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

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: The expression levels of stem cell markers importin13, c-kit, CD146, and telomerase are decreased in endometrial polyps

doi: 10.12659/MSM.881901

Figure Lengend Snippet: The expression levels of IPO13 and caspase3 in the endometrial tissues were detected by immunohistochemistry (400×). ( A ) Control endometrium at the proliferation phase. ( B ) Control endometrium at the secretion phase. ( C ) EP at the proliferation phase. ( D ) EP at the secretion phase. ( E ) Control endometrium at the proliferation phase. ( F ) Control endometrium at the secretion phase. ( G ) EP at the proliferation phase. ( H ) EP at the secretion phase. * p<0.05 ( vs. control endometrium at the proliferation phase); ** p<0.05 ( vs. control endometrium at the secretion phase); error bars, SEM.

Article Snippet: Anti-IPO13 goat polyclonal antibody (1:100, NB100-1369, Novus Biologicals), anti-telomerase rabbit polyclonal antibody (1: 100, ZA-0239, Beijing Golden Bridge Biotechnology Co., Ltd.), anti-CD146 mouse monoclonal antibody (1:50, ZM-0299, Beijing Golden Bridge Biotechnology Co., Ltd.), anti-caspase3 rabbit polyclonal antibody (1:100, BS1518, Bioworld Technology, Inc.), anti-bax rabbit polyclonal antibody (1:100, BS1725, Bioworld Technology, Inc.) and anti-bcl-2 rabbit monoclonal antibody (1:100, bs-0522R, Bioworld Technology, Inc.) were incubated with the sections overnight at 4°C.

Techniques: Expressing, Immunohistochemistry

Journal: Cell Communication and Signaling : CCS

Article Title: Modulation of apoptosis and Inflammasome activation in chondrocytes: co-regulatory role of Chlorogenic acid

doi: 10.1186/s12964-023-01377-w

Figure Lengend Snippet: Role of executioner caspases (i.e., Casp3 and Casp7) under the repercussions of miR-460a and CGA. A The interpretation of Casp3 and Casp7 in chondrocyte apoptosis following transfection was elucidated through Western blot assay and RT-qPCR analyses across different experimental conditions. B Immunofluorescence assay of Casp3 and Casp7, labeled with specific antibodies (200X). The miR-460a mimic and siRNA-based Bcl-2 transfected cells exhibited higher protein expression levels, as shown by the higher fluorescence signal. In contrast, the miR-460a inhibitor and miR-460a inhibitor + siBcl-2 groups showed a significantly lower expression level. C The expression level of Casp3 and Casp7 in chondrocytes through western blot, RT-qPCR, and immunofluorescence assay (Magnification, 200X) in co-delivery of siBcl-2 and CGA. Bar graphs show the proteins and gene expression levels in various groups with varying transfection conditions. The error bars represent the standard deviation of the mean. “*” shows the level of significance among groups. * p < 0.05, ** p < 0.01. “#” shows the non-significance among groups

Article Snippet: The following antibodies were used: anti-Bcl-2 pAb (Bioss, Proteintech Group), anti-Cytochrome C Rabbit pAb (ABclonal Technology), anti-Bax Rabbit pAb (Wanlei Biotechnology Co., Ltd), anti-Bak Rabbit pAb (ABclonal Technology), anti-caspase-3 Rabbit pAb (ABclonal Technology), anti-caspase7/cleaved-caspase7 Rabbit pAb (ABclonal Technology), IL-1β (Wanleibio Biotechnology Co., Ltd), anti-aggrecan Rabbit pAb (ABclonal Technology), Coll1A2 Rabbit pAb (ABclonal Technology), anti-GAPDH Rabbit pAb (ABclonal Technology), HRP goat anti-rabbit IgG (ABclonal Technology), Alexa Fluor® 488 labeled goat anti-rabbit IgG (Servicebio Technology Co., Ltd), Cy3 labeled goat anti-rabbit IgG (Servicebio Technology Co., Ltd).

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Immunofluorescence, Labeling, Expressing, Fluorescence, Standard Deviation

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