Structured Review

Cell Signaling Technology Inc caspase 8
TRAIL receptors and sTRAIL activity on PDAC cell lines. ( a ) Expression of both agonistic (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors by flow cytometry; DR-4 and DcR1 were phycoerithryn (PE) and DR5 and DcR2 allophycocyanin (APC) stained. Proper isotype controls were used for both fluorochromes. ( b ) Tumor cell death was measured by propidium iodide staining. Recombinant human TRAIL (rhTRAIL; 1 μg/ml) was used as positive control while tumor cell lines with unconditioned control medium (CTL) and empty vector (EV) transduced AD-MSC supernatant were used as negative controls. Reported *p values represent significance of rhTRAIL versus the other groups, while **p refers to soluble TRAIL (sTRAIL) versus controls. ( c ) Western blot analysis on whole cell lysates showing Caspase 8 cleavage in treated (sTRAIL and rhTRAIL) and control (CTL and EV) BxPC-3 cells after both 6 and 8 hours. ( d ) Flow cytometry analysis to detect activated Caspase 8 forms in treated (sTRAIL and rhTRAIL) and control (CTL and EV) BxPC-3 cells after 6 hours of treatment with supernatants and CTL media. Full-length blots/gels are presented in Supplementary Fig.   S9 .
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1) Product Images from "Soluble TRAIL Armed Human MSC As Gene Therapy For Pancreatic Cancer"

Article Title: Soluble TRAIL Armed Human MSC As Gene Therapy For Pancreatic Cancer

Journal: Scientific Reports

doi: 10.1038/s41598-018-37433-6

TRAIL receptors and sTRAIL activity on PDAC cell lines. ( a ) Expression of both agonistic (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors by flow cytometry; DR-4 and DcR1 were phycoerithryn (PE) and DR5 and DcR2 allophycocyanin (APC) stained. Proper isotype controls were used for both fluorochromes. ( b ) Tumor cell death was measured by propidium iodide staining. Recombinant human TRAIL (rhTRAIL; 1 μg/ml) was used as positive control while tumor cell lines with unconditioned control medium (CTL) and empty vector (EV) transduced AD-MSC supernatant were used as negative controls. Reported *p values represent significance of rhTRAIL versus the other groups, while **p refers to soluble TRAIL (sTRAIL) versus controls. ( c ) Western blot analysis on whole cell lysates showing Caspase 8 cleavage in treated (sTRAIL and rhTRAIL) and control (CTL and EV) BxPC-3 cells after both 6 and 8 hours. ( d ) Flow cytometry analysis to detect activated Caspase 8 forms in treated (sTRAIL and rhTRAIL) and control (CTL and EV) BxPC-3 cells after 6 hours of treatment with supernatants and CTL media. Full-length blots/gels are presented in Supplementary Fig.   S9 .
Figure Legend Snippet: TRAIL receptors and sTRAIL activity on PDAC cell lines. ( a ) Expression of both agonistic (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors by flow cytometry; DR-4 and DcR1 were phycoerithryn (PE) and DR5 and DcR2 allophycocyanin (APC) stained. Proper isotype controls were used for both fluorochromes. ( b ) Tumor cell death was measured by propidium iodide staining. Recombinant human TRAIL (rhTRAIL; 1 μg/ml) was used as positive control while tumor cell lines with unconditioned control medium (CTL) and empty vector (EV) transduced AD-MSC supernatant were used as negative controls. Reported *p values represent significance of rhTRAIL versus the other groups, while **p refers to soluble TRAIL (sTRAIL) versus controls. ( c ) Western blot analysis on whole cell lysates showing Caspase 8 cleavage in treated (sTRAIL and rhTRAIL) and control (CTL and EV) BxPC-3 cells after both 6 and 8 hours. ( d ) Flow cytometry analysis to detect activated Caspase 8 forms in treated (sTRAIL and rhTRAIL) and control (CTL and EV) BxPC-3 cells after 6 hours of treatment with supernatants and CTL media. Full-length blots/gels are presented in Supplementary Fig.  S9 .

Techniques Used: Activity Assay, Expressing, Flow Cytometry, Cytometry, Staining, Recombinant, Positive Control, CTL Assay, Plasmid Preparation, Western Blot

2) Product Images from "EFFICACY OF PANOBINOSTAT AND MARIZOMIB IN ACUTE MYELOID LEUKEMIA AND BORTEZOMIB-RESISTANT MODELS"

Article Title: EFFICACY OF PANOBINOSTAT AND MARIZOMIB IN ACUTE MYELOID LEUKEMIA AND BORTEZOMIB-RESISTANT MODELS

Journal: Leukemia research

doi: 10.1016/j.leukres.2014.12.014

The combination of panobinostat plus marizomib induces greater and earlier caspase-3 activity and is caspase-8 dependent in ML-1 cells A B) ML-1 cells were pre-treated with inhibitors of caspase-8 (A, IETD-fmk) or caspase-9 (B, LEHD-fmk), followed by treatment with combinations of panobinostat with marizomib or bortezomib. DNA fragmentation was assessed by flow cytometry after propidium iodide staining. C) ML-1 cells were treated for 12 hours with 1 μM panobinostat, 10 nM bortezomib, and 50 nM marizomib. Lysates were probed for cleaved caspase-3. D) ML-1 cells were treated for indicated times with either panobinostat (1 μM) plus marizomib (50 nM) or panobinostat (1 μM) plus bortezomib (10 nM) combinations. Caspase-3/7 activity was measured using the fluorogenic substrate DEVD-amc. E) After 16 hours of treatment with each of the combinations (1 μM panobinostat plus 50 nM marizomib or 1 μM panobinostat plus 10 nM bortezomib), cells were stained with HEt for analysis of superoxide levels. F) ML-1 cells were pretreated for 30 minutes with 24 mM NAC, followed by 24 hours of treatment with diluent, 10 nM bortezomib, 50 nM marizomib, 1 μM panobinostat, or combinations of these agents. DNA fragmentation was assessed by PI staining (*p
Figure Legend Snippet: The combination of panobinostat plus marizomib induces greater and earlier caspase-3 activity and is caspase-8 dependent in ML-1 cells A B) ML-1 cells were pre-treated with inhibitors of caspase-8 (A, IETD-fmk) or caspase-9 (B, LEHD-fmk), followed by treatment with combinations of panobinostat with marizomib or bortezomib. DNA fragmentation was assessed by flow cytometry after propidium iodide staining. C) ML-1 cells were treated for 12 hours with 1 μM panobinostat, 10 nM bortezomib, and 50 nM marizomib. Lysates were probed for cleaved caspase-3. D) ML-1 cells were treated for indicated times with either panobinostat (1 μM) plus marizomib (50 nM) or panobinostat (1 μM) plus bortezomib (10 nM) combinations. Caspase-3/7 activity was measured using the fluorogenic substrate DEVD-amc. E) After 16 hours of treatment with each of the combinations (1 μM panobinostat plus 50 nM marizomib or 1 μM panobinostat plus 10 nM bortezomib), cells were stained with HEt for analysis of superoxide levels. F) ML-1 cells were pretreated for 30 minutes with 24 mM NAC, followed by 24 hours of treatment with diluent, 10 nM bortezomib, 50 nM marizomib, 1 μM panobinostat, or combinations of these agents. DNA fragmentation was assessed by PI staining (*p

Techniques Used: Activity Assay, Flow Cytometry, Cytometry, Staining

3) Product Images from "Modulation of Cell Death and Promotion of Chondrogenic Differentiation by Fas/FasL in Human Dental Pulp Stem Cells (hDPSCs)"

Article Title: Modulation of Cell Death and Promotion of Chondrogenic Differentiation by Fas/FasL in Human Dental Pulp Stem Cells (hDPSCs)

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00279

Evaluation of Fas/FasL pathway in hDPSCs following stimulation with FasL rc. (A) Western Blot analysis of FasL, Fas, FADD, c-FLIP, pro-caspase 8 and cleaved caspase 8 in hDPSCs after stimulation with FasL rc at different concentrations. hDPSCs treated with 1 μM Staurosporine were used as positive control of cleaved caspase 8. At the bottom, histograms represent mean ± SD ( n = 3) of densitometry of FasL, Fas, FADD and c-FLIP; * P
Figure Legend Snippet: Evaluation of Fas/FasL pathway in hDPSCs following stimulation with FasL rc. (A) Western Blot analysis of FasL, Fas, FADD, c-FLIP, pro-caspase 8 and cleaved caspase 8 in hDPSCs after stimulation with FasL rc at different concentrations. hDPSCs treated with 1 μM Staurosporine were used as positive control of cleaved caspase 8. At the bottom, histograms represent mean ± SD ( n = 3) of densitometry of FasL, Fas, FADD and c-FLIP; * P

Techniques Used: Western Blot, Positive Control

4) Product Images from "Caspase-8, association with Alzheimer’s Disease and functional analysis of rare variants"

Article Title: Caspase-8, association with Alzheimer’s Disease and functional analysis of rare variants

Journal: PLoS ONE

doi: 10.1371/journal.pone.0185777

Caspase-8 modeling and expression. ( A ) Schematic illustration of pro-caspase-8 protein and its p43, p18 and p10 fragments resulting from proteolytic processing and activation. ( B ) Protein folding (top) and 3D model (bottom) of caspase-8 DED 2 (left side) and p18 domain (right side). The K 148 and I 298 variants are depicted in red color. F 122 and L 123 of the hydrophobic FL motif within the DED 2 is shown in purple, and critical H 317 and C 360 active site residues within the p18 domain are in green. ( C ) SK-N-BE(2) cells were transfected with expression vectors encoding WT-, K148R-, or I298V-caspase-8 and mock as control. Corresponding immunoblot analysis, 24 h post-transfection, indicating the expression levels for pro-caspase-8 and its p43, p18 and p10 fragments. For the LOAD caspase-8 variant, two clones ( a and b ) are presented.( D ) Representative confocal images of SK-N-BE(2) cells transfected as described in panel C. The cleaved caspase-8 is labeled red and Hoechst counterstained nuclei are blue. Images were taken 24 h after transfection.
Figure Legend Snippet: Caspase-8 modeling and expression. ( A ) Schematic illustration of pro-caspase-8 protein and its p43, p18 and p10 fragments resulting from proteolytic processing and activation. ( B ) Protein folding (top) and 3D model (bottom) of caspase-8 DED 2 (left side) and p18 domain (right side). The K 148 and I 298 variants are depicted in red color. F 122 and L 123 of the hydrophobic FL motif within the DED 2 is shown in purple, and critical H 317 and C 360 active site residues within the p18 domain are in green. ( C ) SK-N-BE(2) cells were transfected with expression vectors encoding WT-, K148R-, or I298V-caspase-8 and mock as control. Corresponding immunoblot analysis, 24 h post-transfection, indicating the expression levels for pro-caspase-8 and its p43, p18 and p10 fragments. For the LOAD caspase-8 variant, two clones ( a and b ) are presented.( D ) Representative confocal images of SK-N-BE(2) cells transfected as described in panel C. The cleaved caspase-8 is labeled red and Hoechst counterstained nuclei are blue. Images were taken 24 h after transfection.

Techniques Used: Expressing, Activation Assay, Transfection, Variant Assay, Clone Assay, Labeling

Caspase-8 enzymatic activity. SK-N-BE(2) cells were transfected with expression vectors encoding WT-, K148R-, or I298V-caspase-8 and mock as control. (A) Caspase-8 (LETDase) and (B) Caspase-3-like (DEVDase) activities were measured 24 h post-transfection. Data are presented as fold over mock untreated. Statistics and error bars: mean±s.d. n = 8 of biological replicates. Data was analyzed as comparison to Caspase-8 WT using two-sided student’s t-test. *P
Figure Legend Snippet: Caspase-8 enzymatic activity. SK-N-BE(2) cells were transfected with expression vectors encoding WT-, K148R-, or I298V-caspase-8 and mock as control. (A) Caspase-8 (LETDase) and (B) Caspase-3-like (DEVDase) activities were measured 24 h post-transfection. Data are presented as fold over mock untreated. Statistics and error bars: mean±s.d. n = 8 of biological replicates. Data was analyzed as comparison to Caspase-8 WT using two-sided student’s t-test. *P

Techniques Used: Activity Assay, Transfection, Expressing

5) Product Images from "Oxidized low density lipoprotein facilitates tumor necrosis factor-α mediated chondrocyte death via autophagy pathway"

Article Title: Oxidized low density lipoprotein facilitates tumor necrosis factor-α mediated chondrocyte death via autophagy pathway

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2017.7786

Autophagy inhibition by 3-MA or Atg-5 siRNA could reverse the effects mediated by TNF-α and ox-LDL co-treatment on chondrocytes. (A) Western blot analysis of the expression of LC3I/II. (B) LC3 pattern analysis by confocal microscopy. (C) Cell death analysis. (D) Western blot analysis of apoptosis related protein cleaved caspase-3 and caspase-8. 3-MA (5 mM) treatment could reverse the LC3II enhancement, punctuate LC3 pattern, increased cell death and cleaved caspase-3 and caspase-8 mediated TNF-α and ox-LDL co-treatment in chondrocytes. (E) Western blot analysis of the expression of LC3I/II. (F) LC3 pattern analysis by confocal microscopy. (G) Cell death analysis. (H) Western blot analysis of apoptosis related protein cleaved caspase-3 and caspase-8. Atg-5 siRNA treatment could reverse the LC3II enhancement, punctuate LC3 pattern, increased cell death and cleaved caspase-3 and caspase-8 mediated TNF-α and ox-LDL co-treatment in chondrocytes. *P
Figure Legend Snippet: Autophagy inhibition by 3-MA or Atg-5 siRNA could reverse the effects mediated by TNF-α and ox-LDL co-treatment on chondrocytes. (A) Western blot analysis of the expression of LC3I/II. (B) LC3 pattern analysis by confocal microscopy. (C) Cell death analysis. (D) Western blot analysis of apoptosis related protein cleaved caspase-3 and caspase-8. 3-MA (5 mM) treatment could reverse the LC3II enhancement, punctuate LC3 pattern, increased cell death and cleaved caspase-3 and caspase-8 mediated TNF-α and ox-LDL co-treatment in chondrocytes. (E) Western blot analysis of the expression of LC3I/II. (F) LC3 pattern analysis by confocal microscopy. (G) Cell death analysis. (H) Western blot analysis of apoptosis related protein cleaved caspase-3 and caspase-8. Atg-5 siRNA treatment could reverse the LC3II enhancement, punctuate LC3 pattern, increased cell death and cleaved caspase-3 and caspase-8 mediated TNF-α and ox-LDL co-treatment in chondrocytes. *P

Techniques Used: Inhibition, Western Blot, Expressing, Confocal Microscopy

Facilitation of TNF-α-mediated chondrocyte death by oxidized low density lipoprotein (ox-LDL). After 30 min preincubation with or without anti-LOX-1 antibody (10 μg/ml), Chondrocytes were co-treated with TNF-α (50 ng/ml) and ox-LDL (20 μg/ml), and harvested at 24 or 48 h for (A) cell death, (B) DNA fragmentation assay, (C) Flow cytometry analysis of the chrondrocyte apoptosis by Annexin V/PI staining and (D) western blot analysis of apoptosis related proteins. (A, B and C) Ox-LDL co-treatment could facilitate TNF-α-mediated chondrocyte death and this process could be blocked by Lox-1 monoclonal antibody pretreatment. (D) Western-blot analysis revealed that increased level of cleaved caspase-8 and caspase-3 in TNF-α and ox-LDL co-treated chondrocytes, and that this effect could be blocked by Lox-1 antibody pretreatment.*P
Figure Legend Snippet: Facilitation of TNF-α-mediated chondrocyte death by oxidized low density lipoprotein (ox-LDL). After 30 min preincubation with or without anti-LOX-1 antibody (10 μg/ml), Chondrocytes were co-treated with TNF-α (50 ng/ml) and ox-LDL (20 μg/ml), and harvested at 24 or 48 h for (A) cell death, (B) DNA fragmentation assay, (C) Flow cytometry analysis of the chrondrocyte apoptosis by Annexin V/PI staining and (D) western blot analysis of apoptosis related proteins. (A, B and C) Ox-LDL co-treatment could facilitate TNF-α-mediated chondrocyte death and this process could be blocked by Lox-1 monoclonal antibody pretreatment. (D) Western-blot analysis revealed that increased level of cleaved caspase-8 and caspase-3 in TNF-α and ox-LDL co-treated chondrocytes, and that this effect could be blocked by Lox-1 antibody pretreatment.*P

Techniques Used: DNA Fragmentation Assay, Flow Cytometry, Cytometry, Staining, Western Blot

6) Product Images from "Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *"

Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.257022

Caspase-8 is required for the proteasome-mediated degradation of IRF-3. A , HT1080 cells were transfected with poly(I:C) in the absence or the presence of an inhibitor of caspase-8 (z-IETD, 10 μ m ) for the indicated times, and cell lysates were
Figure Legend Snippet: Caspase-8 is required for the proteasome-mediated degradation of IRF-3. A , HT1080 cells were transfected with poly(I:C) in the absence or the presence of an inhibitor of caspase-8 (z-IETD, 10 μ m ) for the indicated times, and cell lysates were

Techniques Used: Transfection

Caspase-8 is activated by cytosolic RIG-I-dependent signaling. A , 1080.10 cells were infected with SeV at MOI 10 for the indicated times, and the cell lysates were analyzed for the activation of caspase-8 by Western blot ( FL , full-length; CL , cleaved;
Figure Legend Snippet: Caspase-8 is activated by cytosolic RIG-I-dependent signaling. A , 1080.10 cells were infected with SeV at MOI 10 for the indicated times, and the cell lysates were analyzed for the activation of caspase-8 by Western blot ( FL , full-length; CL , cleaved;

Techniques Used: Infection, Activation Assay, Western Blot

Caspase-8 activity is essential for the cleavage of IRF-3. A , P2.1 cells expressing IRF-3, were pretreated with the inhibitors of multiple caspases (Z, z-VAD, 10 μ m ), caspase-1 (z-WEHD, 10 μ m ), or caspase-8 (z-IETD, 10 μ m ) for
Figure Legend Snippet: Caspase-8 activity is essential for the cleavage of IRF-3. A , P2.1 cells expressing IRF-3, were pretreated with the inhibitors of multiple caspases (Z, z-VAD, 10 μ m ), caspase-1 (z-WEHD, 10 μ m ), or caspase-8 (z-IETD, 10 μ m ) for

Techniques Used: Activity Assay, Expressing

Caspase-8-mediated cleavage of IRF-3 is an intermediate step in its proteasome-mediated degradation. IRF-3 undergoes proteasome-mediated degradation in response to dsRNA-dependent signaling. Stimulation of TLR3 or RIG-I signaling by dsRNA activates caspase-8
Figure Legend Snippet: Caspase-8-mediated cleavage of IRF-3 is an intermediate step in its proteasome-mediated degradation. IRF-3 undergoes proteasome-mediated degradation in response to dsRNA-dependent signaling. Stimulation of TLR3 or RIG-I signaling by dsRNA activates caspase-8

Techniques Used:

Mutation of a caspase-8 recognition motif leads to impaired cleavage and degradation of IRF-3. P2.1 cells expressing Wt or the D121E ( DE ) mutant of IRF-3 were used in these experiments ( A–C ). A , cells were transfected with poly(I:C); cell lysates
Figure Legend Snippet: Mutation of a caspase-8 recognition motif leads to impaired cleavage and degradation of IRF-3. P2.1 cells expressing Wt or the D121E ( DE ) mutant of IRF-3 were used in these experiments ( A–C ). A , cells were transfected with poly(I:C); cell lysates

Techniques Used: Mutagenesis, Expressing, Transfection

7) Product Images from "Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *"

Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.257022

Caspase-8 is required for the proteasome-mediated degradation of IRF-3. A , HT1080 cells were transfected with poly(I:C) in the absence or the presence of an inhibitor of caspase-8 (z-IETD, 10 μ m ) for the indicated times, and cell lysates were
Figure Legend Snippet: Caspase-8 is required for the proteasome-mediated degradation of IRF-3. A , HT1080 cells were transfected with poly(I:C) in the absence or the presence of an inhibitor of caspase-8 (z-IETD, 10 μ m ) for the indicated times, and cell lysates were

Techniques Used: Transfection

Caspase-8 is activated by cytosolic RIG-I-dependent signaling. A , 1080.10 cells were infected with SeV at MOI 10 for the indicated times, and the cell lysates were analyzed for the activation of caspase-8 by Western blot ( FL , full-length; CL , cleaved;
Figure Legend Snippet: Caspase-8 is activated by cytosolic RIG-I-dependent signaling. A , 1080.10 cells were infected with SeV at MOI 10 for the indicated times, and the cell lysates were analyzed for the activation of caspase-8 by Western blot ( FL , full-length; CL , cleaved;

Techniques Used: Infection, Activation Assay, Western Blot

Caspase-8 activity is essential for the cleavage of IRF-3. A , P2.1 cells expressing IRF-3, were pretreated with the inhibitors of multiple caspases (Z, z-VAD, 10 μ m ), caspase-1 (z-WEHD, 10 μ m ), or caspase-8 (z-IETD, 10 μ m ) for
Figure Legend Snippet: Caspase-8 activity is essential for the cleavage of IRF-3. A , P2.1 cells expressing IRF-3, were pretreated with the inhibitors of multiple caspases (Z, z-VAD, 10 μ m ), caspase-1 (z-WEHD, 10 μ m ), or caspase-8 (z-IETD, 10 μ m ) for

Techniques Used: Activity Assay, Expressing

Caspase-8-mediated cleavage of IRF-3 is an intermediate step in its proteasome-mediated degradation. IRF-3 undergoes proteasome-mediated degradation in response to dsRNA-dependent signaling. Stimulation of TLR3 or RIG-I signaling by dsRNA activates caspase-8
Figure Legend Snippet: Caspase-8-mediated cleavage of IRF-3 is an intermediate step in its proteasome-mediated degradation. IRF-3 undergoes proteasome-mediated degradation in response to dsRNA-dependent signaling. Stimulation of TLR3 or RIG-I signaling by dsRNA activates caspase-8

Techniques Used:

Mutation of a caspase-8 recognition motif leads to impaired cleavage and degradation of IRF-3. P2.1 cells expressing Wt or the D121E ( DE ) mutant of IRF-3 were used in these experiments ( A–C ). A , cells were transfected with poly(I:C); cell lysates
Figure Legend Snippet: Mutation of a caspase-8 recognition motif leads to impaired cleavage and degradation of IRF-3. P2.1 cells expressing Wt or the D121E ( DE ) mutant of IRF-3 were used in these experiments ( A–C ). A , cells were transfected with poly(I:C); cell lysates

Techniques Used: Mutagenesis, Expressing, Transfection

8) Product Images from "Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *"

Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.257022

Caspase-8 is required for the proteasome-mediated degradation of IRF-3. A , HT1080 cells were transfected with poly(I:C) in the absence or the presence of an inhibitor of caspase-8 (z-IETD, 10 μ m ) for the indicated times, and cell lysates were
Figure Legend Snippet: Caspase-8 is required for the proteasome-mediated degradation of IRF-3. A , HT1080 cells were transfected with poly(I:C) in the absence or the presence of an inhibitor of caspase-8 (z-IETD, 10 μ m ) for the indicated times, and cell lysates were

Techniques Used: Transfection

Caspase-8 is activated by cytosolic RIG-I-dependent signaling. A , 1080.10 cells were infected with SeV at MOI 10 for the indicated times, and the cell lysates were analyzed for the activation of caspase-8 by Western blot ( FL , full-length; CL , cleaved;
Figure Legend Snippet: Caspase-8 is activated by cytosolic RIG-I-dependent signaling. A , 1080.10 cells were infected with SeV at MOI 10 for the indicated times, and the cell lysates were analyzed for the activation of caspase-8 by Western blot ( FL , full-length; CL , cleaved;

Techniques Used: Infection, Activation Assay, Western Blot

Caspase-8 activity is essential for the cleavage of IRF-3. A , P2.1 cells expressing IRF-3, were pretreated with the inhibitors of multiple caspases (Z, z-VAD, 10 μ m ), caspase-1 (z-WEHD, 10 μ m ), or caspase-8 (z-IETD, 10 μ m ) for
Figure Legend Snippet: Caspase-8 activity is essential for the cleavage of IRF-3. A , P2.1 cells expressing IRF-3, were pretreated with the inhibitors of multiple caspases (Z, z-VAD, 10 μ m ), caspase-1 (z-WEHD, 10 μ m ), or caspase-8 (z-IETD, 10 μ m ) for

Techniques Used: Activity Assay, Expressing

Caspase-8-mediated cleavage of IRF-3 is an intermediate step in its proteasome-mediated degradation. IRF-3 undergoes proteasome-mediated degradation in response to dsRNA-dependent signaling. Stimulation of TLR3 or RIG-I signaling by dsRNA activates caspase-8
Figure Legend Snippet: Caspase-8-mediated cleavage of IRF-3 is an intermediate step in its proteasome-mediated degradation. IRF-3 undergoes proteasome-mediated degradation in response to dsRNA-dependent signaling. Stimulation of TLR3 or RIG-I signaling by dsRNA activates caspase-8

Techniques Used:

Mutation of a caspase-8 recognition motif leads to impaired cleavage and degradation of IRF-3. P2.1 cells expressing Wt or the D121E ( DE ) mutant of IRF-3 were used in these experiments ( A–C ). A , cells were transfected with poly(I:C); cell lysates
Figure Legend Snippet: Mutation of a caspase-8 recognition motif leads to impaired cleavage and degradation of IRF-3. P2.1 cells expressing Wt or the D121E ( DE ) mutant of IRF-3 were used in these experiments ( A–C ). A , cells were transfected with poly(I:C); cell lysates

Techniques Used: Mutagenesis, Expressing, Transfection

9) Product Images from "Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *"

Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.257022

Caspase-8 is required for the proteasome-mediated degradation of IRF-3. A , HT1080 cells were transfected with poly(I:C) in the absence or the presence of an inhibitor of caspase-8 (z-IETD, 10 μ m ) for the indicated times, and cell lysates were
Figure Legend Snippet: Caspase-8 is required for the proteasome-mediated degradation of IRF-3. A , HT1080 cells were transfected with poly(I:C) in the absence or the presence of an inhibitor of caspase-8 (z-IETD, 10 μ m ) for the indicated times, and cell lysates were

Techniques Used: Transfection

Caspase-8 is activated by cytosolic RIG-I-dependent signaling. A , 1080.10 cells were infected with SeV at MOI 10 for the indicated times, and the cell lysates were analyzed for the activation of caspase-8 by Western blot ( FL , full-length; CL , cleaved;
Figure Legend Snippet: Caspase-8 is activated by cytosolic RIG-I-dependent signaling. A , 1080.10 cells were infected with SeV at MOI 10 for the indicated times, and the cell lysates were analyzed for the activation of caspase-8 by Western blot ( FL , full-length; CL , cleaved;

Techniques Used: Infection, Activation Assay, Western Blot

Caspase-8 activity is essential for the cleavage of IRF-3. A , P2.1 cells expressing IRF-3, were pretreated with the inhibitors of multiple caspases (Z, z-VAD, 10 μ m ), caspase-1 (z-WEHD, 10 μ m ), or caspase-8 (z-IETD, 10 μ m ) for
Figure Legend Snippet: Caspase-8 activity is essential for the cleavage of IRF-3. A , P2.1 cells expressing IRF-3, were pretreated with the inhibitors of multiple caspases (Z, z-VAD, 10 μ m ), caspase-1 (z-WEHD, 10 μ m ), or caspase-8 (z-IETD, 10 μ m ) for

Techniques Used: Activity Assay, Expressing

Caspase-8-mediated cleavage of IRF-3 is an intermediate step in its proteasome-mediated degradation. IRF-3 undergoes proteasome-mediated degradation in response to dsRNA-dependent signaling. Stimulation of TLR3 or RIG-I signaling by dsRNA activates caspase-8
Figure Legend Snippet: Caspase-8-mediated cleavage of IRF-3 is an intermediate step in its proteasome-mediated degradation. IRF-3 undergoes proteasome-mediated degradation in response to dsRNA-dependent signaling. Stimulation of TLR3 or RIG-I signaling by dsRNA activates caspase-8

Techniques Used:

Mutation of a caspase-8 recognition motif leads to impaired cleavage and degradation of IRF-3. P2.1 cells expressing Wt or the D121E ( DE ) mutant of IRF-3 were used in these experiments ( A–C ). A , cells were transfected with poly(I:C); cell lysates
Figure Legend Snippet: Mutation of a caspase-8 recognition motif leads to impaired cleavage and degradation of IRF-3. P2.1 cells expressing Wt or the D121E ( DE ) mutant of IRF-3 were used in these experiments ( A–C ). A , cells were transfected with poly(I:C); cell lysates

Techniques Used: Mutagenesis, Expressing, Transfection

10) Product Images from "Following cytochrome c release, autophagy is inhibited during chemotherapy-induced apoptosis by caspase-8-mediated cleavage of Beclin-1"

Article Title: Following cytochrome c release, autophagy is inhibited during chemotherapy-induced apoptosis by caspase-8-mediated cleavage of Beclin-1

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-10-4475

Cleavage of Beclin 1 by caspase 8 at two sites in CPT-induced apoptosis
Figure Legend Snippet: Cleavage of Beclin 1 by caspase 8 at two sites in CPT-induced apoptosis

Techniques Used: Cycling Probe Technology

11) Product Images from "Following cytochrome c release, autophagy is inhibited during chemotherapy-induced apoptosis by caspase-8-mediated cleavage of Beclin-1"

Article Title: Following cytochrome c release, autophagy is inhibited during chemotherapy-induced apoptosis by caspase-8-mediated cleavage of Beclin-1

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-10-4475

Cleavage of Beclin 1 by caspase 8 at two sites in CPT-induced apoptosis
Figure Legend Snippet: Cleavage of Beclin 1 by caspase 8 at two sites in CPT-induced apoptosis

Techniques Used: Cycling Probe Technology

12) Product Images from "Methylmercury (MeHg) elicits mitochondrial-dependent apoptosis in developing hippocampus and acts at low exposures"

Article Title: Methylmercury (MeHg) elicits mitochondrial-dependent apoptosis in developing hippocampus and acts at low exposures

Journal: Neurotoxicology

doi: 10.1016/j.neuro.2011.06.003

Effect of 5 μg/g MeHg exposure on cleaved caspase-8 protein levels in postnatal rat hippocampus
Figure Legend Snippet: Effect of 5 μg/g MeHg exposure on cleaved caspase-8 protein levels in postnatal rat hippocampus

Techniques Used:

13) Product Images from "Short-term pretreatment with atorvastatin attenuates left ventricular dysfunction, reduces infarct size and apoptosis in acute myocardial infarction rats"

Article Title: Short-term pretreatment with atorvastatin attenuates left ventricular dysfunction, reduces infarct size and apoptosis in acute myocardial infarction rats

Journal: International Journal of Clinical and Experimental Medicine

doi:

Effects of atorvastatin on expression of caspase-8 and caspase-3 in AMI rats. A. 60 μg myocardium lysates in sham, model and atorvastatin group were loaded onto an SDS-polyacrylamide gel, and caspase-8 and caspase-3 were detected by Western blotting.
Figure Legend Snippet: Effects of atorvastatin on expression of caspase-8 and caspase-3 in AMI rats. A. 60 μg myocardium lysates in sham, model and atorvastatin group were loaded onto an SDS-polyacrylamide gel, and caspase-8 and caspase-3 were detected by Western blotting.

Techniques Used: Expressing, Western Blot

14) Product Images from "Short-term pretreatment with atorvastatin attenuates left ventricular dysfunction, reduces infarct size and apoptosis in acute myocardial infarction rats"

Article Title: Short-term pretreatment with atorvastatin attenuates left ventricular dysfunction, reduces infarct size and apoptosis in acute myocardial infarction rats

Journal: International Journal of Clinical and Experimental Medicine

doi:

Effects of atorvastatin on expression of caspase-8 and caspase-3 in AMI rats. A. 60 μg myocardium lysates in sham, model and atorvastatin group were loaded onto an SDS-polyacrylamide gel, and caspase-8 and caspase-3 were detected by Western blotting.
Figure Legend Snippet: Effects of atorvastatin on expression of caspase-8 and caspase-3 in AMI rats. A. 60 μg myocardium lysates in sham, model and atorvastatin group were loaded onto an SDS-polyacrylamide gel, and caspase-8 and caspase-3 were detected by Western blotting.

Techniques Used: Expressing, Western Blot

15) Product Images from "The functional domains for BaxΔ2 aggregate-mediated caspase 8-dependent cell death"

Article Title: The functional domains for BaxΔ2 aggregate-mediated caspase 8-dependent cell death

Journal: Experimental cell research

doi: 10.1016/j.yexcr.2017.08.016

BaxΔ2 C-terminus is required for caspase 8 activation, but in a primary sequence independent manner (A) Schematic representation of the C-terminal splicing events for BaxΔ2 and BaxΔ2ω (not scaled). Alternative splicing in BaxΔ2ω causes a partial intron 5 retention (49 bp) and leads to a frameshift that generates a totally different protein sequence of C-terminus (grey color). Stop codon is indicated by an asterisk (*). (B) Cellular colocalization and 3D imaging analysis using the XYZ slicing tool, of transfected GFP-tagged BaxΔ2 and BaxΔ2ω 16 hours after transfection in Bax-negative HCT116 cells. Caspase 8 was detected with anti-caspase 8 antibody (red). Blue, nuclear staining with DAPI. (C) Quantification of cell death of Bax-negative HCT116 cells transfected with GFP, BaxΔ2, BaxΔ2ω and BaxΔ2[Δ141–175] and incubated for 24 hours in the absence (Ctrl) or presence of Caspase 8 Inhibitor (C8I). BaxΔ2[Δ141–175] is BaxΔ2 without the exon 6. ***, p
Figure Legend Snippet: BaxΔ2 C-terminus is required for caspase 8 activation, but in a primary sequence independent manner (A) Schematic representation of the C-terminal splicing events for BaxΔ2 and BaxΔ2ω (not scaled). Alternative splicing in BaxΔ2ω causes a partial intron 5 retention (49 bp) and leads to a frameshift that generates a totally different protein sequence of C-terminus (grey color). Stop codon is indicated by an asterisk (*). (B) Cellular colocalization and 3D imaging analysis using the XYZ slicing tool, of transfected GFP-tagged BaxΔ2 and BaxΔ2ω 16 hours after transfection in Bax-negative HCT116 cells. Caspase 8 was detected with anti-caspase 8 antibody (red). Blue, nuclear staining with DAPI. (C) Quantification of cell death of Bax-negative HCT116 cells transfected with GFP, BaxΔ2, BaxΔ2ω and BaxΔ2[Δ141–175] and incubated for 24 hours in the absence (Ctrl) or presence of Caspase 8 Inhibitor (C8I). BaxΔ2[Δ141–175] is BaxΔ2 without the exon 6. ***, p

Techniques Used: Activation Assay, Sequencing, Imaging, Transfection, Staining, Incubation

Loss of helix α1 is responsible for the behavioral differences between Baxα and BaxΔ2 ) and a computationally predicted model of BaxΔ2 structure using RaptorX and I-Tasser. The structures are color coded by corresponding exons indicated in the key. C, C-terminus. Helix α1 is indicated by an arrow in the Baxα structure, and is missing in the BaxΔ2 model structure. (C) Comparison of the N-terminal protein sequence differences between Baxα and BaxΔ2. Only part of the protein sequence coded by exon 3 is shown in this figure. The amino acids that form helix α1 in Baxα are underlined. The 10 amino acids (aa) coded by the new reading frame caused by the alternative splicing in BaxΔ2 are bolded. L26P, mutation of leucine at position 26 to proline; L27P, mutation of leucine at position 27 to proline. (D) N-terminal disorder probability analysis for Baxα and BaxΔ2 using PrDOS. (E) Prediction of helix α1 helicity for each mutant in comparison to Baxα (“0” point). Helicity was calculated using NetSurfP and GOR4; plotted results represent the average probability values minus Baxα helicity probability values for each mutant. (F) Cell death assay of Bax-negative HCT116 cells after 24 hours of transfection with GFP-tagged constructs and treated without (NT) or with Caspase 8 Inhibitor (C8I). ***, p
Figure Legend Snippet: Loss of helix α1 is responsible for the behavioral differences between Baxα and BaxΔ2 ) and a computationally predicted model of BaxΔ2 structure using RaptorX and I-Tasser. The structures are color coded by corresponding exons indicated in the key. C, C-terminus. Helix α1 is indicated by an arrow in the Baxα structure, and is missing in the BaxΔ2 model structure. (C) Comparison of the N-terminal protein sequence differences between Baxα and BaxΔ2. Only part of the protein sequence coded by exon 3 is shown in this figure. The amino acids that form helix α1 in Baxα are underlined. The 10 amino acids (aa) coded by the new reading frame caused by the alternative splicing in BaxΔ2 are bolded. L26P, mutation of leucine at position 26 to proline; L27P, mutation of leucine at position 27 to proline. (D) N-terminal disorder probability analysis for Baxα and BaxΔ2 using PrDOS. (E) Prediction of helix α1 helicity for each mutant in comparison to Baxα (“0” point). Helicity was calculated using NetSurfP and GOR4; plotted results represent the average probability values minus Baxα helicity probability values for each mutant. (F) Cell death assay of Bax-negative HCT116 cells after 24 hours of transfection with GFP-tagged constructs and treated without (NT) or with Caspase 8 Inhibitor (C8I). ***, p

Techniques Used: Sequencing, Mutagenesis, Transfection, Construct

Disruption of helix α9 significantly reduces BaxΔ2 toxicity (A) Peptide sequence alignment of the C-terminal regions of BaxΔ2 and BaxΔ2ω and the identified conservative motifs are highlighted in yellow. Fully conserved (*), conservative (:), and semi-conservative (.) amino acids are indicated. (B) Detailed list of mutants and the abbreviated names that are used in the following graphs. (C) Immunoblot analysis of expression of BaxΔ2 mutants in Bax-negative HCT116 cells transfected with GFP-tagged constructs for 24 hours, using anti-GFP antibody; anti-actin antibody used as protein-loading control. (D) Prediction of C-terminal helicity of each mutant in comparison to BaxΔ2 (“0” point). Helicity was calculated by subtracting BaxΔ2 helicity probability values from that of each mutant before plotting. (E) Cell death assay of the GFP-tagged BaxΔ2 and its mutants in HCT116 Bax-negative cells 24 hours after transfection and treated without (NT) or with Caspase 8 inhibitor (C8I). Statistical analysis compares the NT and C8I in each individual mutant and then NT of each mutant with the BaxΔ2 control, ***, p
Figure Legend Snippet: Disruption of helix α9 significantly reduces BaxΔ2 toxicity (A) Peptide sequence alignment of the C-terminal regions of BaxΔ2 and BaxΔ2ω and the identified conservative motifs are highlighted in yellow. Fully conserved (*), conservative (:), and semi-conservative (.) amino acids are indicated. (B) Detailed list of mutants and the abbreviated names that are used in the following graphs. (C) Immunoblot analysis of expression of BaxΔ2 mutants in Bax-negative HCT116 cells transfected with GFP-tagged constructs for 24 hours, using anti-GFP antibody; anti-actin antibody used as protein-loading control. (D) Prediction of C-terminal helicity of each mutant in comparison to BaxΔ2 (“0” point). Helicity was calculated by subtracting BaxΔ2 helicity probability values from that of each mutant before plotting. (E) Cell death assay of the GFP-tagged BaxΔ2 and its mutants in HCT116 Bax-negative cells 24 hours after transfection and treated without (NT) or with Caspase 8 inhibitor (C8I). Statistical analysis compares the NT and C8I in each individual mutant and then NT of each mutant with the BaxΔ2 control, ***, p

Techniques Used: Sequencing, Expressing, Transfection, Construct, Mutagenesis

16) Product Images from "The functional domains for BaxΔ2 aggregate-mediated caspase 8-dependent cell death"

Article Title: The functional domains for BaxΔ2 aggregate-mediated caspase 8-dependent cell death

Journal: Experimental cell research

doi: 10.1016/j.yexcr.2017.08.016

BaxΔ2 C-terminus is required for caspase 8 activation, but in a primary sequence independent manner (A) Schematic representation of the C-terminal splicing events for BaxΔ2 and BaxΔ2ω (not scaled). Alternative splicing in BaxΔ2ω causes a partial intron 5 retention (49 bp) and leads to a frameshift that generates a totally different protein sequence of C-terminus (grey color). Stop codon is indicated by an asterisk (*). (B) Cellular colocalization and 3D imaging analysis using the XYZ slicing tool, of transfected GFP-tagged BaxΔ2 and BaxΔ2ω 16 hours after transfection in Bax-negative HCT116 cells. Caspase 8 was detected with anti-caspase 8 antibody (red). Blue, nuclear staining with DAPI. (C) Quantification of cell death of Bax-negative HCT116 cells transfected with GFP, BaxΔ2, BaxΔ2ω and BaxΔ2[Δ141–175] and incubated for 24 hours in the absence (Ctrl) or presence of Caspase 8 Inhibitor (C8I). BaxΔ2[Δ141–175] is BaxΔ2 without the exon 6. ***, p
Figure Legend Snippet: BaxΔ2 C-terminus is required for caspase 8 activation, but in a primary sequence independent manner (A) Schematic representation of the C-terminal splicing events for BaxΔ2 and BaxΔ2ω (not scaled). Alternative splicing in BaxΔ2ω causes a partial intron 5 retention (49 bp) and leads to a frameshift that generates a totally different protein sequence of C-terminus (grey color). Stop codon is indicated by an asterisk (*). (B) Cellular colocalization and 3D imaging analysis using the XYZ slicing tool, of transfected GFP-tagged BaxΔ2 and BaxΔ2ω 16 hours after transfection in Bax-negative HCT116 cells. Caspase 8 was detected with anti-caspase 8 antibody (red). Blue, nuclear staining with DAPI. (C) Quantification of cell death of Bax-negative HCT116 cells transfected with GFP, BaxΔ2, BaxΔ2ω and BaxΔ2[Δ141–175] and incubated for 24 hours in the absence (Ctrl) or presence of Caspase 8 Inhibitor (C8I). BaxΔ2[Δ141–175] is BaxΔ2 without the exon 6. ***, p

Techniques Used: Activation Assay, Sequencing, Imaging, Transfection, Staining, Incubation

Loss of helix α1 is responsible for the behavioral differences between Baxα and BaxΔ2 ) and a computationally predicted model of BaxΔ2 structure using RaptorX and I-Tasser. The structures are color coded by corresponding exons indicated in the key. C, C-terminus. Helix α1 is indicated by an arrow in the Baxα structure, and is missing in the BaxΔ2 model structure. (C) Comparison of the N-terminal protein sequence differences between Baxα and BaxΔ2. Only part of the protein sequence coded by exon 3 is shown in this figure. The amino acids that form helix α1 in Baxα are underlined. The 10 amino acids (aa) coded by the new reading frame caused by the alternative splicing in BaxΔ2 are bolded. L26P, mutation of leucine at position 26 to proline; L27P, mutation of leucine at position 27 to proline. (D) N-terminal disorder probability analysis for Baxα and BaxΔ2 using PrDOS. (E) Prediction of helix α1 helicity for each mutant in comparison to Baxα (“0” point). Helicity was calculated using NetSurfP and GOR4; plotted results represent the average probability values minus Baxα helicity probability values for each mutant. (F) Cell death assay of Bax-negative HCT116 cells after 24 hours of transfection with GFP-tagged constructs and treated without (NT) or with Caspase 8 Inhibitor (C8I). ***, p
Figure Legend Snippet: Loss of helix α1 is responsible for the behavioral differences between Baxα and BaxΔ2 ) and a computationally predicted model of BaxΔ2 structure using RaptorX and I-Tasser. The structures are color coded by corresponding exons indicated in the key. C, C-terminus. Helix α1 is indicated by an arrow in the Baxα structure, and is missing in the BaxΔ2 model structure. (C) Comparison of the N-terminal protein sequence differences between Baxα and BaxΔ2. Only part of the protein sequence coded by exon 3 is shown in this figure. The amino acids that form helix α1 in Baxα are underlined. The 10 amino acids (aa) coded by the new reading frame caused by the alternative splicing in BaxΔ2 are bolded. L26P, mutation of leucine at position 26 to proline; L27P, mutation of leucine at position 27 to proline. (D) N-terminal disorder probability analysis for Baxα and BaxΔ2 using PrDOS. (E) Prediction of helix α1 helicity for each mutant in comparison to Baxα (“0” point). Helicity was calculated using NetSurfP and GOR4; plotted results represent the average probability values minus Baxα helicity probability values for each mutant. (F) Cell death assay of Bax-negative HCT116 cells after 24 hours of transfection with GFP-tagged constructs and treated without (NT) or with Caspase 8 Inhibitor (C8I). ***, p

Techniques Used: Sequencing, Mutagenesis, Transfection, Construct

Disruption of helix α9 significantly reduces BaxΔ2 toxicity (A) Peptide sequence alignment of the C-terminal regions of BaxΔ2 and BaxΔ2ω and the identified conservative motifs are highlighted in yellow. Fully conserved (*), conservative (:), and semi-conservative (.) amino acids are indicated. (B) Detailed list of mutants and the abbreviated names that are used in the following graphs. (C) Immunoblot analysis of expression of BaxΔ2 mutants in Bax-negative HCT116 cells transfected with GFP-tagged constructs for 24 hours, using anti-GFP antibody; anti-actin antibody used as protein-loading control. (D) Prediction of C-terminal helicity of each mutant in comparison to BaxΔ2 (“0” point). Helicity was calculated by subtracting BaxΔ2 helicity probability values from that of each mutant before plotting. (E) Cell death assay of the GFP-tagged BaxΔ2 and its mutants in HCT116 Bax-negative cells 24 hours after transfection and treated without (NT) or with Caspase 8 inhibitor (C8I). Statistical analysis compares the NT and C8I in each individual mutant and then NT of each mutant with the BaxΔ2 control, ***, p
Figure Legend Snippet: Disruption of helix α9 significantly reduces BaxΔ2 toxicity (A) Peptide sequence alignment of the C-terminal regions of BaxΔ2 and BaxΔ2ω and the identified conservative motifs are highlighted in yellow. Fully conserved (*), conservative (:), and semi-conservative (.) amino acids are indicated. (B) Detailed list of mutants and the abbreviated names that are used in the following graphs. (C) Immunoblot analysis of expression of BaxΔ2 mutants in Bax-negative HCT116 cells transfected with GFP-tagged constructs for 24 hours, using anti-GFP antibody; anti-actin antibody used as protein-loading control. (D) Prediction of C-terminal helicity of each mutant in comparison to BaxΔ2 (“0” point). Helicity was calculated by subtracting BaxΔ2 helicity probability values from that of each mutant before plotting. (E) Cell death assay of the GFP-tagged BaxΔ2 and its mutants in HCT116 Bax-negative cells 24 hours after transfection and treated without (NT) or with Caspase 8 inhibitor (C8I). Statistical analysis compares the NT and C8I in each individual mutant and then NT of each mutant with the BaxΔ2 control, ***, p

Techniques Used: Sequencing, Expressing, Transfection, Construct, Mutagenesis

17) Product Images from "Necroptosis of Dendritic Cells Promotes Activation of γδ T cells"

Article Title: Necroptosis of Dendritic Cells Promotes Activation of γδ T cells

Journal: Journal of innate immunity

doi: 10.1159/000446498

IL-4 induces c-FLIP and caspase-8 activity in DC
Figure Legend Snippet: IL-4 induces c-FLIP and caspase-8 activity in DC

Techniques Used: Activity Assay

18) Product Images from "Necroptosis of Dendritic Cells Promotes Activation of γδ T cells"

Article Title: Necroptosis of Dendritic Cells Promotes Activation of γδ T cells

Journal: Journal of innate immunity

doi: 10.1159/000446498

IL-4 induces c-FLIP and caspase-8 activity in DC
Figure Legend Snippet: IL-4 induces c-FLIP and caspase-8 activity in DC

Techniques Used: Activity Assay

19) Product Images from "Cdk1/Cyclin B1 Controls Fas-Mediated Apoptosis by Regulating Caspase-8 Activity ▿"

Article Title: Cdk1/Cyclin B1 Controls Fas-Mediated Apoptosis by Regulating Caspase-8 Activity ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00731-10

Inhibition of Cdk1 activity using the small molecule RO-3306 suppresses the phosphorylation of procaspase-8 at Ser-387 in vitro and in vivo . (a) Procaspase-8 purified from bacteria was subjected to kinase assays with Cdk1/cyclin B1 and increasing concentrations of RO-3306. Samples were immunoblotted for Cdk1, caspase-8, and caspase-8 (pSer387). (b) Treatment of proliferating human SW480 cancer cells with RO-3306 for 14 h led to a complete block of the cell cycle at the G 2 /M border. After releasing SW480 cells from RO-3306 a Western blot analysis was performed for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, and β-actin.
Figure Legend Snippet: Inhibition of Cdk1 activity using the small molecule RO-3306 suppresses the phosphorylation of procaspase-8 at Ser-387 in vitro and in vivo . (a) Procaspase-8 purified from bacteria was subjected to kinase assays with Cdk1/cyclin B1 and increasing concentrations of RO-3306. Samples were immunoblotted for Cdk1, caspase-8, and caspase-8 (pSer387). (b) Treatment of proliferating human SW480 cancer cells with RO-3306 for 14 h led to a complete block of the cell cycle at the G 2 /M border. After releasing SW480 cells from RO-3306 a Western blot analysis was performed for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, and β-actin.

Techniques Used: Inhibition, Activity Assay, In Vitro, In Vivo, Purification, Blocking Assay, Western Blot

The phosphorylation of procaspase-8 correlates with Cdk1/cyclin B1 activity in cell lines of different origin. (a) T47D, SW480, and MDA-MB-231 cells were arrested in G 1 /S using a double-thymidine block (time zero) and released for the times indicated. The cellular lysates were immunoblotted for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, Plk1, and β-actin. (b) Characterization of a cyclin B1-depleted clone using a stably integrated RNAi cassette (Cycl.B1 − -HeLa). Mitotic shake-off cells (wild-type and Cycl.B1 − ) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), cyclin B1, and β-actin (*, unspecific band) (left panel). Characterization of Cdk1-depleted cells by RNAi. Mitotic shake-off cells (wild-type and Cdk1-depleted cells) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), Cdk1, and β-actin (*, unspecific band) (right panel).
Figure Legend Snippet: The phosphorylation of procaspase-8 correlates with Cdk1/cyclin B1 activity in cell lines of different origin. (a) T47D, SW480, and MDA-MB-231 cells were arrested in G 1 /S using a double-thymidine block (time zero) and released for the times indicated. The cellular lysates were immunoblotted for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, Plk1, and β-actin. (b) Characterization of a cyclin B1-depleted clone using a stably integrated RNAi cassette (Cycl.B1 − -HeLa). Mitotic shake-off cells (wild-type and Cycl.B1 − ) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), cyclin B1, and β-actin (*, unspecific band) (left panel). Characterization of Cdk1-depleted cells by RNAi. Mitotic shake-off cells (wild-type and Cdk1-depleted cells) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), Cdk1, and β-actin (*, unspecific band) (right panel).

Techniques Used: Activity Assay, Multiple Displacement Amplification, Blocking Assay, Stable Transfection

Cdk1/cyclin B1 phosphorylates procaspase-8 in vitro at Ser-387. (a) Recombinant GST-fused subdomains of procaspase-8 were analyzed by SDS-PAGE (lower left panel). Subdomains were incubated with active Cdk1/cyclin B1 and [γ- 32 P]ATP, analyzed by SDS-PAGE, and visualized by autoradiography (upper left panel). This was followed by phospho amino acid analysis (lower right panel). (b) Wild-type (WT) and mutated (S387A, S387E) recombinant full-length GST-caspase-8 proteins were subjected to kinase assays using Cdk1/cyclin B1, analyzed by SDS-PAGE, and autoradiography. (c) Recombinant caspase-8 (WT) was incubated with active Cdk1/cyclin B1. Samples were immunoblotted with antibodies specific for caspase-8, for a caspase-8 peptide [ABcasp.-8(pSer387)] containing phospho-Ser387 (YLEMDLSpSPQTRY) and for Cdk1. (d) GST, recombinant caspase-8 (GST-WT), and a mutated form (GST-S387A) were incubated with or without active Cdk1/cyclin B1 and immunoblotted with ABcasp.-8(pSer387) (e) Recombinant caspase-8 was incubated with or without active Cdk1/cyclin B1 in kinase buffer, followed by treatment with 100 U of λ-phosphatase and immunoblotted with ABcasp.-8(pSer387).
Figure Legend Snippet: Cdk1/cyclin B1 phosphorylates procaspase-8 in vitro at Ser-387. (a) Recombinant GST-fused subdomains of procaspase-8 were analyzed by SDS-PAGE (lower left panel). Subdomains were incubated with active Cdk1/cyclin B1 and [γ- 32 P]ATP, analyzed by SDS-PAGE, and visualized by autoradiography (upper left panel). This was followed by phospho amino acid analysis (lower right panel). (b) Wild-type (WT) and mutated (S387A, S387E) recombinant full-length GST-caspase-8 proteins were subjected to kinase assays using Cdk1/cyclin B1, analyzed by SDS-PAGE, and autoradiography. (c) Recombinant caspase-8 (WT) was incubated with active Cdk1/cyclin B1. Samples were immunoblotted with antibodies specific for caspase-8, for a caspase-8 peptide [ABcasp.-8(pSer387)] containing phospho-Ser387 (YLEMDLSpSPQTRY) and for Cdk1. (d) GST, recombinant caspase-8 (GST-WT), and a mutated form (GST-S387A) were incubated with or without active Cdk1/cyclin B1 and immunoblotted with ABcasp.-8(pSer387) (e) Recombinant caspase-8 was incubated with or without active Cdk1/cyclin B1 in kinase buffer, followed by treatment with 100 U of λ-phosphatase and immunoblotted with ABcasp.-8(pSer387).

Techniques Used: In Vitro, Recombinant, SDS Page, Incubation, Autoradiography

A nonphosphorylatable S387 mutant of procaspase-8 sensitizes mitotic BAX-deficient HCT116 cells to Fas-mediated apoptosis. (a) On day 1, wild-type (BAX-positive) and BAX-deficient HCT116 cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, WT, or S387A) on day 2. On day 3, cells were treated overnight with nocodazole, and then a mitotic shake-off was performed on day 4. Subsequently, the cells were reseeded in nocodazole-containing medium and stimulated with 100 ng of FasL/ml. Lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody and BAX. (b) Lysates of BAX-positive and -negative cells that were treated as described in panel a were immunoblotted for PARP. (c and d) Apoptosis (annexin staining) and caspase-8 activity were determined. All experiments were performed in triplicate. Error bars represent the SD.
Figure Legend Snippet: A nonphosphorylatable S387 mutant of procaspase-8 sensitizes mitotic BAX-deficient HCT116 cells to Fas-mediated apoptosis. (a) On day 1, wild-type (BAX-positive) and BAX-deficient HCT116 cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, WT, or S387A) on day 2. On day 3, cells were treated overnight with nocodazole, and then a mitotic shake-off was performed on day 4. Subsequently, the cells were reseeded in nocodazole-containing medium and stimulated with 100 ng of FasL/ml. Lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody and BAX. (b) Lysates of BAX-positive and -negative cells that were treated as described in panel a were immunoblotted for PARP. (c and d) Apoptosis (annexin staining) and caspase-8 activity were determined. All experiments were performed in triplicate. Error bars represent the SD.

Techniques Used: Mutagenesis, Transfection, Staining, Activity Assay

Phosphorylation of procaspase-8 at Ser-387 in primary cells and tissues. (a) Lysates were prepared from human breast cancer tissues, premalignant ductal carcinoma in situ (DCIS) samples and normal breast tissues. Total protein was resolved by SDS-PAGE and immunoblotted for caspase-8 (pSer387), caspase-8, cyclin B1, and β-actin (top panel). Blue bars represent the level of caspase-8 (pSer387) normalized to the level of β-actin expression (lower panel). Red bars represent the level of cyclin B1 expression standardized to the level of β-actin expression. (b) Peripheral blood lymphocytes (PBLs) were treated with PHA for the times indicated. Cell lysates were immunoblotted for caspase-8 (pS387), caspase-8, histone H3 phosphorylated at Ser10 (H3 pS10), cyclin B1, Cdk1, Plk1, CD95/Fas, and β-actin (upper panels). To measure Cdk1/cyclin B1 activity in cell lysates of synchronized cells, immunoprecipitation assays were performed using a cyclin B1-specific antibody, and the purified proteins were subjected to kinase assays with histone H1 as the substrate (lower panel). (c) PBLs were cultured with PHA for 16 h. Cells were then washed three times and cultured for an additional 5 days in the presence of interleukin-2, followed by nocodazole treatment for 16 h and MG132 for 2 h. For the inhibition of Cdk1/cyclin B1, RO-3306 was added.
Figure Legend Snippet: Phosphorylation of procaspase-8 at Ser-387 in primary cells and tissues. (a) Lysates were prepared from human breast cancer tissues, premalignant ductal carcinoma in situ (DCIS) samples and normal breast tissues. Total protein was resolved by SDS-PAGE and immunoblotted for caspase-8 (pSer387), caspase-8, cyclin B1, and β-actin (top panel). Blue bars represent the level of caspase-8 (pSer387) normalized to the level of β-actin expression (lower panel). Red bars represent the level of cyclin B1 expression standardized to the level of β-actin expression. (b) Peripheral blood lymphocytes (PBLs) were treated with PHA for the times indicated. Cell lysates were immunoblotted for caspase-8 (pS387), caspase-8, histone H3 phosphorylated at Ser10 (H3 pS10), cyclin B1, Cdk1, Plk1, CD95/Fas, and β-actin (upper panels). To measure Cdk1/cyclin B1 activity in cell lysates of synchronized cells, immunoprecipitation assays were performed using a cyclin B1-specific antibody, and the purified proteins were subjected to kinase assays with histone H1 as the substrate (lower panel). (c) PBLs were cultured with PHA for 16 h. Cells were then washed three times and cultured for an additional 5 days in the presence of interleukin-2, followed by nocodazole treatment for 16 h and MG132 for 2 h. For the inhibition of Cdk1/cyclin B1, RO-3306 was added.

Techniques Used: In Situ, SDS Page, Expressing, Activity Assay, Immunoprecipitation, Purification, Cell Culture, Inhibition

Inhibition of the extrinsic death pathway in Fas-induced mitotic cells. (a) B lymphoblastoid SKW6.4 cells were enriched in G 1 /S phase by treatment with thymidine (lane 1), in pro-/metaphase with nocodazole for 16 h, followed by MG132 for 2 h (lane 2) or in a G 1 -like state with nocodazole for 16 h, followed by MG132 for 2 h and RO-3306 for the inhibition of Cdk1/cyclin B1 (lane 3) (upper panel). Cell lysates were immunoblotted for cyclin B1, histone H3 phosphorylated at Ser10 (H3 pS10), cyclin E, Plk1, and β-actin. The cell cycle status was monitored by flow cytometry (lower panel). (b) Cells treated as described in panel a were stimulated with 100 ng of FasL/ml, 10 ng of TRIAL/ml, or 10 ng of TNF-α/ml for the times indicated, and total cellular lysates were analyzed by Western blotting with anti-caspase-8 MAb C15. (c) Caspase-8 activity was determined by measuring the cleavage of the luminogenic substrate containing the IETD peptide. All experiments were performed in triplicate. Error bars represent the standard deviation (SD). (d) Apoptosis analyses were performed by using an annexin V kit. All experiments were performed in triplicate. Error bars represent the SD.
Figure Legend Snippet: Inhibition of the extrinsic death pathway in Fas-induced mitotic cells. (a) B lymphoblastoid SKW6.4 cells were enriched in G 1 /S phase by treatment with thymidine (lane 1), in pro-/metaphase with nocodazole for 16 h, followed by MG132 for 2 h (lane 2) or in a G 1 -like state with nocodazole for 16 h, followed by MG132 for 2 h and RO-3306 for the inhibition of Cdk1/cyclin B1 (lane 3) (upper panel). Cell lysates were immunoblotted for cyclin B1, histone H3 phosphorylated at Ser10 (H3 pS10), cyclin E, Plk1, and β-actin. The cell cycle status was monitored by flow cytometry (lower panel). (b) Cells treated as described in panel a were stimulated with 100 ng of FasL/ml, 10 ng of TRIAL/ml, or 10 ng of TNF-α/ml for the times indicated, and total cellular lysates were analyzed by Western blotting with anti-caspase-8 MAb C15. (c) Caspase-8 activity was determined by measuring the cleavage of the luminogenic substrate containing the IETD peptide. All experiments were performed in triplicate. Error bars represent the standard deviation (SD). (d) Apoptosis analyses were performed by using an annexin V kit. All experiments were performed in triplicate. Error bars represent the SD.

Techniques Used: Inhibition, Flow Cytometry, Cytometry, Western Blot, Activity Assay, Standard Deviation

Cdk1/cyclin B1 associates with and phosphorylates procaspase-8 in vitro at Ser-387. (a) The p10 subunit was mutated at different sites (S387A, SS386/387AA, and T419A), and the corresponding GST fusion proteins were incubated with active Cdk1/cyclin B1 and [γ- 32 P]ATP, analyzed by SDS-PAGE and visualized by autoradiography. (b) Bacterially expressed, purified GST-fused full-length caspase-8 (GST-WT), and the N-terminal (GST-NT) and C-terminal (GST-CT) subdomains of caspase-8 were incubated with lysates of HeLa cells that had been transfected with a Flag epitope-tagged Cdk1 (Flag-Cdk1) expression construct for pulldown assays. The Flag-Cdk1 protein that associated with GST-caspase-8 or its subdomains was detected by immunoblot with an anti-Flag antibody. (c) The binding of the GST-fused N-terminal subdomains (GST-2xDED, -DED1+, and -DED1) was also analyzed in pulldown assays. The Flag-Cdk1 protein that associated with GST-fused subdomains of the N-terminal portion of caspase-8 was detected by immunoblotting with an anti-Flag antibody. A 5% total input lane was also run.
Figure Legend Snippet: Cdk1/cyclin B1 associates with and phosphorylates procaspase-8 in vitro at Ser-387. (a) The p10 subunit was mutated at different sites (S387A, SS386/387AA, and T419A), and the corresponding GST fusion proteins were incubated with active Cdk1/cyclin B1 and [γ- 32 P]ATP, analyzed by SDS-PAGE and visualized by autoradiography. (b) Bacterially expressed, purified GST-fused full-length caspase-8 (GST-WT), and the N-terminal (GST-NT) and C-terminal (GST-CT) subdomains of caspase-8 were incubated with lysates of HeLa cells that had been transfected with a Flag epitope-tagged Cdk1 (Flag-Cdk1) expression construct for pulldown assays. The Flag-Cdk1 protein that associated with GST-caspase-8 or its subdomains was detected by immunoblot with an anti-Flag antibody. (c) The binding of the GST-fused N-terminal subdomains (GST-2xDED, -DED1+, and -DED1) was also analyzed in pulldown assays. The Flag-Cdk1 protein that associated with GST-fused subdomains of the N-terminal portion of caspase-8 was detected by immunoblotting with an anti-Flag antibody. A 5% total input lane was also run.

Techniques Used: In Vitro, Incubation, SDS Page, Autoradiography, Purification, Transfection, FLAG-tag, Expressing, Construct, Binding Assay

The phosphorylation of procaspase-8 correlates with Cdk1/cyclin B1 activity in vivo . (a) Lysates from asynchronous (−) or mitotic (+) HeLa cells were immunoprecipitated with caspase-8-, Cdk1-, or cyclin B1-specific antibodies. Anti-mouse IgG was used as a control. The immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted for caspase-8, cyclin B1, and Cdk1. Lanes 1 and 2 show 2.5% total input. (b) HeLa cells were arrested in G 1 /S using a double-thymidine block and released into fresh medium for 10 h. Semisynchronized cells were stained with DAPI (4′,6′-diamidino-2-phenylindole) and subsequently categorized according to their staining patterns. Cells were labeled using ABcasp.-8 (pSer387), caspase-8, or cyclin B1 antibodies for analysis by microscopy. Caspase-8-deficient HeLa cells were arrested in G 1 /S using a double-thymidine block and released into fresh medium for 10 h. Semisynchronized cells were stained with DAPI or labeled using ABcasp.-8 (pSer387) or cyclin B1 antibodies for analysis by microscopy. (c) To enrich for mitotic HeLa cells, an incubation with nocodazole (Noc) for 16 h was carried out; cells were captured by shake-off and then treated with MG132 for 2 h (lane 2). Shake-off cells were treated as described for lane 2, except that, 1 h after MG132 addition, the Cdk1 inhibitor RO-3306 was added (lane 3). The cell lysates were immunoblotted for caspase-8, caspase-8 (pS387), cyclin B1, Cdk1, pH3S10, securin, and β-actin. (d) HeLa cells were arrested in G 1 /S using a double-thymidine block (time zero) and released for the times indicated. The cellular lysates were immunoblotted for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, Plk1, CD95/Fas, and β-actin (upper left panels). To measure the Cdk1/cyclin B1 activity in cell lysates of synchronized cells, immunoprecipitation assays were performed with cyclin B1-specific antibodies, and immunoprecipitated proteins were then subjected to kinase assays with histone H1 as the substrate (lower left panel). The cell cycle status was monitored using flow cytometry (upper right panel). Boxed numbers exhibit the percentage of cells with 4 N DNA content. The mitotic index and distribution of mitotic phases of HeLa cells 10 h after release from the G 1 /S block are shown: the leftmost bar graph indicates 30% mitotic index 10 h after G 1 /S (of these, 46% were in prophase and 25% were in metaphase). The mitotic index was determined by counting 6 times 250 to 300 cells (lower right panel). All experiments were performed in triplicate. The error bars represent the SD. (e) Characterization of mock-transfected HeLa cells and of a cyclin B1-depleted HeLa clone using a stably integrated RNAi cassette (Cycl.B1 − -HeLa). Mitotic shake-off cells (mock-transfected and Cycl.B1 − ) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), cyclin B1, and β-actin (*, unspecific band). To measure Cdk1/cyclin B1 activity, immunoprecipitation assays were performed using cyclin B1-specific antibodies, and the immunoprecipitated proteins were subjected to kinase assays with histone H1 as the substrate (middle panel). Apoptosis analyses were performed by using an annexin V kit (lower panel). All experiments were performed in triplicate. Error bars represent the SD. (f) To enrich for mitotic SKW 6.4 cells, an incubation with nocodazole (Noc) for 16 h was carried out; cells were captured by shake-off and then treated with MG132 for 2 h. For the inhibition of Cdk1 activity, RO-3306 was added 1 h after MG132 treatment. Cells were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the anti-caspase-8 MAb C15. Apoptosis analyses were performed by using an annexin V kit (lower panel). All experiments were performed in triplicate. Error bars represent the SD.
Figure Legend Snippet: The phosphorylation of procaspase-8 correlates with Cdk1/cyclin B1 activity in vivo . (a) Lysates from asynchronous (−) or mitotic (+) HeLa cells were immunoprecipitated with caspase-8-, Cdk1-, or cyclin B1-specific antibodies. Anti-mouse IgG was used as a control. The immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted for caspase-8, cyclin B1, and Cdk1. Lanes 1 and 2 show 2.5% total input. (b) HeLa cells were arrested in G 1 /S using a double-thymidine block and released into fresh medium for 10 h. Semisynchronized cells were stained with DAPI (4′,6′-diamidino-2-phenylindole) and subsequently categorized according to their staining patterns. Cells were labeled using ABcasp.-8 (pSer387), caspase-8, or cyclin B1 antibodies for analysis by microscopy. Caspase-8-deficient HeLa cells were arrested in G 1 /S using a double-thymidine block and released into fresh medium for 10 h. Semisynchronized cells were stained with DAPI or labeled using ABcasp.-8 (pSer387) or cyclin B1 antibodies for analysis by microscopy. (c) To enrich for mitotic HeLa cells, an incubation with nocodazole (Noc) for 16 h was carried out; cells were captured by shake-off and then treated with MG132 for 2 h (lane 2). Shake-off cells were treated as described for lane 2, except that, 1 h after MG132 addition, the Cdk1 inhibitor RO-3306 was added (lane 3). The cell lysates were immunoblotted for caspase-8, caspase-8 (pS387), cyclin B1, Cdk1, pH3S10, securin, and β-actin. (d) HeLa cells were arrested in G 1 /S using a double-thymidine block (time zero) and released for the times indicated. The cellular lysates were immunoblotted for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, Plk1, CD95/Fas, and β-actin (upper left panels). To measure the Cdk1/cyclin B1 activity in cell lysates of synchronized cells, immunoprecipitation assays were performed with cyclin B1-specific antibodies, and immunoprecipitated proteins were then subjected to kinase assays with histone H1 as the substrate (lower left panel). The cell cycle status was monitored using flow cytometry (upper right panel). Boxed numbers exhibit the percentage of cells with 4 N DNA content. The mitotic index and distribution of mitotic phases of HeLa cells 10 h after release from the G 1 /S block are shown: the leftmost bar graph indicates 30% mitotic index 10 h after G 1 /S (of these, 46% were in prophase and 25% were in metaphase). The mitotic index was determined by counting 6 times 250 to 300 cells (lower right panel). All experiments were performed in triplicate. The error bars represent the SD. (e) Characterization of mock-transfected HeLa cells and of a cyclin B1-depleted HeLa clone using a stably integrated RNAi cassette (Cycl.B1 − -HeLa). Mitotic shake-off cells (mock-transfected and Cycl.B1 − ) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), cyclin B1, and β-actin (*, unspecific band). To measure Cdk1/cyclin B1 activity, immunoprecipitation assays were performed using cyclin B1-specific antibodies, and the immunoprecipitated proteins were subjected to kinase assays with histone H1 as the substrate (middle panel). Apoptosis analyses were performed by using an annexin V kit (lower panel). All experiments were performed in triplicate. Error bars represent the SD. (f) To enrich for mitotic SKW 6.4 cells, an incubation with nocodazole (Noc) for 16 h was carried out; cells were captured by shake-off and then treated with MG132 for 2 h. For the inhibition of Cdk1 activity, RO-3306 was added 1 h after MG132 treatment. Cells were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the anti-caspase-8 MAb C15. Apoptosis analyses were performed by using an annexin V kit (lower panel). All experiments were performed in triplicate. Error bars represent the SD.

Techniques Used: Activity Assay, In Vivo, Immunoprecipitation, SDS Page, Blocking Assay, Staining, Labeling, Microscopy, Incubation, Flow Cytometry, Cytometry, Transfection, Stable Transfection, Inhibition

A nonphosphorylatable S387 mutant of procaspase-8 sensitizes mitotic cells to Fas-mediated apoptosis. (a) HeLa cells were transiently transfected with vectors encoding Flag-tagged caspase-8 (Flag-WT, Flag-S387A, and Flag-S387E). At 18 h after transfection, cells were incubated with 100 ng of FasL/ml plus 1 μg of cycloheximide/ml for 3 and 6 h. Lysates were immunoblotted for exogenous caspase-8 with a Flag-specific antibody and for β-actin (upper panels). The signal intensity of immunoblotted, Flag-tagged caspase-8 was standardized to the level of β-actin expression (lower panel). All experiments were performed in triplicate. The error bars represent the SD. (b) Analysis of procaspase-8 processing in HeLa cells expressing different double-tagged (N-terminal Flag, C-terminal V5) forms of procaspase-8 (S387A, S387E). Lysates were immunoblotted for V5 and PARP. (c) Analysis of procaspase-8 processing in caspase-8 knockdown HeLa cell clones (caspase-8 − ) expressing different double-tagged (N-terminal Flag, C-terminal V5) forms of procaspase-8 (S387A, S387E). Lysates were immunoblotted for V5 and caspase-8. (d) On day 1, HeLa cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, WT, or S387A) on day 2. On day 3, the cells were treated overnight with nocodazole, and then a mitotic shake-off was performed on day 4. Subsequently, cells were reseeded in nocodazole-containing medium and stimulated with 100 ng of FasL/ml. The cell cycle status was monitored by flow cytometry (upper panels). Lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody, caspase-8, PARP, and β-actin (middle panels). Caspase-8 activity and apoptosis (annexin staining) were determined (lower panels). All experiments were performed in triplicate. Error bars represent the SD. (e) On day 1 HeLa cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, S387A, or S387E) on day 2. On day 3 cells were stimulated with 100 ng of FasL/ml. After 24 h, the cell numbers were determined, and lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody and β-actin. The experiment was performed in triplicate. Error bars represent the SD.
Figure Legend Snippet: A nonphosphorylatable S387 mutant of procaspase-8 sensitizes mitotic cells to Fas-mediated apoptosis. (a) HeLa cells were transiently transfected with vectors encoding Flag-tagged caspase-8 (Flag-WT, Flag-S387A, and Flag-S387E). At 18 h after transfection, cells were incubated with 100 ng of FasL/ml plus 1 μg of cycloheximide/ml for 3 and 6 h. Lysates were immunoblotted for exogenous caspase-8 with a Flag-specific antibody and for β-actin (upper panels). The signal intensity of immunoblotted, Flag-tagged caspase-8 was standardized to the level of β-actin expression (lower panel). All experiments were performed in triplicate. The error bars represent the SD. (b) Analysis of procaspase-8 processing in HeLa cells expressing different double-tagged (N-terminal Flag, C-terminal V5) forms of procaspase-8 (S387A, S387E). Lysates were immunoblotted for V5 and PARP. (c) Analysis of procaspase-8 processing in caspase-8 knockdown HeLa cell clones (caspase-8 − ) expressing different double-tagged (N-terminal Flag, C-terminal V5) forms of procaspase-8 (S387A, S387E). Lysates were immunoblotted for V5 and caspase-8. (d) On day 1, HeLa cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, WT, or S387A) on day 2. On day 3, the cells were treated overnight with nocodazole, and then a mitotic shake-off was performed on day 4. Subsequently, cells were reseeded in nocodazole-containing medium and stimulated with 100 ng of FasL/ml. The cell cycle status was monitored by flow cytometry (upper panels). Lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody, caspase-8, PARP, and β-actin (middle panels). Caspase-8 activity and apoptosis (annexin staining) were determined (lower panels). All experiments were performed in triplicate. Error bars represent the SD. (e) On day 1 HeLa cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, S387A, or S387E) on day 2. On day 3 cells were stimulated with 100 ng of FasL/ml. After 24 h, the cell numbers were determined, and lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody and β-actin. The experiment was performed in triplicate. Error bars represent the SD.

Techniques Used: Mutagenesis, Transfection, Incubation, Expressing, Clone Assay, Flow Cytometry, Cytometry, Activity Assay, Staining

20) Product Images from "Cdk1/Cyclin B1 Controls Fas-Mediated Apoptosis by Regulating Caspase-8 Activity ▿"

Article Title: Cdk1/Cyclin B1 Controls Fas-Mediated Apoptosis by Regulating Caspase-8 Activity ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00731-10

Inhibition of Cdk1 activity using the small molecule RO-3306 suppresses the phosphorylation of procaspase-8 at Ser-387 in vitro and in vivo . (a) Procaspase-8 purified from bacteria was subjected to kinase assays with Cdk1/cyclin B1 and increasing concentrations of RO-3306. Samples were immunoblotted for Cdk1, caspase-8, and caspase-8 (pSer387). (b) Treatment of proliferating human SW480 cancer cells with RO-3306 for 14 h led to a complete block of the cell cycle at the G 2 /M border. After releasing SW480 cells from RO-3306 a Western blot analysis was performed for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, and β-actin.
Figure Legend Snippet: Inhibition of Cdk1 activity using the small molecule RO-3306 suppresses the phosphorylation of procaspase-8 at Ser-387 in vitro and in vivo . (a) Procaspase-8 purified from bacteria was subjected to kinase assays with Cdk1/cyclin B1 and increasing concentrations of RO-3306. Samples were immunoblotted for Cdk1, caspase-8, and caspase-8 (pSer387). (b) Treatment of proliferating human SW480 cancer cells with RO-3306 for 14 h led to a complete block of the cell cycle at the G 2 /M border. After releasing SW480 cells from RO-3306 a Western blot analysis was performed for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, and β-actin.

Techniques Used: Inhibition, Activity Assay, In Vitro, In Vivo, Purification, Blocking Assay, Western Blot

The phosphorylation of procaspase-8 correlates with Cdk1/cyclin B1 activity in cell lines of different origin. (a) T47D, SW480, and MDA-MB-231 cells were arrested in G 1 /S using a double-thymidine block (time zero) and released for the times indicated. The cellular lysates were immunoblotted for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, Plk1, and β-actin. (b) Characterization of a cyclin B1-depleted clone using a stably integrated RNAi cassette (Cycl.B1 − -HeLa). Mitotic shake-off cells (wild-type and Cycl.B1 − ) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), cyclin B1, and β-actin (*, unspecific band) (left panel). Characterization of Cdk1-depleted cells by RNAi. Mitotic shake-off cells (wild-type and Cdk1-depleted cells) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), Cdk1, and β-actin (*, unspecific band) (right panel).
Figure Legend Snippet: The phosphorylation of procaspase-8 correlates with Cdk1/cyclin B1 activity in cell lines of different origin. (a) T47D, SW480, and MDA-MB-231 cells were arrested in G 1 /S using a double-thymidine block (time zero) and released for the times indicated. The cellular lysates were immunoblotted for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, Plk1, and β-actin. (b) Characterization of a cyclin B1-depleted clone using a stably integrated RNAi cassette (Cycl.B1 − -HeLa). Mitotic shake-off cells (wild-type and Cycl.B1 − ) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), cyclin B1, and β-actin (*, unspecific band) (left panel). Characterization of Cdk1-depleted cells by RNAi. Mitotic shake-off cells (wild-type and Cdk1-depleted cells) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), Cdk1, and β-actin (*, unspecific band) (right panel).

Techniques Used: Activity Assay, Multiple Displacement Amplification, Blocking Assay, Stable Transfection

Cdk1/cyclin B1 phosphorylates procaspase-8 in vitro at Ser-387. (a) Recombinant GST-fused subdomains of procaspase-8 were analyzed by SDS-PAGE (lower left panel). Subdomains were incubated with active Cdk1/cyclin B1 and [γ- 32 P]ATP, analyzed by SDS-PAGE, and visualized by autoradiography (upper left panel). This was followed by phospho amino acid analysis (lower right panel). (b) Wild-type (WT) and mutated (S387A, S387E) recombinant full-length GST-caspase-8 proteins were subjected to kinase assays using Cdk1/cyclin B1, analyzed by SDS-PAGE, and autoradiography. (c) Recombinant caspase-8 (WT) was incubated with active Cdk1/cyclin B1. Samples were immunoblotted with antibodies specific for caspase-8, for a caspase-8 peptide [ABcasp.-8(pSer387)] containing phospho-Ser387 (YLEMDLSpSPQTRY) and for Cdk1. (d) GST, recombinant caspase-8 (GST-WT), and a mutated form (GST-S387A) were incubated with or without active Cdk1/cyclin B1 and immunoblotted with ABcasp.-8(pSer387) (e) Recombinant caspase-8 was incubated with or without active Cdk1/cyclin B1 in kinase buffer, followed by treatment with 100 U of λ-phosphatase and immunoblotted with ABcasp.-8(pSer387).
Figure Legend Snippet: Cdk1/cyclin B1 phosphorylates procaspase-8 in vitro at Ser-387. (a) Recombinant GST-fused subdomains of procaspase-8 were analyzed by SDS-PAGE (lower left panel). Subdomains were incubated with active Cdk1/cyclin B1 and [γ- 32 P]ATP, analyzed by SDS-PAGE, and visualized by autoradiography (upper left panel). This was followed by phospho amino acid analysis (lower right panel). (b) Wild-type (WT) and mutated (S387A, S387E) recombinant full-length GST-caspase-8 proteins were subjected to kinase assays using Cdk1/cyclin B1, analyzed by SDS-PAGE, and autoradiography. (c) Recombinant caspase-8 (WT) was incubated with active Cdk1/cyclin B1. Samples were immunoblotted with antibodies specific for caspase-8, for a caspase-8 peptide [ABcasp.-8(pSer387)] containing phospho-Ser387 (YLEMDLSpSPQTRY) and for Cdk1. (d) GST, recombinant caspase-8 (GST-WT), and a mutated form (GST-S387A) were incubated with or without active Cdk1/cyclin B1 and immunoblotted with ABcasp.-8(pSer387) (e) Recombinant caspase-8 was incubated with or without active Cdk1/cyclin B1 in kinase buffer, followed by treatment with 100 U of λ-phosphatase and immunoblotted with ABcasp.-8(pSer387).

Techniques Used: In Vitro, Recombinant, SDS Page, Incubation, Autoradiography

A nonphosphorylatable S387 mutant of procaspase-8 sensitizes mitotic BAX-deficient HCT116 cells to Fas-mediated apoptosis. (a) On day 1, wild-type (BAX-positive) and BAX-deficient HCT116 cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, WT, or S387A) on day 2. On day 3, cells were treated overnight with nocodazole, and then a mitotic shake-off was performed on day 4. Subsequently, the cells were reseeded in nocodazole-containing medium and stimulated with 100 ng of FasL/ml. Lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody and BAX. (b) Lysates of BAX-positive and -negative cells that were treated as described in panel a were immunoblotted for PARP. (c and d) Apoptosis (annexin staining) and caspase-8 activity were determined. All experiments were performed in triplicate. Error bars represent the SD.
Figure Legend Snippet: A nonphosphorylatable S387 mutant of procaspase-8 sensitizes mitotic BAX-deficient HCT116 cells to Fas-mediated apoptosis. (a) On day 1, wild-type (BAX-positive) and BAX-deficient HCT116 cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, WT, or S387A) on day 2. On day 3, cells were treated overnight with nocodazole, and then a mitotic shake-off was performed on day 4. Subsequently, the cells were reseeded in nocodazole-containing medium and stimulated with 100 ng of FasL/ml. Lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody and BAX. (b) Lysates of BAX-positive and -negative cells that were treated as described in panel a were immunoblotted for PARP. (c and d) Apoptosis (annexin staining) and caspase-8 activity were determined. All experiments were performed in triplicate. Error bars represent the SD.

Techniques Used: Mutagenesis, Transfection, Staining, Activity Assay

Phosphorylation of procaspase-8 at Ser-387 in primary cells and tissues. (a) Lysates were prepared from human breast cancer tissues, premalignant ductal carcinoma in situ (DCIS) samples and normal breast tissues. Total protein was resolved by SDS-PAGE and immunoblotted for caspase-8 (pSer387), caspase-8, cyclin B1, and β-actin (top panel). Blue bars represent the level of caspase-8 (pSer387) normalized to the level of β-actin expression (lower panel). Red bars represent the level of cyclin B1 expression standardized to the level of β-actin expression. (b) Peripheral blood lymphocytes (PBLs) were treated with PHA for the times indicated. Cell lysates were immunoblotted for caspase-8 (pS387), caspase-8, histone H3 phosphorylated at Ser10 (H3 pS10), cyclin B1, Cdk1, Plk1, CD95/Fas, and β-actin (upper panels). To measure Cdk1/cyclin B1 activity in cell lysates of synchronized cells, immunoprecipitation assays were performed using a cyclin B1-specific antibody, and the purified proteins were subjected to kinase assays with histone H1 as the substrate (lower panel). (c) PBLs were cultured with PHA for 16 h. Cells were then washed three times and cultured for an additional 5 days in the presence of interleukin-2, followed by nocodazole treatment for 16 h and MG132 for 2 h. For the inhibition of Cdk1/cyclin B1, RO-3306 was added.
Figure Legend Snippet: Phosphorylation of procaspase-8 at Ser-387 in primary cells and tissues. (a) Lysates were prepared from human breast cancer tissues, premalignant ductal carcinoma in situ (DCIS) samples and normal breast tissues. Total protein was resolved by SDS-PAGE and immunoblotted for caspase-8 (pSer387), caspase-8, cyclin B1, and β-actin (top panel). Blue bars represent the level of caspase-8 (pSer387) normalized to the level of β-actin expression (lower panel). Red bars represent the level of cyclin B1 expression standardized to the level of β-actin expression. (b) Peripheral blood lymphocytes (PBLs) were treated with PHA for the times indicated. Cell lysates were immunoblotted for caspase-8 (pS387), caspase-8, histone H3 phosphorylated at Ser10 (H3 pS10), cyclin B1, Cdk1, Plk1, CD95/Fas, and β-actin (upper panels). To measure Cdk1/cyclin B1 activity in cell lysates of synchronized cells, immunoprecipitation assays were performed using a cyclin B1-specific antibody, and the purified proteins were subjected to kinase assays with histone H1 as the substrate (lower panel). (c) PBLs were cultured with PHA for 16 h. Cells were then washed three times and cultured for an additional 5 days in the presence of interleukin-2, followed by nocodazole treatment for 16 h and MG132 for 2 h. For the inhibition of Cdk1/cyclin B1, RO-3306 was added.

Techniques Used: In Situ, SDS Page, Expressing, Activity Assay, Immunoprecipitation, Purification, Cell Culture, Inhibition

Inhibition of the extrinsic death pathway in Fas-induced mitotic cells. (a) B lymphoblastoid SKW6.4 cells were enriched in G 1 /S phase by treatment with thymidine (lane 1), in pro-/metaphase with nocodazole for 16 h, followed by MG132 for 2 h (lane 2) or in a G 1 -like state with nocodazole for 16 h, followed by MG132 for 2 h and RO-3306 for the inhibition of Cdk1/cyclin B1 (lane 3) (upper panel). Cell lysates were immunoblotted for cyclin B1, histone H3 phosphorylated at Ser10 (H3 pS10), cyclin E, Plk1, and β-actin. The cell cycle status was monitored by flow cytometry (lower panel). (b) Cells treated as described in panel a were stimulated with 100 ng of FasL/ml, 10 ng of TRIAL/ml, or 10 ng of TNF-α/ml for the times indicated, and total cellular lysates were analyzed by Western blotting with anti-caspase-8 MAb C15. (c) Caspase-8 activity was determined by measuring the cleavage of the luminogenic substrate containing the IETD peptide. All experiments were performed in triplicate. Error bars represent the standard deviation (SD). (d) Apoptosis analyses were performed by using an annexin V kit. All experiments were performed in triplicate. Error bars represent the SD.
Figure Legend Snippet: Inhibition of the extrinsic death pathway in Fas-induced mitotic cells. (a) B lymphoblastoid SKW6.4 cells were enriched in G 1 /S phase by treatment with thymidine (lane 1), in pro-/metaphase with nocodazole for 16 h, followed by MG132 for 2 h (lane 2) or in a G 1 -like state with nocodazole for 16 h, followed by MG132 for 2 h and RO-3306 for the inhibition of Cdk1/cyclin B1 (lane 3) (upper panel). Cell lysates were immunoblotted for cyclin B1, histone H3 phosphorylated at Ser10 (H3 pS10), cyclin E, Plk1, and β-actin. The cell cycle status was monitored by flow cytometry (lower panel). (b) Cells treated as described in panel a were stimulated with 100 ng of FasL/ml, 10 ng of TRIAL/ml, or 10 ng of TNF-α/ml for the times indicated, and total cellular lysates were analyzed by Western blotting with anti-caspase-8 MAb C15. (c) Caspase-8 activity was determined by measuring the cleavage of the luminogenic substrate containing the IETD peptide. All experiments were performed in triplicate. Error bars represent the standard deviation (SD). (d) Apoptosis analyses were performed by using an annexin V kit. All experiments were performed in triplicate. Error bars represent the SD.

Techniques Used: Inhibition, Flow Cytometry, Cytometry, Western Blot, Activity Assay, Standard Deviation

Cdk1/cyclin B1 associates with and phosphorylates procaspase-8 in vitro at Ser-387. (a) The p10 subunit was mutated at different sites (S387A, SS386/387AA, and T419A), and the corresponding GST fusion proteins were incubated with active Cdk1/cyclin B1 and [γ- 32 P]ATP, analyzed by SDS-PAGE and visualized by autoradiography. (b) Bacterially expressed, purified GST-fused full-length caspase-8 (GST-WT), and the N-terminal (GST-NT) and C-terminal (GST-CT) subdomains of caspase-8 were incubated with lysates of HeLa cells that had been transfected with a Flag epitope-tagged Cdk1 (Flag-Cdk1) expression construct for pulldown assays. The Flag-Cdk1 protein that associated with GST-caspase-8 or its subdomains was detected by immunoblot with an anti-Flag antibody. (c) The binding of the GST-fused N-terminal subdomains (GST-2xDED, -DED1+, and -DED1) was also analyzed in pulldown assays. The Flag-Cdk1 protein that associated with GST-fused subdomains of the N-terminal portion of caspase-8 was detected by immunoblotting with an anti-Flag antibody. A 5% total input lane was also run.
Figure Legend Snippet: Cdk1/cyclin B1 associates with and phosphorylates procaspase-8 in vitro at Ser-387. (a) The p10 subunit was mutated at different sites (S387A, SS386/387AA, and T419A), and the corresponding GST fusion proteins were incubated with active Cdk1/cyclin B1 and [γ- 32 P]ATP, analyzed by SDS-PAGE and visualized by autoradiography. (b) Bacterially expressed, purified GST-fused full-length caspase-8 (GST-WT), and the N-terminal (GST-NT) and C-terminal (GST-CT) subdomains of caspase-8 were incubated with lysates of HeLa cells that had been transfected with a Flag epitope-tagged Cdk1 (Flag-Cdk1) expression construct for pulldown assays. The Flag-Cdk1 protein that associated with GST-caspase-8 or its subdomains was detected by immunoblot with an anti-Flag antibody. (c) The binding of the GST-fused N-terminal subdomains (GST-2xDED, -DED1+, and -DED1) was also analyzed in pulldown assays. The Flag-Cdk1 protein that associated with GST-fused subdomains of the N-terminal portion of caspase-8 was detected by immunoblotting with an anti-Flag antibody. A 5% total input lane was also run.

Techniques Used: In Vitro, Incubation, SDS Page, Autoradiography, Purification, Transfection, FLAG-tag, Expressing, Construct, Binding Assay

The phosphorylation of procaspase-8 correlates with Cdk1/cyclin B1 activity in vivo . (a) Lysates from asynchronous (−) or mitotic (+) HeLa cells were immunoprecipitated with caspase-8-, Cdk1-, or cyclin B1-specific antibodies. Anti-mouse IgG was used as a control. The immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted for caspase-8, cyclin B1, and Cdk1. Lanes 1 and 2 show 2.5% total input. (b) HeLa cells were arrested in G 1 /S using a double-thymidine block and released into fresh medium for 10 h. Semisynchronized cells were stained with DAPI (4′,6′-diamidino-2-phenylindole) and subsequently categorized according to their staining patterns. Cells were labeled using ABcasp.-8 (pSer387), caspase-8, or cyclin B1 antibodies for analysis by microscopy. Caspase-8-deficient HeLa cells were arrested in G 1 /S using a double-thymidine block and released into fresh medium for 10 h. Semisynchronized cells were stained with DAPI or labeled using ABcasp.-8 (pSer387) or cyclin B1 antibodies for analysis by microscopy. (c) To enrich for mitotic HeLa cells, an incubation with nocodazole (Noc) for 16 h was carried out; cells were captured by shake-off and then treated with MG132 for 2 h (lane 2). Shake-off cells were treated as described for lane 2, except that, 1 h after MG132 addition, the Cdk1 inhibitor RO-3306 was added (lane 3). The cell lysates were immunoblotted for caspase-8, caspase-8 (pS387), cyclin B1, Cdk1, pH3S10, securin, and β-actin. (d) HeLa cells were arrested in G 1 /S using a double-thymidine block (time zero) and released for the times indicated. The cellular lysates were immunoblotted for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, Plk1, CD95/Fas, and β-actin (upper left panels). To measure the Cdk1/cyclin B1 activity in cell lysates of synchronized cells, immunoprecipitation assays were performed with cyclin B1-specific antibodies, and immunoprecipitated proteins were then subjected to kinase assays with histone H1 as the substrate (lower left panel). The cell cycle status was monitored using flow cytometry (upper right panel). Boxed numbers exhibit the percentage of cells with 4 N DNA content. The mitotic index and distribution of mitotic phases of HeLa cells 10 h after release from the G 1 /S block are shown: the leftmost bar graph indicates 30% mitotic index 10 h after G 1 /S (of these, 46% were in prophase and 25% were in metaphase). The mitotic index was determined by counting 6 times 250 to 300 cells (lower right panel). All experiments were performed in triplicate. The error bars represent the SD. (e) Characterization of mock-transfected HeLa cells and of a cyclin B1-depleted HeLa clone using a stably integrated RNAi cassette (Cycl.B1 − -HeLa). Mitotic shake-off cells (mock-transfected and Cycl.B1 − ) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), cyclin B1, and β-actin (*, unspecific band). To measure Cdk1/cyclin B1 activity, immunoprecipitation assays were performed using cyclin B1-specific antibodies, and the immunoprecipitated proteins were subjected to kinase assays with histone H1 as the substrate (middle panel). Apoptosis analyses were performed by using an annexin V kit (lower panel). All experiments were performed in triplicate. Error bars represent the SD. (f) To enrich for mitotic SKW 6.4 cells, an incubation with nocodazole (Noc) for 16 h was carried out; cells were captured by shake-off and then treated with MG132 for 2 h. For the inhibition of Cdk1 activity, RO-3306 was added 1 h after MG132 treatment. Cells were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the anti-caspase-8 MAb C15. Apoptosis analyses were performed by using an annexin V kit (lower panel). All experiments were performed in triplicate. Error bars represent the SD.
Figure Legend Snippet: The phosphorylation of procaspase-8 correlates with Cdk1/cyclin B1 activity in vivo . (a) Lysates from asynchronous (−) or mitotic (+) HeLa cells were immunoprecipitated with caspase-8-, Cdk1-, or cyclin B1-specific antibodies. Anti-mouse IgG was used as a control. The immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted for caspase-8, cyclin B1, and Cdk1. Lanes 1 and 2 show 2.5% total input. (b) HeLa cells were arrested in G 1 /S using a double-thymidine block and released into fresh medium for 10 h. Semisynchronized cells were stained with DAPI (4′,6′-diamidino-2-phenylindole) and subsequently categorized according to their staining patterns. Cells were labeled using ABcasp.-8 (pSer387), caspase-8, or cyclin B1 antibodies for analysis by microscopy. Caspase-8-deficient HeLa cells were arrested in G 1 /S using a double-thymidine block and released into fresh medium for 10 h. Semisynchronized cells were stained with DAPI or labeled using ABcasp.-8 (pSer387) or cyclin B1 antibodies for analysis by microscopy. (c) To enrich for mitotic HeLa cells, an incubation with nocodazole (Noc) for 16 h was carried out; cells were captured by shake-off and then treated with MG132 for 2 h (lane 2). Shake-off cells were treated as described for lane 2, except that, 1 h after MG132 addition, the Cdk1 inhibitor RO-3306 was added (lane 3). The cell lysates were immunoblotted for caspase-8, caspase-8 (pS387), cyclin B1, Cdk1, pH3S10, securin, and β-actin. (d) HeLa cells were arrested in G 1 /S using a double-thymidine block (time zero) and released for the times indicated. The cellular lysates were immunoblotted for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, Plk1, CD95/Fas, and β-actin (upper left panels). To measure the Cdk1/cyclin B1 activity in cell lysates of synchronized cells, immunoprecipitation assays were performed with cyclin B1-specific antibodies, and immunoprecipitated proteins were then subjected to kinase assays with histone H1 as the substrate (lower left panel). The cell cycle status was monitored using flow cytometry (upper right panel). Boxed numbers exhibit the percentage of cells with 4 N DNA content. The mitotic index and distribution of mitotic phases of HeLa cells 10 h after release from the G 1 /S block are shown: the leftmost bar graph indicates 30% mitotic index 10 h after G 1 /S (of these, 46% were in prophase and 25% were in metaphase). The mitotic index was determined by counting 6 times 250 to 300 cells (lower right panel). All experiments were performed in triplicate. The error bars represent the SD. (e) Characterization of mock-transfected HeLa cells and of a cyclin B1-depleted HeLa clone using a stably integrated RNAi cassette (Cycl.B1 − -HeLa). Mitotic shake-off cells (mock-transfected and Cycl.B1 − ) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), cyclin B1, and β-actin (*, unspecific band). To measure Cdk1/cyclin B1 activity, immunoprecipitation assays were performed using cyclin B1-specific antibodies, and the immunoprecipitated proteins were subjected to kinase assays with histone H1 as the substrate (middle panel). Apoptosis analyses were performed by using an annexin V kit (lower panel). All experiments were performed in triplicate. Error bars represent the SD. (f) To enrich for mitotic SKW 6.4 cells, an incubation with nocodazole (Noc) for 16 h was carried out; cells were captured by shake-off and then treated with MG132 for 2 h. For the inhibition of Cdk1 activity, RO-3306 was added 1 h after MG132 treatment. Cells were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the anti-caspase-8 MAb C15. Apoptosis analyses were performed by using an annexin V kit (lower panel). All experiments were performed in triplicate. Error bars represent the SD.

Techniques Used: Activity Assay, In Vivo, Immunoprecipitation, SDS Page, Blocking Assay, Staining, Labeling, Microscopy, Incubation, Flow Cytometry, Cytometry, Transfection, Stable Transfection, Inhibition

A nonphosphorylatable S387 mutant of procaspase-8 sensitizes mitotic cells to Fas-mediated apoptosis. (a) HeLa cells were transiently transfected with vectors encoding Flag-tagged caspase-8 (Flag-WT, Flag-S387A, and Flag-S387E). At 18 h after transfection, cells were incubated with 100 ng of FasL/ml plus 1 μg of cycloheximide/ml for 3 and 6 h. Lysates were immunoblotted for exogenous caspase-8 with a Flag-specific antibody and for β-actin (upper panels). The signal intensity of immunoblotted, Flag-tagged caspase-8 was standardized to the level of β-actin expression (lower panel). All experiments were performed in triplicate. The error bars represent the SD. (b) Analysis of procaspase-8 processing in HeLa cells expressing different double-tagged (N-terminal Flag, C-terminal V5) forms of procaspase-8 (S387A, S387E). Lysates were immunoblotted for V5 and PARP. (c) Analysis of procaspase-8 processing in caspase-8 knockdown HeLa cell clones (caspase-8 − ) expressing different double-tagged (N-terminal Flag, C-terminal V5) forms of procaspase-8 (S387A, S387E). Lysates were immunoblotted for V5 and caspase-8. (d) On day 1, HeLa cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, WT, or S387A) on day 2. On day 3, the cells were treated overnight with nocodazole, and then a mitotic shake-off was performed on day 4. Subsequently, cells were reseeded in nocodazole-containing medium and stimulated with 100 ng of FasL/ml. The cell cycle status was monitored by flow cytometry (upper panels). Lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody, caspase-8, PARP, and β-actin (middle panels). Caspase-8 activity and apoptosis (annexin staining) were determined (lower panels). All experiments were performed in triplicate. Error bars represent the SD. (e) On day 1 HeLa cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, S387A, or S387E) on day 2. On day 3 cells were stimulated with 100 ng of FasL/ml. After 24 h, the cell numbers were determined, and lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody and β-actin. The experiment was performed in triplicate. Error bars represent the SD.
Figure Legend Snippet: A nonphosphorylatable S387 mutant of procaspase-8 sensitizes mitotic cells to Fas-mediated apoptosis. (a) HeLa cells were transiently transfected with vectors encoding Flag-tagged caspase-8 (Flag-WT, Flag-S387A, and Flag-S387E). At 18 h after transfection, cells were incubated with 100 ng of FasL/ml plus 1 μg of cycloheximide/ml for 3 and 6 h. Lysates were immunoblotted for exogenous caspase-8 with a Flag-specific antibody and for β-actin (upper panels). The signal intensity of immunoblotted, Flag-tagged caspase-8 was standardized to the level of β-actin expression (lower panel). All experiments were performed in triplicate. The error bars represent the SD. (b) Analysis of procaspase-8 processing in HeLa cells expressing different double-tagged (N-terminal Flag, C-terminal V5) forms of procaspase-8 (S387A, S387E). Lysates were immunoblotted for V5 and PARP. (c) Analysis of procaspase-8 processing in caspase-8 knockdown HeLa cell clones (caspase-8 − ) expressing different double-tagged (N-terminal Flag, C-terminal V5) forms of procaspase-8 (S387A, S387E). Lysates were immunoblotted for V5 and caspase-8. (d) On day 1, HeLa cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, WT, or S387A) on day 2. On day 3, the cells were treated overnight with nocodazole, and then a mitotic shake-off was performed on day 4. Subsequently, cells were reseeded in nocodazole-containing medium and stimulated with 100 ng of FasL/ml. The cell cycle status was monitored by flow cytometry (upper panels). Lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody, caspase-8, PARP, and β-actin (middle panels). Caspase-8 activity and apoptosis (annexin staining) were determined (lower panels). All experiments were performed in triplicate. Error bars represent the SD. (e) On day 1 HeLa cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, S387A, or S387E) on day 2. On day 3 cells were stimulated with 100 ng of FasL/ml. After 24 h, the cell numbers were determined, and lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody and β-actin. The experiment was performed in triplicate. Error bars represent the SD.

Techniques Used: Mutagenesis, Transfection, Incubation, Expressing, Clone Assay, Flow Cytometry, Cytometry, Activity Assay, Staining

21) Product Images from "Cdk1/Cyclin B1 Controls Fas-Mediated Apoptosis by Regulating Caspase-8 Activity ▿"

Article Title: Cdk1/Cyclin B1 Controls Fas-Mediated Apoptosis by Regulating Caspase-8 Activity ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00731-10

Inhibition of Cdk1 activity using the small molecule RO-3306 suppresses the phosphorylation of procaspase-8 at Ser-387 in vitro and in vivo . (a) Procaspase-8 purified from bacteria was subjected to kinase assays with Cdk1/cyclin B1 and increasing concentrations of RO-3306. Samples were immunoblotted for Cdk1, caspase-8, and caspase-8 (pSer387). (b) Treatment of proliferating human SW480 cancer cells with RO-3306 for 14 h led to a complete block of the cell cycle at the G 2 /M border. After releasing SW480 cells from RO-3306 a Western blot analysis was performed for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, and β-actin.
Figure Legend Snippet: Inhibition of Cdk1 activity using the small molecule RO-3306 suppresses the phosphorylation of procaspase-8 at Ser-387 in vitro and in vivo . (a) Procaspase-8 purified from bacteria was subjected to kinase assays with Cdk1/cyclin B1 and increasing concentrations of RO-3306. Samples were immunoblotted for Cdk1, caspase-8, and caspase-8 (pSer387). (b) Treatment of proliferating human SW480 cancer cells with RO-3306 for 14 h led to a complete block of the cell cycle at the G 2 /M border. After releasing SW480 cells from RO-3306 a Western blot analysis was performed for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, and β-actin.

Techniques Used: Inhibition, Activity Assay, In Vitro, In Vivo, Purification, Blocking Assay, Western Blot

The phosphorylation of procaspase-8 correlates with Cdk1/cyclin B1 activity in cell lines of different origin. (a) T47D, SW480, and MDA-MB-231 cells were arrested in G 1 /S using a double-thymidine block (time zero) and released for the times indicated. The cellular lysates were immunoblotted for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, Plk1, and β-actin. (b) Characterization of a cyclin B1-depleted clone using a stably integrated RNAi cassette (Cycl.B1 − -HeLa). Mitotic shake-off cells (wild-type and Cycl.B1 − ) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), cyclin B1, and β-actin (*, unspecific band) (left panel). Characterization of Cdk1-depleted cells by RNAi. Mitotic shake-off cells (wild-type and Cdk1-depleted cells) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), Cdk1, and β-actin (*, unspecific band) (right panel).
Figure Legend Snippet: The phosphorylation of procaspase-8 correlates with Cdk1/cyclin B1 activity in cell lines of different origin. (a) T47D, SW480, and MDA-MB-231 cells were arrested in G 1 /S using a double-thymidine block (time zero) and released for the times indicated. The cellular lysates were immunoblotted for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, Plk1, and β-actin. (b) Characterization of a cyclin B1-depleted clone using a stably integrated RNAi cassette (Cycl.B1 − -HeLa). Mitotic shake-off cells (wild-type and Cycl.B1 − ) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), cyclin B1, and β-actin (*, unspecific band) (left panel). Characterization of Cdk1-depleted cells by RNAi. Mitotic shake-off cells (wild-type and Cdk1-depleted cells) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), Cdk1, and β-actin (*, unspecific band) (right panel).

Techniques Used: Activity Assay, Multiple Displacement Amplification, Blocking Assay, Stable Transfection

Cdk1/cyclin B1 phosphorylates procaspase-8 in vitro at Ser-387. (a) Recombinant GST-fused subdomains of procaspase-8 were analyzed by SDS-PAGE (lower left panel). Subdomains were incubated with active Cdk1/cyclin B1 and [γ- 32 P]ATP, analyzed by SDS-PAGE, and visualized by autoradiography (upper left panel). This was followed by phospho amino acid analysis (lower right panel). (b) Wild-type (WT) and mutated (S387A, S387E) recombinant full-length GST-caspase-8 proteins were subjected to kinase assays using Cdk1/cyclin B1, analyzed by SDS-PAGE, and autoradiography. (c) Recombinant caspase-8 (WT) was incubated with active Cdk1/cyclin B1. Samples were immunoblotted with antibodies specific for caspase-8, for a caspase-8 peptide [ABcasp.-8(pSer387)] containing phospho-Ser387 (YLEMDLSpSPQTRY) and for Cdk1. (d) GST, recombinant caspase-8 (GST-WT), and a mutated form (GST-S387A) were incubated with or without active Cdk1/cyclin B1 and immunoblotted with ABcasp.-8(pSer387) (e) Recombinant caspase-8 was incubated with or without active Cdk1/cyclin B1 in kinase buffer, followed by treatment with 100 U of λ-phosphatase and immunoblotted with ABcasp.-8(pSer387).
Figure Legend Snippet: Cdk1/cyclin B1 phosphorylates procaspase-8 in vitro at Ser-387. (a) Recombinant GST-fused subdomains of procaspase-8 were analyzed by SDS-PAGE (lower left panel). Subdomains were incubated with active Cdk1/cyclin B1 and [γ- 32 P]ATP, analyzed by SDS-PAGE, and visualized by autoradiography (upper left panel). This was followed by phospho amino acid analysis (lower right panel). (b) Wild-type (WT) and mutated (S387A, S387E) recombinant full-length GST-caspase-8 proteins were subjected to kinase assays using Cdk1/cyclin B1, analyzed by SDS-PAGE, and autoradiography. (c) Recombinant caspase-8 (WT) was incubated with active Cdk1/cyclin B1. Samples were immunoblotted with antibodies specific for caspase-8, for a caspase-8 peptide [ABcasp.-8(pSer387)] containing phospho-Ser387 (YLEMDLSpSPQTRY) and for Cdk1. (d) GST, recombinant caspase-8 (GST-WT), and a mutated form (GST-S387A) were incubated with or without active Cdk1/cyclin B1 and immunoblotted with ABcasp.-8(pSer387) (e) Recombinant caspase-8 was incubated with or without active Cdk1/cyclin B1 in kinase buffer, followed by treatment with 100 U of λ-phosphatase and immunoblotted with ABcasp.-8(pSer387).

Techniques Used: In Vitro, Recombinant, SDS Page, Incubation, Autoradiography

A nonphosphorylatable S387 mutant of procaspase-8 sensitizes mitotic BAX-deficient HCT116 cells to Fas-mediated apoptosis. (a) On day 1, wild-type (BAX-positive) and BAX-deficient HCT116 cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, WT, or S387A) on day 2. On day 3, cells were treated overnight with nocodazole, and then a mitotic shake-off was performed on day 4. Subsequently, the cells were reseeded in nocodazole-containing medium and stimulated with 100 ng of FasL/ml. Lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody and BAX. (b) Lysates of BAX-positive and -negative cells that were treated as described in panel a were immunoblotted for PARP. (c and d) Apoptosis (annexin staining) and caspase-8 activity were determined. All experiments were performed in triplicate. Error bars represent the SD.
Figure Legend Snippet: A nonphosphorylatable S387 mutant of procaspase-8 sensitizes mitotic BAX-deficient HCT116 cells to Fas-mediated apoptosis. (a) On day 1, wild-type (BAX-positive) and BAX-deficient HCT116 cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, WT, or S387A) on day 2. On day 3, cells were treated overnight with nocodazole, and then a mitotic shake-off was performed on day 4. Subsequently, the cells were reseeded in nocodazole-containing medium and stimulated with 100 ng of FasL/ml. Lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody and BAX. (b) Lysates of BAX-positive and -negative cells that were treated as described in panel a were immunoblotted for PARP. (c and d) Apoptosis (annexin staining) and caspase-8 activity were determined. All experiments were performed in triplicate. Error bars represent the SD.

Techniques Used: Mutagenesis, Transfection, Staining, Activity Assay

Phosphorylation of procaspase-8 at Ser-387 in primary cells and tissues. (a) Lysates were prepared from human breast cancer tissues, premalignant ductal carcinoma in situ (DCIS) samples and normal breast tissues. Total protein was resolved by SDS-PAGE and immunoblotted for caspase-8 (pSer387), caspase-8, cyclin B1, and β-actin (top panel). Blue bars represent the level of caspase-8 (pSer387) normalized to the level of β-actin expression (lower panel). Red bars represent the level of cyclin B1 expression standardized to the level of β-actin expression. (b) Peripheral blood lymphocytes (PBLs) were treated with PHA for the times indicated. Cell lysates were immunoblotted for caspase-8 (pS387), caspase-8, histone H3 phosphorylated at Ser10 (H3 pS10), cyclin B1, Cdk1, Plk1, CD95/Fas, and β-actin (upper panels). To measure Cdk1/cyclin B1 activity in cell lysates of synchronized cells, immunoprecipitation assays were performed using a cyclin B1-specific antibody, and the purified proteins were subjected to kinase assays with histone H1 as the substrate (lower panel). (c) PBLs were cultured with PHA for 16 h. Cells were then washed three times and cultured for an additional 5 days in the presence of interleukin-2, followed by nocodazole treatment for 16 h and MG132 for 2 h. For the inhibition of Cdk1/cyclin B1, RO-3306 was added.
Figure Legend Snippet: Phosphorylation of procaspase-8 at Ser-387 in primary cells and tissues. (a) Lysates were prepared from human breast cancer tissues, premalignant ductal carcinoma in situ (DCIS) samples and normal breast tissues. Total protein was resolved by SDS-PAGE and immunoblotted for caspase-8 (pSer387), caspase-8, cyclin B1, and β-actin (top panel). Blue bars represent the level of caspase-8 (pSer387) normalized to the level of β-actin expression (lower panel). Red bars represent the level of cyclin B1 expression standardized to the level of β-actin expression. (b) Peripheral blood lymphocytes (PBLs) were treated with PHA for the times indicated. Cell lysates were immunoblotted for caspase-8 (pS387), caspase-8, histone H3 phosphorylated at Ser10 (H3 pS10), cyclin B1, Cdk1, Plk1, CD95/Fas, and β-actin (upper panels). To measure Cdk1/cyclin B1 activity in cell lysates of synchronized cells, immunoprecipitation assays were performed using a cyclin B1-specific antibody, and the purified proteins were subjected to kinase assays with histone H1 as the substrate (lower panel). (c) PBLs were cultured with PHA for 16 h. Cells were then washed three times and cultured for an additional 5 days in the presence of interleukin-2, followed by nocodazole treatment for 16 h and MG132 for 2 h. For the inhibition of Cdk1/cyclin B1, RO-3306 was added.

Techniques Used: In Situ, SDS Page, Expressing, Activity Assay, Immunoprecipitation, Purification, Cell Culture, Inhibition

Inhibition of the extrinsic death pathway in Fas-induced mitotic cells. (a) B lymphoblastoid SKW6.4 cells were enriched in G 1 /S phase by treatment with thymidine (lane 1), in pro-/metaphase with nocodazole for 16 h, followed by MG132 for 2 h (lane 2) or in a G 1 -like state with nocodazole for 16 h, followed by MG132 for 2 h and RO-3306 for the inhibition of Cdk1/cyclin B1 (lane 3) (upper panel). Cell lysates were immunoblotted for cyclin B1, histone H3 phosphorylated at Ser10 (H3 pS10), cyclin E, Plk1, and β-actin. The cell cycle status was monitored by flow cytometry (lower panel). (b) Cells treated as described in panel a were stimulated with 100 ng of FasL/ml, 10 ng of TRIAL/ml, or 10 ng of TNF-α/ml for the times indicated, and total cellular lysates were analyzed by Western blotting with anti-caspase-8 MAb C15. (c) Caspase-8 activity was determined by measuring the cleavage of the luminogenic substrate containing the IETD peptide. All experiments were performed in triplicate. Error bars represent the standard deviation (SD). (d) Apoptosis analyses were performed by using an annexin V kit. All experiments were performed in triplicate. Error bars represent the SD.
Figure Legend Snippet: Inhibition of the extrinsic death pathway in Fas-induced mitotic cells. (a) B lymphoblastoid SKW6.4 cells were enriched in G 1 /S phase by treatment with thymidine (lane 1), in pro-/metaphase with nocodazole for 16 h, followed by MG132 for 2 h (lane 2) or in a G 1 -like state with nocodazole for 16 h, followed by MG132 for 2 h and RO-3306 for the inhibition of Cdk1/cyclin B1 (lane 3) (upper panel). Cell lysates were immunoblotted for cyclin B1, histone H3 phosphorylated at Ser10 (H3 pS10), cyclin E, Plk1, and β-actin. The cell cycle status was monitored by flow cytometry (lower panel). (b) Cells treated as described in panel a were stimulated with 100 ng of FasL/ml, 10 ng of TRIAL/ml, or 10 ng of TNF-α/ml for the times indicated, and total cellular lysates were analyzed by Western blotting with anti-caspase-8 MAb C15. (c) Caspase-8 activity was determined by measuring the cleavage of the luminogenic substrate containing the IETD peptide. All experiments were performed in triplicate. Error bars represent the standard deviation (SD). (d) Apoptosis analyses were performed by using an annexin V kit. All experiments were performed in triplicate. Error bars represent the SD.

Techniques Used: Inhibition, Flow Cytometry, Cytometry, Western Blot, Activity Assay, Standard Deviation

Cdk1/cyclin B1 associates with and phosphorylates procaspase-8 in vitro at Ser-387. (a) The p10 subunit was mutated at different sites (S387A, SS386/387AA, and T419A), and the corresponding GST fusion proteins were incubated with active Cdk1/cyclin B1 and [γ- 32 P]ATP, analyzed by SDS-PAGE and visualized by autoradiography. (b) Bacterially expressed, purified GST-fused full-length caspase-8 (GST-WT), and the N-terminal (GST-NT) and C-terminal (GST-CT) subdomains of caspase-8 were incubated with lysates of HeLa cells that had been transfected with a Flag epitope-tagged Cdk1 (Flag-Cdk1) expression construct for pulldown assays. The Flag-Cdk1 protein that associated with GST-caspase-8 or its subdomains was detected by immunoblot with an anti-Flag antibody. (c) The binding of the GST-fused N-terminal subdomains (GST-2xDED, -DED1+, and -DED1) was also analyzed in pulldown assays. The Flag-Cdk1 protein that associated with GST-fused subdomains of the N-terminal portion of caspase-8 was detected by immunoblotting with an anti-Flag antibody. A 5% total input lane was also run.
Figure Legend Snippet: Cdk1/cyclin B1 associates with and phosphorylates procaspase-8 in vitro at Ser-387. (a) The p10 subunit was mutated at different sites (S387A, SS386/387AA, and T419A), and the corresponding GST fusion proteins were incubated with active Cdk1/cyclin B1 and [γ- 32 P]ATP, analyzed by SDS-PAGE and visualized by autoradiography. (b) Bacterially expressed, purified GST-fused full-length caspase-8 (GST-WT), and the N-terminal (GST-NT) and C-terminal (GST-CT) subdomains of caspase-8 were incubated with lysates of HeLa cells that had been transfected with a Flag epitope-tagged Cdk1 (Flag-Cdk1) expression construct for pulldown assays. The Flag-Cdk1 protein that associated with GST-caspase-8 or its subdomains was detected by immunoblot with an anti-Flag antibody. (c) The binding of the GST-fused N-terminal subdomains (GST-2xDED, -DED1+, and -DED1) was also analyzed in pulldown assays. The Flag-Cdk1 protein that associated with GST-fused subdomains of the N-terminal portion of caspase-8 was detected by immunoblotting with an anti-Flag antibody. A 5% total input lane was also run.

Techniques Used: In Vitro, Incubation, SDS Page, Autoradiography, Purification, Transfection, FLAG-tag, Expressing, Construct, Binding Assay

The phosphorylation of procaspase-8 correlates with Cdk1/cyclin B1 activity in vivo . (a) Lysates from asynchronous (−) or mitotic (+) HeLa cells were immunoprecipitated with caspase-8-, Cdk1-, or cyclin B1-specific antibodies. Anti-mouse IgG was used as a control. The immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted for caspase-8, cyclin B1, and Cdk1. Lanes 1 and 2 show 2.5% total input. (b) HeLa cells were arrested in G 1 /S using a double-thymidine block and released into fresh medium for 10 h. Semisynchronized cells were stained with DAPI (4′,6′-diamidino-2-phenylindole) and subsequently categorized according to their staining patterns. Cells were labeled using ABcasp.-8 (pSer387), caspase-8, or cyclin B1 antibodies for analysis by microscopy. Caspase-8-deficient HeLa cells were arrested in G 1 /S using a double-thymidine block and released into fresh medium for 10 h. Semisynchronized cells were stained with DAPI or labeled using ABcasp.-8 (pSer387) or cyclin B1 antibodies for analysis by microscopy. (c) To enrich for mitotic HeLa cells, an incubation with nocodazole (Noc) for 16 h was carried out; cells were captured by shake-off and then treated with MG132 for 2 h (lane 2). Shake-off cells were treated as described for lane 2, except that, 1 h after MG132 addition, the Cdk1 inhibitor RO-3306 was added (lane 3). The cell lysates were immunoblotted for caspase-8, caspase-8 (pS387), cyclin B1, Cdk1, pH3S10, securin, and β-actin. (d) HeLa cells were arrested in G 1 /S using a double-thymidine block (time zero) and released for the times indicated. The cellular lysates were immunoblotted for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, Plk1, CD95/Fas, and β-actin (upper left panels). To measure the Cdk1/cyclin B1 activity in cell lysates of synchronized cells, immunoprecipitation assays were performed with cyclin B1-specific antibodies, and immunoprecipitated proteins were then subjected to kinase assays with histone H1 as the substrate (lower left panel). The cell cycle status was monitored using flow cytometry (upper right panel). Boxed numbers exhibit the percentage of cells with 4 N DNA content. The mitotic index and distribution of mitotic phases of HeLa cells 10 h after release from the G 1 /S block are shown: the leftmost bar graph indicates 30% mitotic index 10 h after G 1 /S (of these, 46% were in prophase and 25% were in metaphase). The mitotic index was determined by counting 6 times 250 to 300 cells (lower right panel). All experiments were performed in triplicate. The error bars represent the SD. (e) Characterization of mock-transfected HeLa cells and of a cyclin B1-depleted HeLa clone using a stably integrated RNAi cassette (Cycl.B1 − -HeLa). Mitotic shake-off cells (mock-transfected and Cycl.B1 − ) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), cyclin B1, and β-actin (*, unspecific band). To measure Cdk1/cyclin B1 activity, immunoprecipitation assays were performed using cyclin B1-specific antibodies, and the immunoprecipitated proteins were subjected to kinase assays with histone H1 as the substrate (middle panel). Apoptosis analyses were performed by using an annexin V kit (lower panel). All experiments were performed in triplicate. Error bars represent the SD. (f) To enrich for mitotic SKW 6.4 cells, an incubation with nocodazole (Noc) for 16 h was carried out; cells were captured by shake-off and then treated with MG132 for 2 h. For the inhibition of Cdk1 activity, RO-3306 was added 1 h after MG132 treatment. Cells were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the anti-caspase-8 MAb C15. Apoptosis analyses were performed by using an annexin V kit (lower panel). All experiments were performed in triplicate. Error bars represent the SD.
Figure Legend Snippet: The phosphorylation of procaspase-8 correlates with Cdk1/cyclin B1 activity in vivo . (a) Lysates from asynchronous (−) or mitotic (+) HeLa cells were immunoprecipitated with caspase-8-, Cdk1-, or cyclin B1-specific antibodies. Anti-mouse IgG was used as a control. The immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted for caspase-8, cyclin B1, and Cdk1. Lanes 1 and 2 show 2.5% total input. (b) HeLa cells were arrested in G 1 /S using a double-thymidine block and released into fresh medium for 10 h. Semisynchronized cells were stained with DAPI (4′,6′-diamidino-2-phenylindole) and subsequently categorized according to their staining patterns. Cells were labeled using ABcasp.-8 (pSer387), caspase-8, or cyclin B1 antibodies for analysis by microscopy. Caspase-8-deficient HeLa cells were arrested in G 1 /S using a double-thymidine block and released into fresh medium for 10 h. Semisynchronized cells were stained with DAPI or labeled using ABcasp.-8 (pSer387) or cyclin B1 antibodies for analysis by microscopy. (c) To enrich for mitotic HeLa cells, an incubation with nocodazole (Noc) for 16 h was carried out; cells were captured by shake-off and then treated with MG132 for 2 h (lane 2). Shake-off cells were treated as described for lane 2, except that, 1 h after MG132 addition, the Cdk1 inhibitor RO-3306 was added (lane 3). The cell lysates were immunoblotted for caspase-8, caspase-8 (pS387), cyclin B1, Cdk1, pH3S10, securin, and β-actin. (d) HeLa cells were arrested in G 1 /S using a double-thymidine block (time zero) and released for the times indicated. The cellular lysates were immunoblotted for caspase-8 (pS387), caspase-8, pH3S10, cyclin B1, Cdk1, Plk1, CD95/Fas, and β-actin (upper left panels). To measure the Cdk1/cyclin B1 activity in cell lysates of synchronized cells, immunoprecipitation assays were performed with cyclin B1-specific antibodies, and immunoprecipitated proteins were then subjected to kinase assays with histone H1 as the substrate (lower left panel). The cell cycle status was monitored using flow cytometry (upper right panel). Boxed numbers exhibit the percentage of cells with 4 N DNA content. The mitotic index and distribution of mitotic phases of HeLa cells 10 h after release from the G 1 /S block are shown: the leftmost bar graph indicates 30% mitotic index 10 h after G 1 /S (of these, 46% were in prophase and 25% were in metaphase). The mitotic index was determined by counting 6 times 250 to 300 cells (lower right panel). All experiments were performed in triplicate. The error bars represent the SD. (e) Characterization of mock-transfected HeLa cells and of a cyclin B1-depleted HeLa clone using a stably integrated RNAi cassette (Cycl.B1 − -HeLa). Mitotic shake-off cells (mock-transfected and Cycl.B1 − ) were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the caspase-8 MAb C15 and antibodies for caspase-8 (pS387), cyclin B1, and β-actin (*, unspecific band). To measure Cdk1/cyclin B1 activity, immunoprecipitation assays were performed using cyclin B1-specific antibodies, and the immunoprecipitated proteins were subjected to kinase assays with histone H1 as the substrate (middle panel). Apoptosis analyses were performed by using an annexin V kit (lower panel). All experiments were performed in triplicate. Error bars represent the SD. (f) To enrich for mitotic SKW 6.4 cells, an incubation with nocodazole (Noc) for 16 h was carried out; cells were captured by shake-off and then treated with MG132 for 2 h. For the inhibition of Cdk1 activity, RO-3306 was added 1 h after MG132 treatment. Cells were stimulated with 100 ng of FasL/ml for the times indicated. Lysates were immunoblotted to analyze procaspase-8 processing using the anti-caspase-8 MAb C15. Apoptosis analyses were performed by using an annexin V kit (lower panel). All experiments were performed in triplicate. Error bars represent the SD.

Techniques Used: Activity Assay, In Vivo, Immunoprecipitation, SDS Page, Blocking Assay, Staining, Labeling, Microscopy, Incubation, Flow Cytometry, Cytometry, Transfection, Stable Transfection, Inhibition

A nonphosphorylatable S387 mutant of procaspase-8 sensitizes mitotic cells to Fas-mediated apoptosis. (a) HeLa cells were transiently transfected with vectors encoding Flag-tagged caspase-8 (Flag-WT, Flag-S387A, and Flag-S387E). At 18 h after transfection, cells were incubated with 100 ng of FasL/ml plus 1 μg of cycloheximide/ml for 3 and 6 h. Lysates were immunoblotted for exogenous caspase-8 with a Flag-specific antibody and for β-actin (upper panels). The signal intensity of immunoblotted, Flag-tagged caspase-8 was standardized to the level of β-actin expression (lower panel). All experiments were performed in triplicate. The error bars represent the SD. (b) Analysis of procaspase-8 processing in HeLa cells expressing different double-tagged (N-terminal Flag, C-terminal V5) forms of procaspase-8 (S387A, S387E). Lysates were immunoblotted for V5 and PARP. (c) Analysis of procaspase-8 processing in caspase-8 knockdown HeLa cell clones (caspase-8 − ) expressing different double-tagged (N-terminal Flag, C-terminal V5) forms of procaspase-8 (S387A, S387E). Lysates were immunoblotted for V5 and caspase-8. (d) On day 1, HeLa cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, WT, or S387A) on day 2. On day 3, the cells were treated overnight with nocodazole, and then a mitotic shake-off was performed on day 4. Subsequently, cells were reseeded in nocodazole-containing medium and stimulated with 100 ng of FasL/ml. The cell cycle status was monitored by flow cytometry (upper panels). Lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody, caspase-8, PARP, and β-actin (middle panels). Caspase-8 activity and apoptosis (annexin staining) were determined (lower panels). All experiments were performed in triplicate. Error bars represent the SD. (e) On day 1 HeLa cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, S387A, or S387E) on day 2. On day 3 cells were stimulated with 100 ng of FasL/ml. After 24 h, the cell numbers were determined, and lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody and β-actin. The experiment was performed in triplicate. Error bars represent the SD.
Figure Legend Snippet: A nonphosphorylatable S387 mutant of procaspase-8 sensitizes mitotic cells to Fas-mediated apoptosis. (a) HeLa cells were transiently transfected with vectors encoding Flag-tagged caspase-8 (Flag-WT, Flag-S387A, and Flag-S387E). At 18 h after transfection, cells were incubated with 100 ng of FasL/ml plus 1 μg of cycloheximide/ml for 3 and 6 h. Lysates were immunoblotted for exogenous caspase-8 with a Flag-specific antibody and for β-actin (upper panels). The signal intensity of immunoblotted, Flag-tagged caspase-8 was standardized to the level of β-actin expression (lower panel). All experiments were performed in triplicate. The error bars represent the SD. (b) Analysis of procaspase-8 processing in HeLa cells expressing different double-tagged (N-terminal Flag, C-terminal V5) forms of procaspase-8 (S387A, S387E). Lysates were immunoblotted for V5 and PARP. (c) Analysis of procaspase-8 processing in caspase-8 knockdown HeLa cell clones (caspase-8 − ) expressing different double-tagged (N-terminal Flag, C-terminal V5) forms of procaspase-8 (S387A, S387E). Lysates were immunoblotted for V5 and caspase-8. (d) On day 1, HeLa cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, WT, or S387A) on day 2. On day 3, the cells were treated overnight with nocodazole, and then a mitotic shake-off was performed on day 4. Subsequently, cells were reseeded in nocodazole-containing medium and stimulated with 100 ng of FasL/ml. The cell cycle status was monitored by flow cytometry (upper panels). Lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody, caspase-8, PARP, and β-actin (middle panels). Caspase-8 activity and apoptosis (annexin staining) were determined (lower panels). All experiments were performed in triplicate. Error bars represent the SD. (e) On day 1 HeLa cells were transfected with siRNA targeting the untranslated region of caspase-8, followed by the transfection of Flag-tagged caspase-8 (mock, S387A, or S387E) on day 2. On day 3 cells were stimulated with 100 ng of FasL/ml. After 24 h, the cell numbers were determined, and lysates were immunoblotted for exogenous caspase-8 using a Flag-specific antibody and β-actin. The experiment was performed in triplicate. Error bars represent the SD.

Techniques Used: Mutagenesis, Transfection, Incubation, Expressing, Clone Assay, Flow Cytometry, Cytometry, Activity Assay, Staining

22) Product Images from "FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes"

Article Title: FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1302839

FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome
Figure Legend Snippet: FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome

Techniques Used:

In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection
Figure Legend Snippet: In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection

Techniques Used: In Vivo, Infection

FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli
Figure Legend Snippet: FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli

Techniques Used:

Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome

Techniques Used: Activation Assay

Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation
Figure Legend Snippet: Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation

Techniques Used: Activation Assay

Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome

Techniques Used: Activation Assay

23) Product Images from "FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes"

Article Title: FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1302839

FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome
Figure Legend Snippet: FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome

Techniques Used:

In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection
Figure Legend Snippet: In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection

Techniques Used: In Vivo, Infection

FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli
Figure Legend Snippet: FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli

Techniques Used:

Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome

Techniques Used: Activation Assay

Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation
Figure Legend Snippet: Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation

Techniques Used: Activation Assay

Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome

Techniques Used: Activation Assay

24) Product Images from "FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes"

Article Title: FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1302839

FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome
Figure Legend Snippet: FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome

Techniques Used:

In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection
Figure Legend Snippet: In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection

Techniques Used: In Vivo, Infection

FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli
Figure Legend Snippet: FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli

Techniques Used:

Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome

Techniques Used: Activation Assay

Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation
Figure Legend Snippet: Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation

Techniques Used: Activation Assay

Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome

Techniques Used: Activation Assay

25) Product Images from "FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes"

Article Title: FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1302839

FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome
Figure Legend Snippet: FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome

Techniques Used:

In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection
Figure Legend Snippet: In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection

Techniques Used: In Vivo, Infection

FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli
Figure Legend Snippet: FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli

Techniques Used:

Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome

Techniques Used: Activation Assay

Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation
Figure Legend Snippet: Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation

Techniques Used: Activation Assay

Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome

Techniques Used: Activation Assay

26) Product Images from "FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes"

Article Title: FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1302839

FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome
Figure Legend Snippet: FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome

Techniques Used:

In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection
Figure Legend Snippet: In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection

Techniques Used: In Vivo, Infection

FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli
Figure Legend Snippet: FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli

Techniques Used:

Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome

Techniques Used: Activation Assay

Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation
Figure Legend Snippet: Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation

Techniques Used: Activation Assay

Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome

Techniques Used: Activation Assay

27) Product Images from "Retraction: Fatty acid synthase is a novel therapeutic target in multiple myeloma"

Article Title: Retraction: Fatty acid synthase is a novel therapeutic target in multiple myeloma

Journal: British Journal of Haematology

doi: 10.1111/j.1365-2141.2008.07114.x

Cerulenin induces apoptosis via activation of caspase-independent pathway. (A) MM.1S cells were cultured for 24 h with Cerulenin at the indicated doses. Induction of apoptosis by Cerulenin was determined by Apo2·7 staining and flow-cytometric analysis. (B) MM.1S cells were cultured with Cerulenin (50 μmol/l) for the indicated times (left panel), and preincubated with or without Z-VAD-FMK (50 μmol/l) for 1 h prior to treatment with Cerulenin for 12 h at the indicated doses (right panel). Total cell lysates (20 μg /lane) were subjected to Western blotting using anti-caspase -8, -9, -3, PARP, and α-tubulin Abs. FL, CF indicate the full length and cleaved form, respectively. (C, D) MM.1S cells were treated with the indicated dose of Cerulenin for 24 h, with or without Z-VAD-FMK (25 μmol/l or 50 μmol/l) 1 h pretreatment. Cytotoxicity was determined by MTT assay (C). Values represent mean ± SD of quadruplicate cultures. The percentage of apoptotic cells was determined by flow-cytometric analysis for APO2·7 staining (D). (E) Mitochondrial proteins AIF and Endo G were released into the cytosolic fraction from mitochondria after Cerulenin (50 μmol/l) treatment in MM.1S cells. Total cell lysates (20 μg/lane) were subjected to Western blotting using anti-AIF, Endo G, VDAC and α-tubulin Abs.
Figure Legend Snippet: Cerulenin induces apoptosis via activation of caspase-independent pathway. (A) MM.1S cells were cultured for 24 h with Cerulenin at the indicated doses. Induction of apoptosis by Cerulenin was determined by Apo2·7 staining and flow-cytometric analysis. (B) MM.1S cells were cultured with Cerulenin (50 μmol/l) for the indicated times (left panel), and preincubated with or without Z-VAD-FMK (50 μmol/l) for 1 h prior to treatment with Cerulenin for 12 h at the indicated doses (right panel). Total cell lysates (20 μg /lane) were subjected to Western blotting using anti-caspase -8, -9, -3, PARP, and α-tubulin Abs. FL, CF indicate the full length and cleaved form, respectively. (C, D) MM.1S cells were treated with the indicated dose of Cerulenin for 24 h, with or without Z-VAD-FMK (25 μmol/l or 50 μmol/l) 1 h pretreatment. Cytotoxicity was determined by MTT assay (C). Values represent mean ± SD of quadruplicate cultures. The percentage of apoptotic cells was determined by flow-cytometric analysis for APO2·7 staining (D). (E) Mitochondrial proteins AIF and Endo G were released into the cytosolic fraction from mitochondria after Cerulenin (50 μmol/l) treatment in MM.1S cells. Total cell lysates (20 μg/lane) were subjected to Western blotting using anti-AIF, Endo G, VDAC and α-tubulin Abs.

Techniques Used: Activation Assay, Cell Culture, Staining, Flow Cytometry, Western Blot, MTT Assay

28) Product Images from "Bortezomib Congeners Induce Apoptosis of Hepatocellular Carcinoma via CIP2A Inhibition"

Article Title: Bortezomib Congeners Induce Apoptosis of Hepatocellular Carcinoma via CIP2A Inhibition

Journal: Molecules

doi: 10.3390/molecules181215398

Western blot analysis of Akt (Akt1), p-Akt (Ser473), caspases and PARP levels. Sk-hep1 cells were exposed to bortezomib and derivatives at 500 nM in DMEM with 5% FBS for 24 h. Cell lysates were prepared for Akt (Akt1), p-Akt (Ser473), caspase-8, caspase-9, caspase-3, and PARP. CF, cleaved form (activated form).
Figure Legend Snippet: Western blot analysis of Akt (Akt1), p-Akt (Ser473), caspases and PARP levels. Sk-hep1 cells were exposed to bortezomib and derivatives at 500 nM in DMEM with 5% FBS for 24 h. Cell lysates were prepared for Akt (Akt1), p-Akt (Ser473), caspase-8, caspase-9, caspase-3, and PARP. CF, cleaved form (activated form).

Techniques Used: Western Blot

29) Product Images from "Deletion of caveolin‐1 attenuates LPS/GalN‐induced acute liver injury in mice, et al. Deletion of caveolin‐1 attenuates LPS/GalN‐induced acute liver injury in mice"

Article Title: Deletion of caveolin‐1 attenuates LPS/GalN‐induced acute liver injury in mice, et al. Deletion of caveolin‐1 attenuates LPS/GalN‐induced acute liver injury in mice

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13831

Reduced cell apoptosis and activation of caspase‐3, caspase‐8 and caspase‐9 in livers of LPS /GalN‐treated Cav‐1 −/− mice. WT and Cav‐1 −/− mice were challenged with LPS /GalN for 5 hours. A, TUNEL staining (green) and confocal microscopy of apoptotic cells in WT and Cav‐1 −/− liver. Nuclei were stained with DAPI . B and C,. Immunohistochemistry of cleaved (activated) caspase‐3 (B) and caspase‐8 (C) in livers. Ratio of area of cleaved caspase‐3‐ and caspase‐8‐positive cells were calculated (n = 5). D, Western blot analysis of protein levels of cleaved caspase‐3 and caspase‐8 in liver lysates. E, ELISA of activated caspase‐9 in livers (n = 6). Bar, 200 μm. Data are mean ± SEM . * P
Figure Legend Snippet: Reduced cell apoptosis and activation of caspase‐3, caspase‐8 and caspase‐9 in livers of LPS /GalN‐treated Cav‐1 −/− mice. WT and Cav‐1 −/− mice were challenged with LPS /GalN for 5 hours. A, TUNEL staining (green) and confocal microscopy of apoptotic cells in WT and Cav‐1 −/− liver. Nuclei were stained with DAPI . B and C,. Immunohistochemistry of cleaved (activated) caspase‐3 (B) and caspase‐8 (C) in livers. Ratio of area of cleaved caspase‐3‐ and caspase‐8‐positive cells were calculated (n = 5). D, Western blot analysis of protein levels of cleaved caspase‐3 and caspase‐8 in liver lysates. E, ELISA of activated caspase‐9 in livers (n = 6). Bar, 200 μm. Data are mean ± SEM . * P

Techniques Used: Activation Assay, Mouse Assay, TUNEL Assay, Staining, Confocal Microscopy, Immunohistochemistry, Western Blot, Enzyme-linked Immunosorbent Assay

30) Product Images from "FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes"

Article Title: FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1302839

FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome
Figure Legend Snippet: FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome

Techniques Used:

In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection
Figure Legend Snippet: In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection

Techniques Used: In Vivo, Infection

FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli
Figure Legend Snippet: FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli

Techniques Used:

Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome

Techniques Used: Activation Assay

Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation
Figure Legend Snippet: Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation

Techniques Used: Activation Assay

Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome

Techniques Used: Activation Assay

31) Product Images from "FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes"

Article Title: FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1302839

FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome
Figure Legend Snippet: FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome

Techniques Used:

In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection
Figure Legend Snippet: In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection

Techniques Used: In Vivo, Infection

FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli
Figure Legend Snippet: FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli

Techniques Used:

Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome

Techniques Used: Activation Assay

Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation
Figure Legend Snippet: Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation

Techniques Used: Activation Assay

Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome

Techniques Used: Activation Assay

32) Product Images from "FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes"

Article Title: FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1302839

FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome
Figure Legend Snippet: FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome

Techniques Used:

In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection
Figure Legend Snippet: In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection

Techniques Used: In Vivo, Infection

FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli
Figure Legend Snippet: FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli

Techniques Used:

Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome

Techniques Used: Activation Assay

Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation
Figure Legend Snippet: Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation

Techniques Used: Activation Assay

Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome

Techniques Used: Activation Assay

33) Product Images from "FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes"

Article Title: FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1302839

FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome
Figure Legend Snippet: FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome

Techniques Used:

In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection
Figure Legend Snippet: In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection

Techniques Used: In Vivo, Infection

FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli
Figure Legend Snippet: FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli

Techniques Used:

Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome

Techniques Used: Activation Assay

Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation
Figure Legend Snippet: Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation

Techniques Used: Activation Assay

Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome

Techniques Used: Activation Assay

34) Product Images from "FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes"

Article Title: FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1302839

FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome
Figure Legend Snippet: FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome

Techniques Used:

In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection
Figure Legend Snippet: In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection

Techniques Used: In Vivo, Infection

FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli
Figure Legend Snippet: FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli

Techniques Used:

Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome

Techniques Used: Activation Assay

Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation
Figure Legend Snippet: Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation

Techniques Used: Activation Assay

Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome

Techniques Used: Activation Assay

35) Product Images from "FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes"

Article Title: FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1302839

FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome
Figure Legend Snippet: FADD and caspase-8 mediate transcriptional priming of the Nlrp3 inflammasome

Techniques Used:

In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection
Figure Legend Snippet: In vivo role of caspase-8 and FADD in inflammasome responses during LPS-induced endotoxemia and C. rodentium infection

Techniques Used: In Vivo, Infection

FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli
Figure Legend Snippet: FADD is required for caspase-8 maturation in response to canonical and non-canonical Nlrp3 stimuli

Techniques Used:

Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for efficient activation of the canonical Nlrp3 inflammasome

Techniques Used: Activation Assay

Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation
Figure Legend Snippet: Caspase-8 mediates enteropathogen-induced Nlrp3 inflammasome activation

Techniques Used: Activation Assay

Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome
Figure Legend Snippet: Caspase-8 is required for post-translational activation of the Nlrp3 inflammasome

Techniques Used: Activation Assay

36) Product Images from "Inhibition of apoptosis prevents West Nile virus induced cell death"

Article Title: Inhibition of apoptosis prevents West Nile virus induced cell death

Journal: BMC Microbiology

doi: 10.1186/1471-2180-7-49

Caspase inhibitor peptides inhibit WNV-induced PARP-cleavage. Effect on WNV-induced PARP cleavage of peptides z-IETD-fmk (caspase-8 inhibitor), z-LEHD-fmk (caspase-9 inhibitor) and z-VAD-fmk (pancaspase inhibitor) at 48 h p.i. Results are expressed as percentage of values obtained from WNV-infected cultures (MOI 1) without any inhibitor added. Values represent mean (± SD) from three independent experiments. *p
Figure Legend Snippet: Caspase inhibitor peptides inhibit WNV-induced PARP-cleavage. Effect on WNV-induced PARP cleavage of peptides z-IETD-fmk (caspase-8 inhibitor), z-LEHD-fmk (caspase-9 inhibitor) and z-VAD-fmk (pancaspase inhibitor) at 48 h p.i. Results are expressed as percentage of values obtained from WNV-infected cultures (MOI 1) without any inhibitor added. Values represent mean (± SD) from three independent experiments. *p

Techniques Used: Infection

37) Product Images from "Upregulation of p21 activates the intrinsic apoptotic pathway in ?-cells"

Article Title: Upregulation of p21 activates the intrinsic apoptotic pathway in ?-cells

Journal: American Journal of Physiology - Endocrinology and Metabolism

doi: 10.1152/ajpendo.00663.2012

p21-mediated apoptosis is not regulated through the extrinsic mitochondrial death pathway or by a change in Bcl-2 family member expression. A : Western blot analysis of caspase-8 (Cl casp-8) protein levels in whole cell lysates from 832/13 cells transduced
Figure Legend Snippet: p21-mediated apoptosis is not regulated through the extrinsic mitochondrial death pathway or by a change in Bcl-2 family member expression. A : Western blot analysis of caspase-8 (Cl casp-8) protein levels in whole cell lysates from 832/13 cells transduced

Techniques Used: Expressing, Western Blot

38) Product Images from "Staurosporine induces apoptosis in pancreatic carcinoma cells PaTu 8988t and Panc-1 via the intrinsic signaling pathway"

Article Title: Staurosporine induces apoptosis in pancreatic carcinoma cells PaTu 8988t and Panc-1 via the intrinsic signaling pathway

Journal: European Journal of Medical Research

doi: 10.1186/s40001-019-0365-x

Time-dependent immunoblotting and proof of endogenic expression of Bcl2, BAX, Bad, caspase-8, caspase-9, and ß-actin in colorectal carcinoma cells (SW 480) and pancreatic cancer cells (PaTu 8988t and Panc-1) after stimulation with staurosporine
Figure Legend Snippet: Time-dependent immunoblotting and proof of endogenic expression of Bcl2, BAX, Bad, caspase-8, caspase-9, and ß-actin in colorectal carcinoma cells (SW 480) and pancreatic cancer cells (PaTu 8988t and Panc-1) after stimulation with staurosporine

Techniques Used: Expressing

Immunblotting and proof of endogenic expression of Bcl2, BAX, Bad, caspase-8, caspase-9, and ß-actin in colorectal cancer cells (SW 480) and pancreatic cancer cells (PaTu 8988t and Panc-1)
Figure Legend Snippet: Immunblotting and proof of endogenic expression of Bcl2, BAX, Bad, caspase-8, caspase-9, and ß-actin in colorectal cancer cells (SW 480) and pancreatic cancer cells (PaTu 8988t and Panc-1)

Techniques Used: Expressing

Apoptotic signaling pathway. Caspase-8 is activated via the extrinsic signaling pathway by the binding of a ligand to the death receptor. The intrinsic signaling pathway is activated by changes in the mitochondrial membrane potential and the subsequent release of cytochrome C, which influences the pro-apoptotic factors BAK, Bad, and BAX as well as the anti-apoptotic factors Bcl2 and Bcl-xl and thus triggers caspase-9. Both pathways merge into a common pathway, in which effector caspase-3 finally induces apoptosis
Figure Legend Snippet: Apoptotic signaling pathway. Caspase-8 is activated via the extrinsic signaling pathway by the binding of a ligand to the death receptor. The intrinsic signaling pathway is activated by changes in the mitochondrial membrane potential and the subsequent release of cytochrome C, which influences the pro-apoptotic factors BAK, Bad, and BAX as well as the anti-apoptotic factors Bcl2 and Bcl-xl and thus triggers caspase-9. Both pathways merge into a common pathway, in which effector caspase-3 finally induces apoptosis

Techniques Used: Binding Assay

39) Product Images from "Distinct combinatorial effects of the plant polyphenols curcumin, carnosic acid and silibinin on proliferation and apoptosis in acute myeloid leukemia cells"

Article Title: Distinct combinatorial effects of the plant polyphenols curcumin, carnosic acid and silibinin on proliferation and apoptosis in acute myeloid leukemia cells

Journal: Nutrition and cancer

doi: 10.1080/01635581003693082

Both caspase-8 and caspase-9 are involved in CUR/CA-induced apoptosis
Figure Legend Snippet: Both caspase-8 and caspase-9 are involved in CUR/CA-induced apoptosis

Techniques Used:

Time-course of caspase-8, -9, and -3 and PARP processing in CUR/CA-treated cells
Figure Legend Snippet: Time-course of caspase-8, -9, and -3 and PARP processing in CUR/CA-treated cells

Techniques Used:

40) Product Images from "Distinct combinatorial effects of the plant polyphenols curcumin, carnosic acid and silibinin on proliferation and apoptosis in acute myeloid leukemia cells"

Article Title: Distinct combinatorial effects of the plant polyphenols curcumin, carnosic acid and silibinin on proliferation and apoptosis in acute myeloid leukemia cells

Journal: Nutrition and cancer

doi: 10.1080/01635581003693082

Both caspase-8 and caspase-9 are involved in CUR/CA-induced apoptosis
Figure Legend Snippet: Both caspase-8 and caspase-9 are involved in CUR/CA-induced apoptosis

Techniques Used:

Time-course of caspase-8, -9, and -3 and PARP processing in CUR/CA-treated cells
Figure Legend Snippet: Time-course of caspase-8, -9, and -3 and PARP processing in CUR/CA-treated cells

Techniques Used:

Related Articles

Positive Control:

Article Title: Modulation of Cell Death and Promotion of Chondrogenic Differentiation by Fas/FasL in Human Dental Pulp Stem Cells (hDPSCs)
Article Snippet: .. The expression of FasL, Fas, Caspase 8 (Cell Signaling Technology, Trask Lane Danvers, MA, United States), c-FLIP (R & D systems, McKinley Place NE, Minneapolis, MN, United States), FADD (mouse anti-FADD ab; Santa Cruz Biotechnology, Dallas, TX, United States) was evaluated in hDPSCs after exposure to 0.1 ng/ml, 0.5 ng/ml FasL rc and to 0.5 ng/ml FasL rc + 500 ng/ml FasL inb for 24 h. To this regard, hDPSCs exposed to 1 μM Staurosporine were used as positive control of apoptosis. ..

Mutagenesis:

Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *
Article Snippet: .. Moreover, the expression of the mutant IRF-3 did not affect the activation of caspase-8; this was tested by the cleavage of Bid, a substrate of caspase-8, in the cells expressing Wt or the mutant forms of IRF-3 ( C ). .. To determine whether the cleavage-deficient mutant of IRF-3 (D121E) was protected from proteasome-mediated degradation, we expressed this mutant protein in P2.1 cells and analyzed its degradation in response to dsRNA-dependent signaling.

Infection:

Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *
Article Snippet: .. SeV infection, which is known to trigger RIG-I dependent signaling, caused rapid activation of caspase-8 in HT1080 cells. .. Caspase activation was analyzed by auto-proteolysis of pro-caspase-8, the precursor form, which generates the active caspase-8 enzyme.

Activation Assay:

Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *
Article Snippet: .. Moreover, the expression of the mutant IRF-3 did not affect the activation of caspase-8; this was tested by the cleavage of Bid, a substrate of caspase-8, in the cells expressing Wt or the mutant forms of IRF-3 ( C ). .. To determine whether the cleavage-deficient mutant of IRF-3 (D121E) was protected from proteasome-mediated degradation, we expressed this mutant protein in P2.1 cells and analyzed its degradation in response to dsRNA-dependent signaling.

Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *
Article Snippet: .. SeV infection, which is known to trigger RIG-I dependent signaling, caused rapid activation of caspase-8 in HT1080 cells. .. Caspase activation was analyzed by auto-proteolysis of pro-caspase-8, the precursor form, which generates the active caspase-8 enzyme.

Article Title: EFFICACY OF PANOBINOSTAT AND MARIZOMIB IN ACUTE MYELOID LEUKEMIA AND BORTEZOMIB-RESISTANT MODELS
Article Snippet: .. To verify the role for caspase-8 activation as an early event in panobinostat-induced cell death, we measured cleavage of caspase-8 in RPMI-8226vr10 cells ( ). .. Panobinostat single treatment and its combinations caused activation of caspase-8, indicated by the 43-kDa cleavage fragment.

other:

Article Title: Caspase-8, association with Alzheimer’s Disease and functional analysis of rare variants
Article Snippet: To look closer at APP processing by caspase-8, an antibody against the caspase cleavage product of APP, APPΔC31, was employed.

Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *
Article Snippet: Having established the role of caspase-8 in the proteasome-mediated degradation of IRF-3, we asked how caspase-8 was activated by dsRNA-signaling.

Expressing:

Article Title: Caspase-8-mediated Cleavage Inhibits IRF-3 Protein by Facilitating Its Proteasome-mediated Degradation *
Article Snippet: .. Moreover, the expression of the mutant IRF-3 did not affect the activation of caspase-8; this was tested by the cleavage of Bid, a substrate of caspase-8, in the cells expressing Wt or the mutant forms of IRF-3 ( C ). .. To determine whether the cleavage-deficient mutant of IRF-3 (D121E) was protected from proteasome-mediated degradation, we expressed this mutant protein in P2.1 cells and analyzed its degradation in response to dsRNA-dependent signaling.

Article Title: Modulation of Cell Death and Promotion of Chondrogenic Differentiation by Fas/FasL in Human Dental Pulp Stem Cells (hDPSCs)
Article Snippet: .. The expression of FasL, Fas, Caspase 8 (Cell Signaling Technology, Trask Lane Danvers, MA, United States), c-FLIP (R & D systems, McKinley Place NE, Minneapolis, MN, United States), FADD (mouse anti-FADD ab; Santa Cruz Biotechnology, Dallas, TX, United States) was evaluated in hDPSCs after exposure to 0.1 ng/ml, 0.5 ng/ml FasL rc and to 0.5 ng/ml FasL rc + 500 ng/ml FasL inb for 24 h. To this regard, hDPSCs exposed to 1 μM Staurosporine were used as positive control of apoptosis. ..

Western Blot:

Article Title: Oxidized low density lipoprotein facilitates tumor necrosis factor-α mediated chondrocyte death via autophagy pathway
Article Snippet: .. In addition, the western-blotting analysis of caspase-8 and caspase-3 also verified that both 3-MA and ATG5 siRNA treatment could decreased the level of cleaved caspase-8 and capase-3 ( for 3-MA; for ATG5 siRNA). .. Taken together, by using small molecule inhibitor 3-MA and gene manipulating ATG5 knockdown, we confirmed the autophagy inhibition could reverse the cell death process mediated by TNF-α and ox-LDL.

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    Cell Signaling Technology Inc caspase 8
    TRAIL receptors and sTRAIL activity on PDAC cell lines. ( a ) Expression of both agonistic (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors by flow cytometry; DR-4 and DcR1 were phycoerithryn (PE) and DR5 and DcR2 allophycocyanin (APC) stained. Proper isotype controls were used for both fluorochromes. ( b ) Tumor cell death was measured by propidium iodide staining. Recombinant human TRAIL (rhTRAIL; 1 μg/ml) was used as positive control while tumor cell lines with unconditioned control medium (CTL) and empty vector (EV) transduced AD-MSC supernatant were used as negative controls. Reported *p values represent significance of rhTRAIL versus the other groups, while **p refers to soluble TRAIL (sTRAIL) versus controls. ( c ) Western blot analysis on whole cell lysates showing Caspase 8 cleavage in treated (sTRAIL and rhTRAIL) and control (CTL and EV) BxPC-3 cells after both 6 and 8 hours. ( d ) Flow cytometry analysis to detect activated Caspase 8 forms in treated (sTRAIL and rhTRAIL) and control (CTL and EV) BxPC-3 cells after 6 hours of treatment with supernatants and CTL media. Full-length blots/gels are presented in Supplementary Fig.   S9 .
    Caspase 8, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 670 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Podocyte injury and renal apoptosis in the glomerulus and tubule. (A) Podocyte injury detected in glomeruli by immunohistochemical staining for desmin. The black arrows indicate podocytes. Original magnification, 400×. Scoring of desmin expression in renal tissue is shown on the right. (B) TUNEL staining in renal tissues at day 7, 14, and 28. Original magnification, 400×. The arrows indicate positively stained cells. The scoring is shown on the right. (C) Representative Western blot for the active forms of caspase-3, <t>caspase-9,</t> Bax and Bcl-2, with β-actin as the internal control. (D-F) Active caspase-3/β-actin ratio (D), active caspase-9/β-actin ratio (E), and Bax/Bcl-2 ratio (F). In the histograms, the data are the mean±SEM for seven mice per group. * p
    Caspase 9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TRAIL receptors and sTRAIL activity on PDAC cell lines. ( a ) Expression of both agonistic (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors by flow cytometry; DR-4 and DcR1 were phycoerithryn (PE) and DR5 and DcR2 allophycocyanin (APC) stained. Proper isotype controls were used for both fluorochromes. ( b ) Tumor cell death was measured by propidium iodide staining. Recombinant human TRAIL (rhTRAIL; 1 μg/ml) was used as positive control while tumor cell lines with unconditioned control medium (CTL) and empty vector (EV) transduced AD-MSC supernatant were used as negative controls. Reported *p values represent significance of rhTRAIL versus the other groups, while **p refers to soluble TRAIL (sTRAIL) versus controls. ( c ) Western blot analysis on whole cell lysates showing Caspase 8 cleavage in treated (sTRAIL and rhTRAIL) and control (CTL and EV) BxPC-3 cells after both 6 and 8 hours. ( d ) Flow cytometry analysis to detect activated Caspase 8 forms in treated (sTRAIL and rhTRAIL) and control (CTL and EV) BxPC-3 cells after 6 hours of treatment with supernatants and CTL media. Full-length blots/gels are presented in Supplementary Fig.   S9 .

    Journal: Scientific Reports

    Article Title: Soluble TRAIL Armed Human MSC As Gene Therapy For Pancreatic Cancer

    doi: 10.1038/s41598-018-37433-6

    Figure Lengend Snippet: TRAIL receptors and sTRAIL activity on PDAC cell lines. ( a ) Expression of both agonistic (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors by flow cytometry; DR-4 and DcR1 were phycoerithryn (PE) and DR5 and DcR2 allophycocyanin (APC) stained. Proper isotype controls were used for both fluorochromes. ( b ) Tumor cell death was measured by propidium iodide staining. Recombinant human TRAIL (rhTRAIL; 1 μg/ml) was used as positive control while tumor cell lines with unconditioned control medium (CTL) and empty vector (EV) transduced AD-MSC supernatant were used as negative controls. Reported *p values represent significance of rhTRAIL versus the other groups, while **p refers to soluble TRAIL (sTRAIL) versus controls. ( c ) Western blot analysis on whole cell lysates showing Caspase 8 cleavage in treated (sTRAIL and rhTRAIL) and control (CTL and EV) BxPC-3 cells after both 6 and 8 hours. ( d ) Flow cytometry analysis to detect activated Caspase 8 forms in treated (sTRAIL and rhTRAIL) and control (CTL and EV) BxPC-3 cells after 6 hours of treatment with supernatants and CTL media. Full-length blots/gels are presented in Supplementary Fig.  S9 .

    Article Snippet: After the transfer two blots have been performed: the first for the detection of Caspase-8 using Caspase 8 (1C12) Mouse mAb (Cell Signaling Technologies, Beverly, MA, USA) as primary antibody and goat anti-mouse IRDye 800CW (LI-COR) as secondary antibody.

    Techniques: Activity Assay, Expressing, Flow Cytometry, Cytometry, Staining, Recombinant, Positive Control, CTL Assay, Plasmid Preparation, Western Blot

    The combination of panobinostat plus marizomib induces greater and earlier caspase-3 activity and is caspase-8 dependent in ML-1 cells A B) ML-1 cells were pre-treated with inhibitors of caspase-8 (A, IETD-fmk) or caspase-9 (B, LEHD-fmk), followed by treatment with combinations of panobinostat with marizomib or bortezomib. DNA fragmentation was assessed by flow cytometry after propidium iodide staining. C) ML-1 cells were treated for 12 hours with 1 μM panobinostat, 10 nM bortezomib, and 50 nM marizomib. Lysates were probed for cleaved caspase-3. D) ML-1 cells were treated for indicated times with either panobinostat (1 μM) plus marizomib (50 nM) or panobinostat (1 μM) plus bortezomib (10 nM) combinations. Caspase-3/7 activity was measured using the fluorogenic substrate DEVD-amc. E) After 16 hours of treatment with each of the combinations (1 μM panobinostat plus 50 nM marizomib or 1 μM panobinostat plus 10 nM bortezomib), cells were stained with HEt for analysis of superoxide levels. F) ML-1 cells were pretreated for 30 minutes with 24 mM NAC, followed by 24 hours of treatment with diluent, 10 nM bortezomib, 50 nM marizomib, 1 μM panobinostat, or combinations of these agents. DNA fragmentation was assessed by PI staining (*p

    Journal: Leukemia research

    Article Title: EFFICACY OF PANOBINOSTAT AND MARIZOMIB IN ACUTE MYELOID LEUKEMIA AND BORTEZOMIB-RESISTANT MODELS

    doi: 10.1016/j.leukres.2014.12.014

    Figure Lengend Snippet: The combination of panobinostat plus marizomib induces greater and earlier caspase-3 activity and is caspase-8 dependent in ML-1 cells A B) ML-1 cells were pre-treated with inhibitors of caspase-8 (A, IETD-fmk) or caspase-9 (B, LEHD-fmk), followed by treatment with combinations of panobinostat with marizomib or bortezomib. DNA fragmentation was assessed by flow cytometry after propidium iodide staining. C) ML-1 cells were treated for 12 hours with 1 μM panobinostat, 10 nM bortezomib, and 50 nM marizomib. Lysates were probed for cleaved caspase-3. D) ML-1 cells were treated for indicated times with either panobinostat (1 μM) plus marizomib (50 nM) or panobinostat (1 μM) plus bortezomib (10 nM) combinations. Caspase-3/7 activity was measured using the fluorogenic substrate DEVD-amc. E) After 16 hours of treatment with each of the combinations (1 μM panobinostat plus 50 nM marizomib or 1 μM panobinostat plus 10 nM bortezomib), cells were stained with HEt for analysis of superoxide levels. F) ML-1 cells were pretreated for 30 minutes with 24 mM NAC, followed by 24 hours of treatment with diluent, 10 nM bortezomib, 50 nM marizomib, 1 μM panobinostat, or combinations of these agents. DNA fragmentation was assessed by PI staining (*p

    Article Snippet: To verify the role for caspase-8 activation as an early event in panobinostat-induced cell death, we measured cleavage of caspase-8 in RPMI-8226vr10 cells ( ).

    Techniques: Activity Assay, Flow Cytometry, Cytometry, Staining

    Depletion of HGF in LX-2 CM block its effects on promotion of the EMT, CSC phenotypes and cisplatin resistance. (A) Real-time PCR and (B) Western blot analysis of E-cadherin, N-cadherin and Vimentin expressions. Compared to LX-2 CM exposure, E-cadherin level was higher, while N-cadherin and Vimentin levels were lower in Hep3B cells upon HGF-depleted LX-2 CM exposure. (C) Real-time PCR and (D) Western blot analysis of Bmi1 and Klf4 expressions. Compared to LX-2 CM exposure, Bmi1 and Klf4 expressions were inhibited in Hep3B cells with HGF-depleted LX-2 CM exposure. (E) CCK8 analysis of cell viability. (F) Western blot analysis of active caspase 8 and 3 levels. In response to cisplatin treatment, Hep3B cells upon HGF-depleted LX-2 CM exposure showed lower cell viability and higher active caspase 8 and 3 levels than that upon LX-2 CM exposure. *p

    Journal: PLoS ONE

    Article Title: Hepatic Stellate Cells Secreted Hepatocyte Growth Factor Contributes to the Chemoresistance of Hepatocellular Carcinoma

    doi: 10.1371/journal.pone.0073312

    Figure Lengend Snippet: Depletion of HGF in LX-2 CM block its effects on promotion of the EMT, CSC phenotypes and cisplatin resistance. (A) Real-time PCR and (B) Western blot analysis of E-cadherin, N-cadherin and Vimentin expressions. Compared to LX-2 CM exposure, E-cadherin level was higher, while N-cadherin and Vimentin levels were lower in Hep3B cells upon HGF-depleted LX-2 CM exposure. (C) Real-time PCR and (D) Western blot analysis of Bmi1 and Klf4 expressions. Compared to LX-2 CM exposure, Bmi1 and Klf4 expressions were inhibited in Hep3B cells with HGF-depleted LX-2 CM exposure. (E) CCK8 analysis of cell viability. (F) Western blot analysis of active caspase 8 and 3 levels. In response to cisplatin treatment, Hep3B cells upon HGF-depleted LX-2 CM exposure showed lower cell viability and higher active caspase 8 and 3 levels than that upon LX-2 CM exposure. *p

    Article Snippet: Meanwhile, in response to cisplatin treatment, Hep3B cells with HGF-depleted LX-2 CM exposure showed lower cell viability and higher active caspase 8 and 3 than that of LX-2 CM exposure( ).

    Techniques: Blocking Assay, Real-time Polymerase Chain Reaction, Western Blot

    Induction of cisplatin resistance in Hep3B cells by LX-2 cells. (A) The effect of LX-2 CM on cisplatin-induced cell proliferation arrest. Hep3B cells were treated with cisplatin at the concentration of 10 µg/ml for 12, 24 and 48 h with or without LX-2 CM exposure. Cell viability was measured by using a CCK8 assay. Compared to Hep3B cells cultured in normal medium, cisplatin induced cytotoxicity was markedly attenuated in Hep3B cells upon LX-2 CM exposure. (B) Anti-apoptotic effect of LX-2 CM on cisplatin-induced apoptosis. Hep3B cells with or without LX-2 CM exposure were treated with cisplatin at the concentration of 10 µg/ml for 24h. The apoptosis rate was determined by using flow cytometry with Annexin V-FITC and PI double staining. In response to cisplatin, Hep3B cells incubated in LX-2 CM showed lower apoptosis rate than that in normal medium. (C) Western blot analysis of active caspase 8 and 3, GAPDH as internal control. Cisplatin induced caspase 8 and 3 activation products were profoundly reduced when it collaborated with LX-2 CM exposure. (D) HSCs modulate chemoresistance of HCC in vivo. Hep3B cells were subcutaneously injected or co-injected with LX-2 cells into nude mice to form xenografts. In response to cisplatin treatment, tumors from mice of co-injection with Hep3B cells and LX-2 cells were larger and heavier than that of single injection with Hep3B cells. *p

    Journal: PLoS ONE

    Article Title: Hepatic Stellate Cells Secreted Hepatocyte Growth Factor Contributes to the Chemoresistance of Hepatocellular Carcinoma

    doi: 10.1371/journal.pone.0073312

    Figure Lengend Snippet: Induction of cisplatin resistance in Hep3B cells by LX-2 cells. (A) The effect of LX-2 CM on cisplatin-induced cell proliferation arrest. Hep3B cells were treated with cisplatin at the concentration of 10 µg/ml for 12, 24 and 48 h with or without LX-2 CM exposure. Cell viability was measured by using a CCK8 assay. Compared to Hep3B cells cultured in normal medium, cisplatin induced cytotoxicity was markedly attenuated in Hep3B cells upon LX-2 CM exposure. (B) Anti-apoptotic effect of LX-2 CM on cisplatin-induced apoptosis. Hep3B cells with or without LX-2 CM exposure were treated with cisplatin at the concentration of 10 µg/ml for 24h. The apoptosis rate was determined by using flow cytometry with Annexin V-FITC and PI double staining. In response to cisplatin, Hep3B cells incubated in LX-2 CM showed lower apoptosis rate than that in normal medium. (C) Western blot analysis of active caspase 8 and 3, GAPDH as internal control. Cisplatin induced caspase 8 and 3 activation products were profoundly reduced when it collaborated with LX-2 CM exposure. (D) HSCs modulate chemoresistance of HCC in vivo. Hep3B cells were subcutaneously injected or co-injected with LX-2 cells into nude mice to form xenografts. In response to cisplatin treatment, tumors from mice of co-injection with Hep3B cells and LX-2 cells were larger and heavier than that of single injection with Hep3B cells. *p

    Article Snippet: Meanwhile, in response to cisplatin treatment, Hep3B cells with HGF-depleted LX-2 CM exposure showed lower cell viability and higher active caspase 8 and 3 than that of LX-2 CM exposure( ).

    Techniques: Concentration Assay, CCK-8 Assay, Cell Culture, Flow Cytometry, Cytometry, Double Staining, Incubation, Western Blot, Activation Assay, In Vivo, Injection, Mouse Assay

    Podocyte injury and renal apoptosis in the glomerulus and tubule. (A) Podocyte injury detected in glomeruli by immunohistochemical staining for desmin. The black arrows indicate podocytes. Original magnification, 400×. Scoring of desmin expression in renal tissue is shown on the right. (B) TUNEL staining in renal tissues at day 7, 14, and 28. Original magnification, 400×. The arrows indicate positively stained cells. The scoring is shown on the right. (C) Representative Western blot for the active forms of caspase-3, caspase-9, Bax and Bcl-2, with β-actin as the internal control. (D-F) Active caspase-3/β-actin ratio (D), active caspase-9/β-actin ratio (E), and Bax/Bcl-2 ratio (F). In the histograms, the data are the mean±SEM for seven mice per group. * p

    Journal: PLoS ONE

    Article Title: Citral Is Renoprotective for Focal Segmental Glomerulosclerosis by Inhibiting Oxidative Stress and Apoptosis and Activating Nrf2 Pathway in Mice

    doi: 10.1371/journal.pone.0074871

    Figure Lengend Snippet: Podocyte injury and renal apoptosis in the glomerulus and tubule. (A) Podocyte injury detected in glomeruli by immunohistochemical staining for desmin. The black arrows indicate podocytes. Original magnification, 400×. Scoring of desmin expression in renal tissue is shown on the right. (B) TUNEL staining in renal tissues at day 7, 14, and 28. Original magnification, 400×. The arrows indicate positively stained cells. The scoring is shown on the right. (C) Representative Western blot for the active forms of caspase-3, caspase-9, Bax and Bcl-2, with β-actin as the internal control. (D-F) Active caspase-3/β-actin ratio (D), active caspase-9/β-actin ratio (E), and Bax/Bcl-2 ratio (F). In the histograms, the data are the mean±SEM for seven mice per group. * p

    Article Snippet: Furthermore, Citral administration resulted in decreased renal levels of activated caspase-3 and caspase-9 (but not of activated caspase-8) as well as Bax/Bcl-2 ratio ( ).

    Techniques: Immunohistochemistry, Staining, Expressing, TUNEL Assay, Western Blot, Mouse Assay