Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Role of Apoptotic Proteins in REC-2006 Mediated Radiation Protection in Hepatoma Cell Lines

doi: 10.1093/ecam/neq059

Figure Lengend Snippet: Effect of optimal concentration of drug (REC-2006), that is, 10 −5 μ g mL −1 given 2 h prior to irradiation (LD 80 of respective cell lines) on expression of (a) ICAD; (b) cytoplasmic CAD; (c) nuclear CAD in HepG2 and Hep3B cell lines. Asterisk indicates significant radiomodulatory activity (increase/decrease in expression) in HepG2 cell lines, whereas double asterisks indicate the corresponding effect in Hep3B cell lines with respect to radiation (LD 80 ) controls.

Article Snippet: Mouse monoclonal antibodies for the detection of human p53, ATM, PARP-1, AIF, cytochrome c , caspase-3, caspase-9, ICAD, CAD and β -actin proteins and alkaline phosphate-conjugated secondary anti-mouse antibodies were procured from Santa Cruz Biotech (Santa Cruz, CA, USA).

Techniques: Concentration Assay, Irradiation, Expressing, Activity Assay

Journal: Molecular Biology of the Cell

Article Title: Cell survival, DNA damage, and oncogenic transformation after a transient and reversible apoptotic response

doi: 10.1091/mbc.E11-11-0926

Figure Lengend Snippet: Damage of DNA in dying cells before reversal of apoptosis. (A) Fluorescence micrographs showing the subcellular localization of AIF (green), EndoG (red), and nuclei (blue) in primary liver cells that were untreated (Untreated) or treated with 4.5% ethanol for 5 h (Treated) and treated cells that were then cultured for 24 h in fresh medium after removal of the ethanol (Washed) from cells. Quantification of the corresponding fluorescence signals of AIF, EndoG, and nucleus along the dotted line as indicated in their respective images. Scale bar, 10 μm. (B) Percentage of liver and NIH 3T3 cells that displayed nuclear accumulation of AIF and EndoG. Treated liver cells were exposed to 4.5% ethanol for 5 h. NIH 3T3 cells were treated with 10% DMSO for 20 h. Treated cells that were then washed to remove apoptotic inducers were further cultured for 24 h (Washed). *p < 0.01; n = 3 independent experiments. Error bars denote SD. (C, D) Western blot analysis of total cell lysates of untreated, treated, and washed liver and NIH 3T3 cells were probed for (C) PARP and (D) ICAD. c, cleaved form. (E) Fluorescence microscopy for the SYBR-stained DNA of untreated, treated, and washed liver cells subjected to the comet assay for DNA damage. Cells embedded in agarose were subjected to electrophoresis. Broken DNA forms comet tails such as the one indicated by an arrow. Intact DNA remains within the nuclear envelop. (F) Percentage of untreated, treated, and washed liver and NIH 3T3 cells that displayed a comet tail. **p < 0.01; n = 3 independent experiments. Error bars denote SD. (G) Fluorescence microscopy on nuclear morphology of untreated as well as treated and washed primary liver cells 16 h after removal of apoptotic inducer. Cytokinesis was blocked with cytochalasin B (Cyto B). Arrows indicate micronuclei in the washed cells. Scale bar, 10 μm. (H) Percentage of untreated, as well as treated and then washed, liver, NIH 3T3, and HeLa cells that displayed micronuclei. Apoptotic inductions for the liver and NIH 3T3 cells were as described in (B) and for HeLa as in (J). See Materials and Methods for detail. *p < 0.05; **p < 0.01; n = 3 independent experiments. Error bars denote SD. (I) Proposed model for the formation of micronuclei in cells that divide after reversal of apoptosis, likely as a result of unrepaired DNA damage. (J) Time-lapse live-cell fluorescence microscopy of HeLa cells before, during, and after exposure to ethanol. The same cell before ethanol induction (Untreated, i), induced with 4.3% ethanol in culture medium for 5 h (Treated, ii), and then washed and further cultured with fresh medium (Washed, iii–x). Monochromatic images of nuclei stained with Hoechst 33342. Arrows indicate some of the micronuclei. Time presented as h:min. Scale bar, 30 μm. See Supplemental Figure S5 and Supplemental Video S4 for images and video with fluorescence microscopy for the corresponding mitochondria and nuclei and phase contrast microscopy for cell morphology of the same cells. Cytochalasin B was not present in this experiment.

Article Snippet: Primary antibodies used were as follows: anti-caspase-3, anti-PARP (Cell Signaling Technology, Danvers, MA), and anti-ICAD (BD PharMingen, BD Biosciences, Le Pont de Clax, France).

Techniques: Fluorescence, Cell Culture, Western Blot, Microscopy, Staining, Single Cell Gel Electrophoresis, Electrophoresis

Journal:

Article Title: Constitutive nuclear localization and initial cytoplasmic apoptotic activation of endogenous caspase-3 evidenced by confocal microscopy

doi: 10.1046/j.1365-2613.2003.00339.x

Figure Lengend Snippet: Procaspase-3 (a,b), active-caspase-3 (c,d), and ICAD (e,f) immunolocalization (green fluorescence) in Jurkat cells after 1 h of treatment by 7C11 anti-FAS mAb. After 1 h of treatment, cells were stained as described in ‘Materials and methods’. The chromatin is labelled in red by 7AAD. The ‘basic’ stage pattern (a,c,e) was defined by an homogeneous nuclear staining. In ‘early’ stage apoptotic cells (b,d,f), the nuclei exhibited peripheral chromatin condensation. Procaspase-3 and ICAD were both nuclear and cytoplasmic in ‘basic’ stage cells (a and e, respectively), that reflected their constitutive location in non-treated cells. Cytoplasmic active-caspase-3 was noticed in some ‘basic’ stage cells (c), reflecting the initial cytoplasmic activation of the procaspase-3. In ‘early’ stage cells, a faint nuclear staining of ICAD was noticed, whereas its cytoplasmic staining seemed unchanged (f). At the same time, active-caspase-3 was predominantly cytoplasmic, but appeared also in the nucleus (d), thereby suggesting that the caspase-3-dependent ICAD proteolysis took place in the nucleus.

Article Snippet: Slides were then incubated with the respective primary antibodies: anti-caspase-3 mAb (clone 19, Transduction Laboratories, Lexington KY, USA), anti-ICAD mAb (clone 6B8, MBL, Nagoya, Japan) or anti-active-caspase-3 mAb (clone C92-605, BD-Pharmingen Europe, Erembodegem, Belgium) for 20 min at room temperature.

Techniques: Fluorescence, Staining, Activation Assay

Journal:

Article Title: Constitutive nuclear localization and initial cytoplasmic apoptotic activation of endogenous caspase-3 evidenced by confocal microscopy

doi: 10.1046/j.1365-2613.2003.00339.x

Figure Lengend Snippet: Subcellular distribution of the procaspase-3, active-caspase-3 and ICAD in non-apoptotic and apoptotic Jurkat cells. (a) The upper panel shows the procaspase-3 and ICAD subcellular distribution in non-treated cells, ‘basic’ and ‘early’ stage apoptotic cells. For each molecule, the N/(N + C) ratio was positively correlated with the amount of nuclear protein. Procaspase-3 and ICAD were mainly nuclear in non-treated and ‘basic’ stage cells. In ‘early’ stage cells, the procaspase-3 was mainly nuclear, whereas ICAD was equally distributed between nucleus and cytoplasm. (b) The lower panel shows the active-caspase-3 subcellular distribution in apoptotic cells. In most ‘basic’ stage cells, fluorescence intensity was so low that N/(N + C) estimation was unreliable. The active-caspase-3 amount increased from ‘basic’ to ‘early’ stage cells. Some ‘basic’ stage cells showed intermediate levels of mainly cytoplasmic active-caspase-3. In ‘early’ stage cells, active-caspase-3 was both nuclear and cytoplasmic. N = nuclear fluorescence intensity (arbitrary units); N + C = whole cell fluorescence intensity (arbitrary units); N/(N + C) = N vs. N + C intensity ratio in each cell.

Article Snippet: Slides were then incubated with the respective primary antibodies: anti-caspase-3 mAb (clone 19, Transduction Laboratories, Lexington KY, USA), anti-ICAD mAb (clone 6B8, MBL, Nagoya, Japan) or anti-active-caspase-3 mAb (clone C92-605, BD-Pharmingen Europe, Erembodegem, Belgium) for 20 min at room temperature.

Techniques: Fluorescence