Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: Induction of apoptosis in 70Z/3 cells treated with 3CPA at 250 μ M and 500 μ M . ( A ) Cytochrome c release into the cytosol after exposure to 3CPA for indicated time periods measured by Western blot analysis. ( B ) Pro-caspase-9 processing determined in cytosolic fractions after exposure to 3CPA. Actin was used as an internal standard of the protein load and the results shown are representatives of two separate experiments. ( C ) Caspase inhibitors reduce 3CPA-induced apoptosis in 70Z/3 cells. Apoptosis, measured as AnnexinV + 7AAD − cells, was determined after exposure to 3CPA at 250 μ M (left) and 500 μ M (right) for 24 h. The pan-caspase inhibitor ZVAD-fmk (50 μ M , n =4), the caspase-9 inhibitor LEHD-fmk (50 μ M , n =3), or the caspase-8 inhibitor IETD-fmk (50 μ M , n =3) were added 1 h before treatment with 3CPA. The level of apoptosis is expressed relative to the level in cell cultures treated with the inhibitor only. Results show mean±s.d., and (*) indicates a statistical difference compared to 3CPA only (paired t -test, P <0.05).

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Western Blot

Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: Over-expression of Bcl-2 in 70Z/3 cells inhibits 3CPA-induced apoptosis. ( A ) FACS-analysis of 70Z/3 (upper panel) and 70Z/3 Bcl-2+ (lower panel) cells after exposure to 3CPA (500 μ M ) for 18 h. 7AAD negative (viable) cells were gated (gate A) and analysed for Annexin V + expression (% of gated cells). ( B ) Levels of Bcl-2 and cytochrome c expression were determined by Western blot analysis in mitochondrial enriched fractions after 18 h incubation with 3CPA at the indicated concentrations (30 μg protein per lane). ( C ) Pro-caspase-9 processing into active caspase-9 was determined in the cytosolic fraction after 18 h incubation with 3CPA at the indicated concentrations. Actin was used as an internal standard to control the amount of cytosolic protein loaded in each lane. Results shown are representatives of two independent experiments.

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Over Expression, Expressing, Western Blot, Incubation

Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: N-substituted benzamides induce cytochrome c release and activation of caspase-9 in HL60 cells. ( A ) Cytochrome c release induced by MCA (500 μ M ) and 3CPA (250 μ M ) in HL60 cells after 24 and 48 h, measured by Western blot analysis of mitochondria or cytosol fractions (30 μg protein/lane). Cells were incubated with etoposide for 6 and 18 h at 100 μ M as a positive control. ( B ) Activation of caspase-9 after treatment of HL60 cells with 3CPA at 250 and 500 μ M for 12 and 18 h (48 μg protein per lane). One representative experiment out of two is shown.

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Activation Assay, Western Blot, Incubation, Positive Control

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: Mechanism for Pre-B Cell Loss in V H -Mutant Rabbits

doi: 10.4049/jimmunol.1101778

Figure Lengend Snippet: Flow cytometric analysis of ali/ali and wt B-lineage cell survival 2–6 d after transfer of pro-B cells to OP9 stroma in the absence of IL-7. A, Relative number of B-lineage (CD79a+) cells normalized to ali/ali cultures using bead-based counting and gating strategy in Fig. 2A. Error bars show SD from three independent experiments. B, Live/Dead staining of total lymphocytes. C, Cross-reactivity of anti–caspase-3 Ab with a UV-irradiated rabbit B cell line (55D1). Left panel, UV-treated 55D1 B cells immediately after irradiation (t0) (gray filled) or 3 h postirradiation stained with anti–caspase-3 (black dashed line) or isotype control for 3 h time point (solid black line) (identical result was observed for isotype control staining at t0); number is percentage of caspase-3+ cells at 3 h. Right panel, CD79a expression in caspase-3+ (dashed line) and caspase-3neg (solid line) cells 3 h postirradiation. D, Frequency of active caspase-3+ B-lineage cells (CD79a+). Data for B and D are representative of three independent experiments.

Article Snippet: Abs and flow cytometry Abs used: mouse anti-human CD79a-PE (catalog number 555935) or APC (catalog number 551134), rabbit anti-human caspase-3–FITC (catalog number 559341), mouse anti-human spleen tyrosine kinase (Syk)-P-Y348 (I1s0-722), mouse anti-rabbit–μ (367.2-bio), and rat anti-mouse pre-BCR (SL-156-bio) from BD Pharmingen (San Diego, CA); rat anti-mouse–μ-PE and PE-Cy7 (II/41) from eBioscience (San Diego, CA); goat F(ab′) 2 anti-human IgH+L, rabbit F(ab)-FITC anti-goat IgG H+L, and allophycocyanin-conjugated streptavidin from Jackson ImmunoResearch Laboratories (West Grove, PA); and biotinylated rabbit anti–a2-allotype and FITC-rabbit anti–a2-allotype IgG (generated by K.L.K.’s laboratory).

Techniques: Staining, Irradiation, Expressing