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: The Journal of Biological Chemistry

Article Title: Histamine Induces ATP Release from Human Subcutaneous Fibroblasts, via Pannexin-1 Hemichannels, Leading to Ca 2+ Mobilization and Cell Proliferation *

doi: 10.1074/jbc.M113.460865

Figure Lengend Snippet: Histamine-induced [Ca2+]i mobilization depends on pannexin-1 hemichannel activity in cultured human subcutaneous fibroblasts. The effect of Hist (100 μm) was tested in the presence of inhibitors of connexin- and/or pannexin-containing hemichannels, namely MFQ (3 μm) (A, i), 2-octanol (1 mm) (A, ii), CBX (300 μm) (A, iii), 10Panx (100 μm) (A, iv), probenecid (150 μm) (A, v), and H1152 (3 μm) (A, vi) as well as upon blocking vesicle exocytosis with BFA (20 μm) (C). Cells were preincubated with the fluorescent calcium indicator Fluo-4 NW (see “Experimental Procedures”); [Ca2+]i transients were calibrated to the maximal calcium load produced by ionomycin (5 μm; 100% response). Black arrows, time of drug application. None of the inhibitors significantly changed base-line fluorescence when applied alone. Each point represents pooled data from n experiments. Vertical bars, S.E., shown when they exceed the symbols in size. *, p < 0.05, significant differences from Hist (100 μm) alone. B, representative confocal micrographs and blots of Cx43 and Panx1 hemichannel immunoreactivity in cultured human subcutaneous fibroblasts from several distinct individuals (a–g). β-Tubulin was used as a reference protein. Image scale bars, 60 μm.

Article Snippet: Membranes were subsequently incubated with goat anti-human P2Y 1 (1:200) (Santa Cruz Biotechnology, Inc.), rabbit anti-human P2Y 12 (1:200) (Alomone), rabbit anti-human P2Y 13 (1:200) (Alomone), rabbit anti-human Panx1 (1:250) (Novex, Invitrogen), and rabbit anti-human Cx43 (1:6000) (Abcam) in the above blocking buffer overnight at 4 °C.

Techniques: Activity Assay, Cell Culture, Blocking Assay, Produced, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Histamine Induces ATP Release from Human Subcutaneous Fibroblasts, via Pannexin-1 Hemichannels, Leading to Ca 2+ Mobilization and Cell Proliferation *

doi: 10.1074/jbc.M113.460865

Figure Lengend Snippet: Histamine-induced single-cell [Ca2+]i oscillations and To-Pro3 dye uptake by cultured fibroblasts of the human subcutaneous tissue evaluated by time-lapse fluorescence confocal microscopy. A, fluorescence variation in cells preloaded with the fluorescent calcium indicator Fluo-4 NW (see “Experimental Procedures”); To-Pro3 was added to the superfusion fluid 6 min before Hist (100 μm) (dashed vertical line). Results are in arbitrary units (a.u.) of fluorescence; individual [Ca2+]i transients were calibrated to the corresponding maximal calcium load produced by ionomycin (5 μm; 100% response) in the same cell. B, bars represent the effect of Hist (100 μm) in the absence and in the presence of pannexin-1 hemichannel permeability blockers, CBX (300 μm) and H1152 (3 μm). Vertical bars, S.E. from n experiments. *, p < 0.05, significant differences from control values obtained in the absence of tested drugs; **, p < 0.05, significant differences compared with the effect of Hist (100 μm) alone.

Article Snippet: Membranes were subsequently incubated with goat anti-human P2Y 1 (1:200) (Santa Cruz Biotechnology, Inc.), rabbit anti-human P2Y 12 (1:200) (Alomone), rabbit anti-human P2Y 13 (1:200) (Alomone), rabbit anti-human Panx1 (1:250) (Novex, Invitrogen), and rabbit anti-human Cx43 (1:6000) (Abcam) in the above blocking buffer overnight at 4 °C.

Techniques: Cell Culture, Fluorescence, Confocal Microscopy, Produced, Permeability