Journal: Experimental and Therapeutic Medicine

Article Title: Jinmaitong decreases sciatic nerve DNA oxidative damage and apoptosis in a streptozotocin-induced diabetic rat model

doi: 10.3892/etm.2015.2543

Figure Lengend Snippet: Primers for the reverse transcription-quantitative polymerase chain reaction sequences.

Article Snippet: SC783) or rabbit anti-caspase 3 (17 kDa) polyclonal antibody (1:100; Bioworld Technology Inc., Nanjing, China; cat.no.

Techniques: Polymerase Chain Reaction, Sequencing

Journal: Experimental and Therapeutic Medicine

Article Title: Jinmaitong decreases sciatic nerve DNA oxidative damage and apoptosis in a streptozotocin-induced diabetic rat model

doi: 10.3892/etm.2015.2543

Figure Lengend Snippet: Effect of JMT on caspase-3 mRNA and protein expression in the sciatic nerve in a diabetic rat model. (A) Caspase-3 mRNA expression was determined by reverse transcription-quantitative polymerase chain reaction analysis; β-actin was used as a reference gene. (B) Active caspase-3 protein expression was determined by immunohistochemistry (magnification, x200). The data are shown as the mean ± standard deviation (n=5/group). **P<0.01 vs. Con; #P<0.05 and ##P<0.01 vs. DM; &P<0.05 and &&P<0.01 vs. VC; %P<0.05 vs. JMT-L. Con, normal control; DM, diabetic model control; JMT, Jinmaitong; -L, -low-dosage; -M, medium-dosage; -H, -high-dosage; VC, vitamin C; IOD, integrated optical density.

Article Snippet: SC783) or rabbit anti-caspase 3 (17 kDa) polyclonal antibody (1:100; Bioworld Technology Inc., Nanjing, China; cat.no.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Transforming growth factor-β1 reduces apoptosis via autophagy activation in hepatic stellate cells

doi: 10.3892/mmr.2014.2383

Figure Lengend Snippet: TGF-β1 ameliorates HSC-T6 cell apoptosis induced by serum deprivation. (A) Cell viability assessed by MTS assay; (B) western blot analysis of caspase-3 protein expression in cells with or without TGF-β1 treatment; and (C) cell apoptosis levels assessed by flow cytometry following incubation with 10% FBS, serum-deprived medium or serum-deprived medium with TGF-β1 (10 ng/ml) treatment for 6 h. * P<0.05 vs. control (10% FBS); # P<0.05 vs. serum deprivation without TGF-β1 treatment. FBS, fetal bovine serum; TGF-β1, transforming growth factor β1; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.

Article Snippet: Primary antibodies were as follows: Rabbit polyclonal anti-LC3B (1/1,000; Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti-GAPDH (1/1,000) and rabbit polyclonal anti-cleaved caspase-3 (1/1,000) (Shanghai ExCell Biology, Inc.).

Techniques: MTS Assay, Western Blot, Expressing, Flow Cytometry, Incubation

Journal: Molecular Cancer

Article Title: Arecoline induces HA22T/VGH hepatoma cells to undergo anoikis - involvement of STAT3 and RhoA activation

doi: 10.1186/1476-4598-9-126

Figure Lengend Snippet: Arecoline alters the expression of apoptosis-related proteins and caspase activity in HA22T/VGH cells . (A) HA22T/VGH cells were treated with 0, 30, or 100 μg/ml of arecoline for 24 h, then the cells were harvested and proteins extracted and used for Western blotting for Bcl-2, Bcl-X L , Bax, cytochrome c, or procaspase-9. β-actin was used as the internal control. The values shown are the quantitative density analysis expressed as the relative density compared to that in untreated cells (control), taken as 100%. The results are expressed as the mean ± S.D. for three separate experiments. (B) Caspase-3 activity was detected using RPE-conjugated anti-active caspase-3 antibody by flow cytometric analysis. The values shown are the percentage of cells with active caspase-3 and are the mean ± S.D. of three independent experiments. The red filled area is the untreated control and the black lines the treated groups. *: p < 0.05 as compared to the untreated control.

Article Snippet: R-phycoerythrin (RPE)-conjugated rabbit anti-active caspase-3 polyclonal antibodies, RPE-conjugated mouse anti-human β1-integrin monoclonal antibody and the RPE-conjugated mouse IgG isotype control were purchased from BD Pharmingen Inc. (San Diego, CA, USA).

Techniques: Expressing, Activity Assay, Western Blot

Journal: Molecular Cancer

Article Title: Arecoline induces HA22T/VGH hepatoma cells to undergo anoikis - involvement of STAT3 and RhoA activation

doi: 10.1186/1476-4598-9-126

Figure Lengend Snippet: Schematic representation of the arecoline-stimulated signaling pathways for detachment and apoptosis of HA22T/VGH cells . Arecoline treatment decreases IL-6 levels, but does not change gp130 of IL-6 receptor. In addition, phosphorylation/activation of STAT3, which provides protection against anoikis, is inhibited and levels of its downstream signals IL-6, Bcl-2, and Bcl-X L are decreased, while Bax levels, mitochondrial cytochrome c release, caspase-9 levels, and caspase-3 activity are increased. Phosphorylation/activation of p190RhoGAP, a RhoA inhibitor, and its upstream regulator, SHP2, are inhibited, while the activation/cleavage of Rock-1, a RhoA downstream kinase, and actin stress fiber formation are increased, contributing to anoikis.

Article Snippet: R-phycoerythrin (RPE)-conjugated rabbit anti-active caspase-3 polyclonal antibodies, RPE-conjugated mouse anti-human β1-integrin monoclonal antibody and the RPE-conjugated mouse IgG isotype control were purchased from BD Pharmingen Inc. (San Diego, CA, USA).

Techniques: Activation Assay, Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Isolation of peptides blocking the function of anti-apoptotic Livin protein

doi: 10.1007/s00018-010-0300-3

Figure Lengend Snippet: Expression of Livin-binding peptides sensitizes Livin-expressing tumor cells toward pro-apoptotic stimuli. Livin-positive HeLa, Caki-1, and MeWo, and Livin-negative H1299 cells were transfected with pIRESneo vectors expressing the indicated peptides, and exposed to 50 J/m2 UV-irradiation or 50 μg/ml etoposide. a Detection of apoptotic cells by TUNEL assays. Cell nuclei were visualized by DAPI staining. Magnification: 200-fold. b Relative increase in number of TUNEL-positive cells after etoposide or UV treatment. The number of apoptotic cells after transfection of the negative control peptide K22 was set at 1.0. Standard deviations and p-values are indicated. Asterisk p = 0.0529. c Western-blot analysis of caspase-3 activation in HeLa cells, after expression of indicated peptides and etoposide treatment

Article Snippet: The following antibodies were employed: monoclonal anti-Livin antibody (Active Motif, Carlsbad, CA, USA), monoclonal anti-Livin antibody no. 6 [ 9 ], monoclonal anti-α-Tubulin antibody (Calbiochem, Darmstadt, Germany), and polyclonal rabbit anti-caspase-3 antibody Pab CM1 (BD Biosciences, Heidelberg, Germany).

Techniques: Expressing, Binding Assay, Transfection, Irradiation, TUNEL Assay, Staining, Negative Control, Western Blot, Activation Assay

Journal: The Journal of Cell Biology

Article Title: Activation of Membrane-associated Procaspase-3 Is Regulated by Bcl-2

doi:

Figure Lengend Snippet: Neo-membranes and Bcl-2-membranes contain similar amounts of procaspase-3. (A) Immunoblot of heavy membrane and cytosolic fractions from 697-neo and 697-Bcl-2 cells using an affinity-purified rabbit polyclonal antibody to caspase-3. The arrowheads indicate the migration of protein size markers (Rainbow Markers; Novex); the arrow indicates procaspase-3. HM, heavy membrane fractions; S100, cytosolic fraction. Note: The immunoreactive procaspase-3 band in heavy membrane fractions migrates more slowly than the cytosolic form of the protein. (B) Activation of membrane-associated acDEVD-amc cleavage activity by exogenous caspase-1. Heavy membrane fractions (containing 50 μg total protein) from 697-Bcl-2 and 697-neo cells were resuspended and treated with murine caspase-1 or buffer for 1 h at room temperature. After centrifugation, the acDEVD-amc cleavage activity of the resulting supernatant was measured. The acDEVD-amc cleavage activity of caspase-1–treated samples was corrected for exogenous caspase-1 activity by subtracting the fluorescence of control samples containing only caspase-1 from the observed fluorescence. The error bars represent the standard deviation of the observed values in three independent experiments.

Article Snippet: Cells were then incubated with affinity-purified anti-caspase-3 rabbit polyclonal antibody CSP3 ( ) or purified rabbit IgG ( PharMingen ; 0.3–1.2 μg/ml), plus anti-cytochrome c mouse monoclonal antibody (clone 6H2.B4; PharMingen , 0.25 μg/ml) diluted in blocking buffer, for 1 h at room temperature.

Techniques: Western Blot, Affinity Purification, Migration, Activation Assay, Activity Assay, Centrifugation, Fluorescence, Standard Deviation