Journal: Molecular Medicine

Article Title: Apoptosis in Caspase-inhibited Neurons

doi: 10.1007/bf03401837

Figure Lengend Snippet: Fig. 1. Caspase-inhibited neurons undergo delayed chromatin condensation and cell death. (A) Neurons were treated with colchicine (1 M) in the presence or absence of N- benzyloxycarbonyl-Val-Ala-aspartyl-fluoromethyl ketone (zVAD- fmk; 100 M), or with zVAD-fmk alone. At the indicated time points, nuclei were stained with H-33342. Then, phase contrast and chromatin fluorescence were imaged simultaneously by confocal microscopy (63, NA 1.32 lens). The width of the scale bar corresponds to 40 m. Cultures from wild-type mice (wt) or Bcl-2-overexpressing mice (Bcl-2) were treated with colchicine alone (B) or with colchicine plus zVAD-fmk (C). At the time points indicated, cultures were stained with H-33342, and the percentage of nuclei with condensed chromatin was counted. Data are means standard deviation (SD) from seven determinations.

Article Snippet: Pefabloc and the caspase substrates Ac-Asp-Glu-Val-aspartyl(DEVD)-aminotrifluoromethyl coumarine (-afc) came from Biomol (Hamburg, Germany) Ac-Val-Asp-ValAla-aspartyl-(VDVAD)-afc from California Peptide Research (Napa, CA), and ( )-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK801) came from RBI (Biotrend Chemi kalien GmbH, Köln, Germany).

Techniques: Staining, Confocal Microscopy, Standard Deviation

Journal: Molecular Medicine

Article Title: Apoptosis in Caspase-inhibited Neurons

doi: 10.1007/bf03401837

Figure Lengend Snippet: Fig. 2. Caspase inhibitor zVAD-fmk is stable and prevents caspase activity. Medium alone (A) or neuronal cultures (B) were supplemented with N-benzyloxycarbonyl-Val-Ala- aspartyl-fluoromethyl ketone (zVAD-fmk; 100 M). After the indicated time points, supernatant was removed and incubated in different dilutions with recombinant caspase-3 (30 ng/ml). Then, caspase-3 activity was measured by Ac-Asp-Glu-Val- aspartyl-aminotrifluoromethyl coumarin (DEVD-afc) cleavage. Data are means from five determinations. Error bars are smaller than the data symbols. (C) Enzymatic activities of recombinant caspase-2 and caspase-3 (0.6 g/ml) were measured with the

Article Snippet: Pefabloc and the caspase substrates Ac-Asp-Glu-Val-aspartyl(DEVD)-aminotrifluoromethyl coumarine (-afc) came from Biomol (Hamburg, Germany) Ac-Val-Asp-ValAla-aspartyl-(VDVAD)-afc from California Peptide Research (Napa, CA), and ( )-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK801) came from RBI (Biotrend Chemi kalien GmbH, Köln, Germany).

Techniques: Activity Assay, Incubation, Recombinant

Journal: Molecular Medicine

Article Title: Apoptosis in Caspase-inhibited Neurons

doi: 10.1007/bf03401837

Figure Lengend Snippet: Fig. 3. Nuclei of caspase-inhibited neurons display apop- totic morphological changes. Neurons were treated with colchicine (1 M) in the absence (left panel) or presence (right panel) of N-benzyloxycarbonyl-Val-Ala-aspartyl-fluoromethyl ketone (zVAD-fmk; 100 M). At different time points, cultures were fixed with 2.5% glutaraldehyde for transmission electron microscopy analysis. In colchicine-treated neurons, chromatin condensed to crescent- and sphere-shaped structures. Early stages (18 hr) of chromatin condensation in caspase-inhibited neurons were characterized by compaction of DNA under the nuclear envelope. At later time points (24 hr), lumpy, highly condensed chromatin structures were formed. The width of scale bars corresponds to2 m.

Article Snippet: Pefabloc and the caspase substrates Ac-Asp-Glu-Val-aspartyl(DEVD)-aminotrifluoromethyl coumarine (-afc) came from Biomol (Hamburg, Germany) Ac-Val-Asp-ValAla-aspartyl-(VDVAD)-afc from California Peptide Research (Napa, CA), and ( )-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK801) came from RBI (Biotrend Chemi kalien GmbH, Köln, Germany).

Techniques: Transmission Assay, Electron Microscopy

Journal: Molecular Medicine

Article Title: Apoptosis in Caspase-inhibited Neurons

doi: 10.1007/bf03401837

Figure Lengend Snippet: Fig. 4. Caspase-inhibited neurons display chromatin degradation. Neurons were incubated with colchicine (1 M) in the presence or absence of N-benzyloxycarbonyl- Val-Ala-aspartyl-fluoromethyl ketone (zVAD-fmk; 100 M) or with zVAD-fmk alone. After different time points, the percentage of neurons retaining plasma membrane integrity was determined by counting calcein acetoxymethyl ester (calcein-AM)-positive cells. Then, DNA fragmentation was analyzed by field-inversion gel electrophoresis. The arrow indicates high molecular weight DNA fragments of 50 kbp.

Article Snippet: Pefabloc and the caspase substrates Ac-Asp-Glu-Val-aspartyl(DEVD)-aminotrifluoromethyl coumarine (-afc) came from Biomol (Hamburg, Germany) Ac-Val-Asp-ValAla-aspartyl-(VDVAD)-afc from California Peptide Research (Napa, CA), and ( )-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK801) came from RBI (Biotrend Chemi kalien GmbH, Köln, Germany).

Techniques: Incubation, Clinical Proteomics, Membrane, Nucleic Acid Electrophoresis, High Molecular Weight

Journal: Molecular Medicine

Article Title: Apoptosis in Caspase-inhibited Neurons

doi: 10.1007/bf03401837

Figure Lengend Snippet: Fig. 5. Caspase-inhibited neurons display PS-translocation. Neurons were incubated with colchicine (1 M) and N-benzy- loxycarbonyl-Val-Ala-aspartyl-fluoromethyl ketone (zVAD-fmk; 100 M) for 24 hr and stained with fluorescent annexin V [phosphatidylserine (PS)-translocation], calcein acetoxymethyl ester (calcein-AM; plasma membrane integrity) and H-33342 (chromatin structure). Fluorescence images were collected by confocal microscopy (63, NA 1.32 lens). Top: xy-section through the cell bodies. Arrows indicate condensed nuclei and an asterisk indicates an annexin V-positive neuron. The width of the scale bar corresponds to 10 m. Middle: xy-section through the annexin V-positive neuron in higher magnification. Bottom: transversal section (xz-plane) demonstrates that the entire sur- face of the neuron was surface stained with annexin V. The width of the scale bar corresponds to 9 m. Data are representa- tive of five experiments in three independent cell preparations.

Article Snippet: Pefabloc and the caspase substrates Ac-Asp-Glu-Val-aspartyl(DEVD)-aminotrifluoromethyl coumarine (-afc) came from Biomol (Hamburg, Germany) Ac-Val-Asp-ValAla-aspartyl-(VDVAD)-afc from California Peptide Research (Napa, CA), and ( )-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK801) came from RBI (Biotrend Chemi kalien GmbH, Köln, Germany).

Techniques: Translocation Assay, Incubation, Staining, Clinical Proteomics, Membrane, Fluorescence, Confocal Microscopy

Journal: Molecular Medicine

Article Title: Apoptosis in Caspase-inhibited Neurons

doi: 10.1007/bf03401837

Figure Lengend Snippet: Fig. 7. Mitochondrial cytochrome c release is not pre- vented by caspase inhibition. Cytochrome c release into the cytosol (the cytosolic fraction is indicated by an S for super- natant) and the cytochrome c retained in the organelle fraction (indicated as P for pellet) were determined by Western blots. As control for maximal cyto-chrome c release, neurons were treated with 0.3% Triton X-100. CHX, cycloheximide; zVAD-fmk, N- benzyloxycarbonyl-Val-Ala-aspartyl-fluoromethyl ketone; cyt c, cytochrome c.

Article Snippet: Pefabloc and the caspase substrates Ac-Asp-Glu-Val-aspartyl(DEVD)-aminotrifluoromethyl coumarine (-afc) came from Biomol (Hamburg, Germany) Ac-Val-Asp-ValAla-aspartyl-(VDVAD)-afc from California Peptide Research (Napa, CA), and ( )-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK801) came from RBI (Biotrend Chemi kalien GmbH, Köln, Germany).

Techniques: Inhibition, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Dietary Apigenin Exerts Immune-Regulatory Activity in Vivo by Reducing NF-κB Activity, Halting Leukocyte Infiltration and Restoring Normal Metabolic Function

doi: 10.3390/ijms17030323

Figure Lengend Snippet: Apigenin reduces LPS-induced apoptosis in the lungs. Lungs from mice treated with LPS, control, apigenin (Api) or apigenin 3 h prior to LPS (Api + LPS) were obtained at 24 h. ( A ) The number of apoptotic cells was determined by the TUNEL assay and expressed as the mean ± SEM of the numbers of stained cells per 1000× microscopic field (cpf) ( N = 4 mice for each biological condition; * p < 0.01 vs. LPS), scale bars: 25 μm for all figures; ( B , C ) Caspase-3 activity was determined by the DEVD-AFC assay using lung lysates as described in materials and methods. Values represent the means ± SEM ( N = 4 mice for each biological condition; * p < 0.05 vs. LPS); ( D ) Lung tissue immuno-histochemistry using antibodies specific for active caspase-3 in combination with anti-CD31, anti-cytokeratin or anti-7/4 antibodies specific for epithelial, endothelial and neutrophils, respectively. Scale bars, 100 μm.

Article Snippet: Protein lysates were incubated in a cytobuffer containing 20 µM DEVD-AFC (tetrapeptide Ac-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin flurogenic caspase-3 substrate from MP Biomedicals, Santa Ana, CA, USA) to determine caspase-3 activity, as previously described [ ].

Techniques: TUNEL Assay, Staining, Activity Assay, Immunohistochemistry