2-AP), NF-κB p65 (cat. no. 10745-1-AP), IκB (cat. no. 10268-1-AP), GAPDH (cat. no. 10494-1-AP), lamin B (cat. no. 12987-1-AP), caspase-3 (cat. no. 19677-1-AP), poly-(ADP-ribose) polymerase (PARP; cat. no. 13371-1-AP) and inhibitor of caspase-activated DNase (ICAD; cat. no. 10191-2-AP) were obtained from ProteinTech Group, Inc. Phosphorylated (p)-PI3K (cat. no. 4228), p-Akt (cat. no. 4060) and p-NF-κB p65 (cat. no. 3033) antibodies were from Cell Signaling Technology, Inc. Horseradish peroxidase (HRP)-conjugated secondary antibodies (whole IgG affinity-purified antibodies, cat. no. 115-035-003) were obtained from Jackson ImmunoResearch Laboratories, Inc.

Techniques: Expressing, Western Blot, Standard Deviation