caspase lysis buffer  (Roche)


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    Roche caspase lysis buffer
    Cleavage of <t>caspase-7</t> and PARP is inhibited in influenza virus-infected BAD knockdown cells. (A) Nontargeting shRNA control (NSi) and BAD knockdown cells were infected with NY55 at an MOI of 3, and cells were harvested at specific times points. Whole-cell
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    Images

    1) Product Images from "Influenza Virus Induces Apoptosis via BAD-Mediated Mitochondrial Dysregulation"

    Article Title: Influenza Virus Induces Apoptosis via BAD-Mediated Mitochondrial Dysregulation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02017-12

    Cleavage of caspase-7 and PARP is inhibited in influenza virus-infected BAD knockdown cells. (A) Nontargeting shRNA control (NSi) and BAD knockdown cells were infected with NY55 at an MOI of 3, and cells were harvested at specific times points. Whole-cell
    Figure Legend Snippet: Cleavage of caspase-7 and PARP is inhibited in influenza virus-infected BAD knockdown cells. (A) Nontargeting shRNA control (NSi) and BAD knockdown cells were infected with NY55 at an MOI of 3, and cells were harvested at specific times points. Whole-cell

    Techniques Used: Infection, shRNA

    BAD is required for influenza virus-induced caspase-3 and caspase-7 activity. (A) Measurement of caspase-3/7 activity using Caspase-Glo 3/7 assay at 72 hpi in A549 cells infected with NY55 at an MOI of 1 or at 24 h after treatment of cells with 1 μM
    Figure Legend Snippet: BAD is required for influenza virus-induced caspase-3 and caspase-7 activity. (A) Measurement of caspase-3/7 activity using Caspase-Glo 3/7 assay at 72 hpi in A549 cells infected with NY55 at an MOI of 1 or at 24 h after treatment of cells with 1 μM

    Techniques Used: Activity Assay, Caspase-Glo Assay, Infection

    2) Product Images from "Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps"

    Article Title: Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198203

    XIAP downregulation restores TRAIL sensitivity in Bcl-2 overexpressing cells. (A) Downregulation of XIAP by siRNA treatment. Cells were left untreated (UT) or were transiently transfected with non-targeting (siControl) or siRNA specific for XIAP (siXIAP). Protein expression was determined 48 h post transfection from whole cell lysates via immunoblot assay using an XIAP specific antibody. As control c-IAP1 and survivin were included. (B) and (C) NCI-H460 wild type (B) and NCI-H460/Bcl-2 cells (C) had been pretreated as in (A) were treated with serial dilutions of Db-scTRAIL (open symbols). NCI-H460/Bcl-2 cells transfected with siXIAP were incubated with 50 μM z-VAD-fmk and 1 nM Db-scTRAIL (filled triangle). After 24 h viable cells were stained with crystal violet. All values were normalized to those from unstimulated cells. The experiment was performed in triplicates and the data shown are representative of three independent experiments. (D) NCI-H460/Bcl-2 cells had been pretreated as in (A) were treated with Db-scTRAIL (1 nM) for 2 and 4 h and analyzed by immunoblotting using antibodies for cleaved caspase-8, -9 and -3. Tubulin-α was used as loading control. Blot shown is representative of three independent experiments.
    Figure Legend Snippet: XIAP downregulation restores TRAIL sensitivity in Bcl-2 overexpressing cells. (A) Downregulation of XIAP by siRNA treatment. Cells were left untreated (UT) or were transiently transfected with non-targeting (siControl) or siRNA specific for XIAP (siXIAP). Protein expression was determined 48 h post transfection from whole cell lysates via immunoblot assay using an XIAP specific antibody. As control c-IAP1 and survivin were included. (B) and (C) NCI-H460 wild type (B) and NCI-H460/Bcl-2 cells (C) had been pretreated as in (A) were treated with serial dilutions of Db-scTRAIL (open symbols). NCI-H460/Bcl-2 cells transfected with siXIAP were incubated with 50 μM z-VAD-fmk and 1 nM Db-scTRAIL (filled triangle). After 24 h viable cells were stained with crystal violet. All values were normalized to those from unstimulated cells. The experiment was performed in triplicates and the data shown are representative of three independent experiments. (D) NCI-H460/Bcl-2 cells had been pretreated as in (A) were treated with Db-scTRAIL (1 nM) for 2 and 4 h and analyzed by immunoblotting using antibodies for cleaved caspase-8, -9 and -3. Tubulin-α was used as loading control. Blot shown is representative of three independent experiments.

    Techniques Used: Transfection, Expressing, Incubation, Staining

    Bcl-2 overexpression strongly inhibits caspase activity but affects caspase cleavage preferentially at late activation steps. (A) Cells were stimulated with Db-scTRAIL (1 nM) for the indicated time periods. Whole protein lysates were incubated with fluorogenic caspase substrates Ac-IEPD-AMC (caspase-8), Ac-DMQD-AMC (caspase-3) and Ac-LEHD-AMC (caspase-9), respectively. Increasing fluorescence values were measured every 2 min for 2 h at λ = 460 nm. Data points shown, representing slopes, are mean values ± SD calculated from 3 independent experiments, normalized to the highest value of each experiment. (B) Cells were stimulated with Db-scTRAIL (1 nM) up to 8 h, one group was left untreated as a control. Equal amounts of whole cell lysates were subjected to immunoblot analysis using antibodies specific for cleaved caspase-8, cleaved caspase-3 and caspase-9 followed by HRP-conjugated secondary antibody.
    Figure Legend Snippet: Bcl-2 overexpression strongly inhibits caspase activity but affects caspase cleavage preferentially at late activation steps. (A) Cells were stimulated with Db-scTRAIL (1 nM) for the indicated time periods. Whole protein lysates were incubated with fluorogenic caspase substrates Ac-IEPD-AMC (caspase-8), Ac-DMQD-AMC (caspase-3) and Ac-LEHD-AMC (caspase-9), respectively. Increasing fluorescence values were measured every 2 min for 2 h at λ = 460 nm. Data points shown, representing slopes, are mean values ± SD calculated from 3 independent experiments, normalized to the highest value of each experiment. (B) Cells were stimulated with Db-scTRAIL (1 nM) up to 8 h, one group was left untreated as a control. Equal amounts of whole cell lysates were subjected to immunoblot analysis using antibodies specific for cleaved caspase-8, cleaved caspase-3 and caspase-9 followed by HRP-conjugated secondary antibody.

    Techniques Used: Over Expression, Activity Assay, Activation Assay, Incubation, Fluorescence

    Caspase-9 plays only a minor role in TRAIL-induced apoptosis in NCI-H460 cells. (A) Cells were preincubated with caspase-9 inhibitor (z-LEHD-fmk, 50 μM) for 1 h, followed by stimulation with serial dilutions of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and absorbance was measured at 550 nm. (B) Western blot showing efficient downregulation of procaspase-9 by siRNA treatment. NCI-H460 cells were transfected with siRNAs (siControl and siCaspase-9) and harvested after 48 h. (C) NCI-H460 cells with downregulated caspase-9 do not show increased TRAIL resistance. Cells transfected with either non-targeting (siControl) or caspase-9 (siCaspase-9) siRNAs were stimulated 48 h post transfection with serial dilutions of Db-scTRAIL for further 24 h. (D) Cells pretreated as in (B) were treated for 2 and 4 h with Db-scTRAIL (1 nM), control cells were left untreated. Whole cell lysates were subjected to western blot analysis using antibodies specific for caspase-9 and caspase-3, α-tubulin was used to confirm equal loading.
    Figure Legend Snippet: Caspase-9 plays only a minor role in TRAIL-induced apoptosis in NCI-H460 cells. (A) Cells were preincubated with caspase-9 inhibitor (z-LEHD-fmk, 50 μM) for 1 h, followed by stimulation with serial dilutions of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and absorbance was measured at 550 nm. (B) Western blot showing efficient downregulation of procaspase-9 by siRNA treatment. NCI-H460 cells were transfected with siRNAs (siControl and siCaspase-9) and harvested after 48 h. (C) NCI-H460 cells with downregulated caspase-9 do not show increased TRAIL resistance. Cells transfected with either non-targeting (siControl) or caspase-9 (siCaspase-9) siRNAs were stimulated 48 h post transfection with serial dilutions of Db-scTRAIL for further 24 h. (D) Cells pretreated as in (B) were treated for 2 and 4 h with Db-scTRAIL (1 nM), control cells were left untreated. Whole cell lysates were subjected to western blot analysis using antibodies specific for caspase-9 and caspase-3, α-tubulin was used to confirm equal loading.

    Techniques Used: Staining, Western Blot, Transfection

    Smac is a potent regulator of TRAIL-induced apoptosis in NCI-H460 cells. (A) and (B) Smac mimetic SM83 sensitizes NCI-H460/Bcl-2 cells for TRAIL-induced apoptosis. NCI-H460 wild type (A) and NCI-H460/Bcl-2 cells (B) were preincubated with SM83 (1 μM) followed by stimulation with increasing concentrations of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and the absorbance was determined at 550 nm. Values from stimulated cells were normalized to unstimulated cells. Shown are mean values ± SD calculated from triplicates. One representative experiment out of three is shown. (C) Downregulation of Smac protein by siRNA treatment. NCI-H460 cells were transfected with siRNAs directed against Smac (siSmac) or non-targeting (siControl). Expression levels of Smac were determined 48 h later by western blotting. (D) Reduced caspase-3 processing after Smac downregulation is rescued by proteasome inhibition. Cells from (C) were preincubated with MG132 (25 μM) or Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3. (E) and (F) NCI-H460 cells were preincubated with Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3 (E) and apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry (F).
    Figure Legend Snippet: Smac is a potent regulator of TRAIL-induced apoptosis in NCI-H460 cells. (A) and (B) Smac mimetic SM83 sensitizes NCI-H460/Bcl-2 cells for TRAIL-induced apoptosis. NCI-H460 wild type (A) and NCI-H460/Bcl-2 cells (B) were preincubated with SM83 (1 μM) followed by stimulation with increasing concentrations of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and the absorbance was determined at 550 nm. Values from stimulated cells were normalized to unstimulated cells. Shown are mean values ± SD calculated from triplicates. One representative experiment out of three is shown. (C) Downregulation of Smac protein by siRNA treatment. NCI-H460 cells were transfected with siRNAs directed against Smac (siSmac) or non-targeting (siControl). Expression levels of Smac were determined 48 h later by western blotting. (D) Reduced caspase-3 processing after Smac downregulation is rescued by proteasome inhibition. Cells from (C) were preincubated with MG132 (25 μM) or Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3. (E) and (F) NCI-H460 cells were preincubated with Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3 (E) and apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry (F).

    Techniques Used: Staining, Transfection, Expressing, Western Blot, Inhibition, Flow Cytometry, Cytometry

    3) Product Images from "Identification of the PANoptosome: A Molecular Platform Triggering Pyroptosis, Apoptosis, and Necroptosis (PANoptosis)"

    Article Title: Identification of the PANoptosome: A Molecular Platform Triggering Pyroptosis, Apoptosis, and Necroptosis (PANoptosis)

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2020.00237

    Inhibition of TAK1 promotes PANoptosis and PANoptosome formation. (A) Representative cell death images of BMDMs lacking different components of pyroptosis, apoptosis, or necroptosis after LPS priming and inhibition of TAK1. (B) Western blot analysis of PANoptosis activation after LPS priming and TAK1 inhibition. (C) Co-immunoprecipitation of PANoptosome components from primary BMDMs after TAK1 and caspase inhibition. Red asterisks denote a non-specific band.
    Figure Legend Snippet: Inhibition of TAK1 promotes PANoptosis and PANoptosome formation. (A) Representative cell death images of BMDMs lacking different components of pyroptosis, apoptosis, or necroptosis after LPS priming and inhibition of TAK1. (B) Western blot analysis of PANoptosis activation after LPS priming and TAK1 inhibition. (C) Co-immunoprecipitation of PANoptosome components from primary BMDMs after TAK1 and caspase inhibition. Red asterisks denote a non-specific band.

    Techniques Used: Inhibition, Western Blot, Activation Assay, Immunoprecipitation

    4) Product Images from "Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps"

    Article Title: Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198203

    XIAP downregulation restores TRAIL sensitivity in Bcl-2 overexpressing cells. (A) Downregulation of XIAP by siRNA treatment. Cells were left untreated (UT) or were transiently transfected with non-targeting (siControl) or siRNA specific for XIAP (siXIAP). Protein expression was determined 48 h post transfection from whole cell lysates via immunoblot assay using an XIAP specific antibody. As control c-IAP1 and survivin were included. (B) and (C) NCI-H460 wild type (B) and NCI-H460/Bcl-2 cells (C) had been pretreated as in (A) were treated with serial dilutions of Db-scTRAIL (open symbols). NCI-H460/Bcl-2 cells transfected with siXIAP were incubated with 50 μM z-VAD-fmk and 1 nM Db-scTRAIL (filled triangle). After 24 h viable cells were stained with crystal violet. All values were normalized to those from unstimulated cells. The experiment was performed in triplicates and the data shown are representative of three independent experiments. (D) NCI-H460/Bcl-2 cells had been pretreated as in (A) were treated with Db-scTRAIL (1 nM) for 2 and 4 h and analyzed by immunoblotting using antibodies for cleaved caspase-8, -9 and -3. Tubulin-α was used as loading control. Blot shown is representative of three independent experiments.
    Figure Legend Snippet: XIAP downregulation restores TRAIL sensitivity in Bcl-2 overexpressing cells. (A) Downregulation of XIAP by siRNA treatment. Cells were left untreated (UT) or were transiently transfected with non-targeting (siControl) or siRNA specific for XIAP (siXIAP). Protein expression was determined 48 h post transfection from whole cell lysates via immunoblot assay using an XIAP specific antibody. As control c-IAP1 and survivin were included. (B) and (C) NCI-H460 wild type (B) and NCI-H460/Bcl-2 cells (C) had been pretreated as in (A) were treated with serial dilutions of Db-scTRAIL (open symbols). NCI-H460/Bcl-2 cells transfected with siXIAP were incubated with 50 μM z-VAD-fmk and 1 nM Db-scTRAIL (filled triangle). After 24 h viable cells were stained with crystal violet. All values were normalized to those from unstimulated cells. The experiment was performed in triplicates and the data shown are representative of three independent experiments. (D) NCI-H460/Bcl-2 cells had been pretreated as in (A) were treated with Db-scTRAIL (1 nM) for 2 and 4 h and analyzed by immunoblotting using antibodies for cleaved caspase-8, -9 and -3. Tubulin-α was used as loading control. Blot shown is representative of three independent experiments.

    Techniques Used: Transfection, Expressing, Incubation, Staining

    Bcl-2 overexpression strongly inhibits caspase activity but affects caspase cleavage preferentially at late activation steps. (A) Cells were stimulated with Db-scTRAIL (1 nM) for the indicated time periods. Whole protein lysates were incubated with fluorogenic caspase substrates Ac-IEPD-AMC (caspase-8), Ac-DMQD-AMC (caspase-3) and Ac-LEHD-AMC (caspase-9), respectively. Increasing fluorescence values were measured every 2 min for 2 h at λ = 460 nm. Data points shown, representing slopes, are mean values ± SD calculated from 3 independent experiments, normalized to the highest value of each experiment. (B) Cells were stimulated with Db-scTRAIL (1 nM) up to 8 h, one group was left untreated as a control. Equal amounts of whole cell lysates were subjected to immunoblot analysis using antibodies specific for cleaved caspase-8, cleaved caspase-3 and caspase-9 followed by HRP-conjugated secondary antibody.
    Figure Legend Snippet: Bcl-2 overexpression strongly inhibits caspase activity but affects caspase cleavage preferentially at late activation steps. (A) Cells were stimulated with Db-scTRAIL (1 nM) for the indicated time periods. Whole protein lysates were incubated with fluorogenic caspase substrates Ac-IEPD-AMC (caspase-8), Ac-DMQD-AMC (caspase-3) and Ac-LEHD-AMC (caspase-9), respectively. Increasing fluorescence values were measured every 2 min for 2 h at λ = 460 nm. Data points shown, representing slopes, are mean values ± SD calculated from 3 independent experiments, normalized to the highest value of each experiment. (B) Cells were stimulated with Db-scTRAIL (1 nM) up to 8 h, one group was left untreated as a control. Equal amounts of whole cell lysates were subjected to immunoblot analysis using antibodies specific for cleaved caspase-8, cleaved caspase-3 and caspase-9 followed by HRP-conjugated secondary antibody.

    Techniques Used: Over Expression, Activity Assay, Activation Assay, Incubation, Fluorescence

    Caspase-9 plays only a minor role in TRAIL-induced apoptosis in NCI-H460 cells. (A) Cells were preincubated with caspase-9 inhibitor (z-LEHD-fmk, 50 μM) for 1 h, followed by stimulation with serial dilutions of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and absorbance was measured at 550 nm. (B) Western blot showing efficient downregulation of procaspase-9 by siRNA treatment. NCI-H460 cells were transfected with siRNAs (siControl and siCaspase-9) and harvested after 48 h. (C) NCI-H460 cells with downregulated caspase-9 do not show increased TRAIL resistance. Cells transfected with either non-targeting (siControl) or caspase-9 (siCaspase-9) siRNAs were stimulated 48 h post transfection with serial dilutions of Db-scTRAIL for further 24 h. (D) Cells pretreated as in (B) were treated for 2 and 4 h with Db-scTRAIL (1 nM), control cells were left untreated. Whole cell lysates were subjected to western blot analysis using antibodies specific for caspase-9 and caspase-3, α-tubulin was used to confirm equal loading.
    Figure Legend Snippet: Caspase-9 plays only a minor role in TRAIL-induced apoptosis in NCI-H460 cells. (A) Cells were preincubated with caspase-9 inhibitor (z-LEHD-fmk, 50 μM) for 1 h, followed by stimulation with serial dilutions of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and absorbance was measured at 550 nm. (B) Western blot showing efficient downregulation of procaspase-9 by siRNA treatment. NCI-H460 cells were transfected with siRNAs (siControl and siCaspase-9) and harvested after 48 h. (C) NCI-H460 cells with downregulated caspase-9 do not show increased TRAIL resistance. Cells transfected with either non-targeting (siControl) or caspase-9 (siCaspase-9) siRNAs were stimulated 48 h post transfection with serial dilutions of Db-scTRAIL for further 24 h. (D) Cells pretreated as in (B) were treated for 2 and 4 h with Db-scTRAIL (1 nM), control cells were left untreated. Whole cell lysates were subjected to western blot analysis using antibodies specific for caspase-9 and caspase-3, α-tubulin was used to confirm equal loading.

    Techniques Used: Staining, Western Blot, Transfection

    Smac is a potent regulator of TRAIL-induced apoptosis in NCI-H460 cells. (A) and (B) Smac mimetic SM83 sensitizes NCI-H460/Bcl-2 cells for TRAIL-induced apoptosis. NCI-H460 wild type (A) and NCI-H460/Bcl-2 cells (B) were preincubated with SM83 (1 μM) followed by stimulation with increasing concentrations of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and the absorbance was determined at 550 nm. Values from stimulated cells were normalized to unstimulated cells. Shown are mean values ± SD calculated from triplicates. One representative experiment out of three is shown. (C) Downregulation of Smac protein by siRNA treatment. NCI-H460 cells were transfected with siRNAs directed against Smac (siSmac) or non-targeting (siControl). Expression levels of Smac were determined 48 h later by western blotting. (D) Reduced caspase-3 processing after Smac downregulation is rescued by proteasome inhibition. Cells from (C) were preincubated with MG132 (25 μM) or Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3. (E) and (F) NCI-H460 cells were preincubated with Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3 (E) and apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry (F).
    Figure Legend Snippet: Smac is a potent regulator of TRAIL-induced apoptosis in NCI-H460 cells. (A) and (B) Smac mimetic SM83 sensitizes NCI-H460/Bcl-2 cells for TRAIL-induced apoptosis. NCI-H460 wild type (A) and NCI-H460/Bcl-2 cells (B) were preincubated with SM83 (1 μM) followed by stimulation with increasing concentrations of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and the absorbance was determined at 550 nm. Values from stimulated cells were normalized to unstimulated cells. Shown are mean values ± SD calculated from triplicates. One representative experiment out of three is shown. (C) Downregulation of Smac protein by siRNA treatment. NCI-H460 cells were transfected with siRNAs directed against Smac (siSmac) or non-targeting (siControl). Expression levels of Smac were determined 48 h later by western blotting. (D) Reduced caspase-3 processing after Smac downregulation is rescued by proteasome inhibition. Cells from (C) were preincubated with MG132 (25 μM) or Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3. (E) and (F) NCI-H460 cells were preincubated with Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3 (E) and apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry (F).

    Techniques Used: Staining, Transfection, Expressing, Western Blot, Inhibition, Flow Cytometry, Cytometry

    5) Product Images from "Mdm2 inhibition confers protection of p53-proficient cells from the cytotoxic effects of Wee1 inhibitors"

    Article Title: Mdm2 inhibition confers protection of p53-proficient cells from the cytotoxic effects of Wee1 inhibitors

    Journal: Oncotarget

    doi:

    Nutlin prevents caspase activation and γH2AX accumulation in response to Wee1 inhibitor and/or gemcitabine A . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, and/or 8μM Nutlin in the absence and presence of 50μM ZVAD-FMK for another 24 hrs. Cells were harvested and immunoblot analysis was performed to detect poly-ADP ribose polymerase (PARP) and γH2AX. B ., C . U2OS cells were treated as in (A). The cells were then fixed and stained for γH2AX by immunofluorescence. Detection and analysis was performed using automated immunofluorescence microscopy (BD Pathway). Figure panel (B) shows images of γH2AX staining for each treatment condition. Quantitation of γH2AX intensities was done using the BD pathway analysis tool and depicted in figure panel (C). Error bars represent the SD, n=3. D . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, 8μM Nutlin in the absence and presence ( Supplementary Figure 1 ) of 50μM ZVAD-FMK for another 24 hrs. The cells were harvested and lysed for caspase activity assay. Fluorescent intensity measurements were obtained for each treatment. The activity (arbitrary units of fluorescence/min) was calculated for each treatment at the linear part of the curve (cf. Supplementary Figure 1 ). Error bars represent the S.D, n=3.
    Figure Legend Snippet: Nutlin prevents caspase activation and γH2AX accumulation in response to Wee1 inhibitor and/or gemcitabine A . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, and/or 8μM Nutlin in the absence and presence of 50μM ZVAD-FMK for another 24 hrs. Cells were harvested and immunoblot analysis was performed to detect poly-ADP ribose polymerase (PARP) and γH2AX. B ., C . U2OS cells were treated as in (A). The cells were then fixed and stained for γH2AX by immunofluorescence. Detection and analysis was performed using automated immunofluorescence microscopy (BD Pathway). Figure panel (B) shows images of γH2AX staining for each treatment condition. Quantitation of γH2AX intensities was done using the BD pathway analysis tool and depicted in figure panel (C). Error bars represent the SD, n=3. D . U2OS cells were treated with 8μM Nutlin for 24 hrs, followed by treatment with 1μM Wee1 inhibitor, 300nM gemcitabine, 8μM Nutlin in the absence and presence ( Supplementary Figure 1 ) of 50μM ZVAD-FMK for another 24 hrs. The cells were harvested and lysed for caspase activity assay. Fluorescent intensity measurements were obtained for each treatment. The activity (arbitrary units of fluorescence/min) was calculated for each treatment at the linear part of the curve (cf. Supplementary Figure 1 ). Error bars represent the S.D, n=3.

    Techniques Used: Activation Assay, Staining, Immunofluorescence, Microscopy, Quantitation Assay, Caspase Activity Assay, Activity Assay, Fluorescence

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    Article Snippet: Proteins using TNT Quick Coupled Transcription/Transcription System (Promega, Charbonnières-les-Bains, France) were produced as follows: 1 μ g of template plasmid DNA was added to the reaction mixture, which was afterwards incubated at 30 °C for 90 min. Twenty microliters of the in vitro translated proteins were used for immunoprecipitation. .. Immunoprecipitation and immunoblotting Cells were lysed in lysis buffer (50 mM Hepes pH 7.4, 140 mM NaCl, 5 mM EDTA, 0.2% NP40, 10 mM sodium fluoride, protease inhibitor cocktail (Roche, Neuilly sur Seine, France)). .. In all, 800 μ g of each lysate was incubated with 3 μ g of CK2α , CK2β (Santa Cruz), Par-4 (Sigma-Aldrich), GFP-tag (Millipore) or HA-tag (Biomol, Hamburg, Germany) antibodies with constant agitation at 4 °C.

    Article Title: Combination of IAP antagonist and IFNγ activates novel caspase-10- and RIPK1-dependent cell death pathways
    Article Snippet: .. For co-immunoprecipitation, HT29 cells were lysed in DISC lysis buffer (1% (v/v) Triton X-100, 150 mM NaCl, 20 mM Tris, pH 7.5, 10% (v/v) glycerol, 2 mM EDTA) with complete protease inhibitor cocktail (Roche, Dee Why, NSW, Australia), phosphatase inhibitors (2 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM sodium molybdate, 5 mM β -glycerophosphate, 2 mM sodium pyrophosphate) and 1 mM NEM ( N -ethylmaleimide; Sigma, Castle Hill, NSW, Australia). .. Lysates were incubated overnight with 2 μ g caspase-8 antibody (Santa Cruz, Santa Cruz, CA, USA; sc6136) and 20 μ l packed Sepharose Protein G beads were incubated overnight with 1% BSA in PBS.

    Article Title: p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3 *
    Article Snippet: Quantification was performed using ImageJ. .. IP of endogenous caspase-2 was done using cell lysates in RIPA lysis buffer containing 150 m m NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 50 m m Tris-HCl, and 0.1% SDS, pH 8.0, supplemented with protease inhibitor mixture (Roche Applied Science) essentially as described ( , ). .. Preclearing was done followed by an overnight incubation with anti-caspase-2 antibody.

    Protease Inhibitor:

    Article Title: A new experimental model of acid- and endotoxin-induced acute lung injury in rats
    Article Snippet: Thus, the resulting lung injury score was derived from the following calculation: LIS: {[(20 × A) + (14 × B) + (7 × C) + (7 × D) + (2 × E) + (2 × F)]/(number of fields × 100)} × 10. .. For cytokine and caspase-3 measurements, the right lung was homogenized in lysis buffer containing 1 mM sodium orthovanadate, protease inhibitor cocktail tablets (1 tablet for 250 mg of lung tissue) (Roche; Mannheim, Germany), 0.5% Triton X-100, 150 mM NaCl, 15 mM Tris, 1 mM CaCl2 , and 50 mM MgCl2 (pH 7.4) using a hand-held homogenizer. .. The homogenates were incubated for 30 min at 4°C, centrifuged at 12,000 rpm at 4° C for 20 min, and then filtered with 0.45 μm Nanosep filters (Pall Life Sciences; Madrid, Spain).

    Article Title: Stress-induced Skeletal Muscle Gadd45a Expression Reprograms Myonuclei and Causes Muscle Atrophy *
    Article Snippet: Mouse TA muscles were snap frozen in liquid N2 , and homogenized in 1 ml of cold lysis buffer solution containing 50 m m Tris (pH 7.4), 150 m m NaCl, 0.1% (v/v) Triton X-100, 1.5 m m MgCl2 , 1 m m sodium EDTA, 1 m m sodium EGTA, and cOmplete Mini protease inhibitor mixture (Roche Applied Science) using a polytron (Tissue Master 240, Omni International) for 1 min on setting number 8. .. C2C12 myotube homogenates were prepared by scrapping PBS-washed myotubes into cold lysis buffer solution (above) that contained cOmplete Mini protease inhibitor mixture (Roche Applied Science) then lysed with 10 passes through a 22-gauge needle. .. Muscle and myotube homogenates were centrifuged at 4 °C and 10,000 × g for 10 min, and caspase activity assays were set-up in white-walled 96-well plates; each assay contained 20 μg of protein from the sample supernatant mixed with an equal volume of caspase reagent (Promega, Madison, WI).

    Article Title: Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells
    Article Snippet: Proteins using TNT Quick Coupled Transcription/Transcription System (Promega, Charbonnières-les-Bains, France) were produced as follows: 1 μ g of template plasmid DNA was added to the reaction mixture, which was afterwards incubated at 30 °C for 90 min. Twenty microliters of the in vitro translated proteins were used for immunoprecipitation. .. Immunoprecipitation and immunoblotting Cells were lysed in lysis buffer (50 mM Hepes pH 7.4, 140 mM NaCl, 5 mM EDTA, 0.2% NP40, 10 mM sodium fluoride, protease inhibitor cocktail (Roche, Neuilly sur Seine, France)). .. In all, 800 μ g of each lysate was incubated with 3 μ g of CK2α , CK2β (Santa Cruz), Par-4 (Sigma-Aldrich), GFP-tag (Millipore) or HA-tag (Biomol, Hamburg, Germany) antibodies with constant agitation at 4 °C.

    Article Title: Combination of IAP antagonist and IFNγ activates novel caspase-10- and RIPK1-dependent cell death pathways
    Article Snippet: .. For co-immunoprecipitation, HT29 cells were lysed in DISC lysis buffer (1% (v/v) Triton X-100, 150 mM NaCl, 20 mM Tris, pH 7.5, 10% (v/v) glycerol, 2 mM EDTA) with complete protease inhibitor cocktail (Roche, Dee Why, NSW, Australia), phosphatase inhibitors (2 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM sodium molybdate, 5 mM β -glycerophosphate, 2 mM sodium pyrophosphate) and 1 mM NEM ( N -ethylmaleimide; Sigma, Castle Hill, NSW, Australia). .. Lysates were incubated overnight with 2 μ g caspase-8 antibody (Santa Cruz, Santa Cruz, CA, USA; sc6136) and 20 μ l packed Sepharose Protein G beads were incubated overnight with 1% BSA in PBS.

    Article Title: p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3 *
    Article Snippet: Quantification was performed using ImageJ. .. IP of endogenous caspase-2 was done using cell lysates in RIPA lysis buffer containing 150 m m NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 50 m m Tris-HCl, and 0.1% SDS, pH 8.0, supplemented with protease inhibitor mixture (Roche Applied Science) essentially as described ( , ). .. Preclearing was done followed by an overnight incubation with anti-caspase-2 antibody.

    Caspase-Glo Assay:

    Article Title: Novel agent DMAMCL suppresses osteosarcoma growth and decreases the stemness of osteosarcoma stem cell
    Article Snippet: .. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt assay (MTS; inner salt assay) and Caspase-Glo 3/7 Assay Kit were purchased from Promega (America), RNase A and Propidium iodide (PI) were from Sigma (America), Annexin V-FITC Apoptosis Detection Kit was from Dojindo laboratories (Japan), the primary antibodies against PARP, BAK, BAX, BCL2, and MCL1 were from CST (America), antibodies against CyclinB1, CDC2, CD133, and Nanog were from Wanleibio (China), antibodies againstα-Tubulin was from Biotechnology (China), goat anti-rabbit antibody and goat anti-mouse antibody were from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd (China), Lysis buffer and Hoechst33342 Staining Solution for Living Cells were from Beyotime (China), TUNEL Apoptosis Assay Kit was from Roche (Switzerland), Giemsa Stain solution (10× Stock solution) was from Solarbio (China), Matrigel was from BD (America), DMEM medium, Fetal bovine serum, Glutamine, Antibiotics (penicillin 100 units/ml, streptomycin 100 μg/ml), and PBS were from Bioind (Israel), KnockOut™ Serum Replacement was from Gibco (America). ..

    Staining:

    Article Title: Novel agent DMAMCL suppresses osteosarcoma growth and decreases the stemness of osteosarcoma stem cell
    Article Snippet: .. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt assay (MTS; inner salt assay) and Caspase-Glo 3/7 Assay Kit were purchased from Promega (America), RNase A and Propidium iodide (PI) were from Sigma (America), Annexin V-FITC Apoptosis Detection Kit was from Dojindo laboratories (Japan), the primary antibodies against PARP, BAK, BAX, BCL2, and MCL1 were from CST (America), antibodies against CyclinB1, CDC2, CD133, and Nanog were from Wanleibio (China), antibodies againstα-Tubulin was from Biotechnology (China), goat anti-rabbit antibody and goat anti-mouse antibody were from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd (China), Lysis buffer and Hoechst33342 Staining Solution for Living Cells were from Beyotime (China), TUNEL Apoptosis Assay Kit was from Roche (Switzerland), Giemsa Stain solution (10× Stock solution) was from Solarbio (China), Matrigel was from BD (America), DMEM medium, Fetal bovine serum, Glutamine, Antibiotics (penicillin 100 units/ml, streptomycin 100 μg/ml), and PBS were from Bioind (Israel), KnockOut™ Serum Replacement was from Gibco (America). ..

    TUNEL Assay:

    Article Title: Novel agent DMAMCL suppresses osteosarcoma growth and decreases the stemness of osteosarcoma stem cell
    Article Snippet: .. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt assay (MTS; inner salt assay) and Caspase-Glo 3/7 Assay Kit were purchased from Promega (America), RNase A and Propidium iodide (PI) were from Sigma (America), Annexin V-FITC Apoptosis Detection Kit was from Dojindo laboratories (Japan), the primary antibodies against PARP, BAK, BAX, BCL2, and MCL1 were from CST (America), antibodies against CyclinB1, CDC2, CD133, and Nanog were from Wanleibio (China), antibodies againstα-Tubulin was from Biotechnology (China), goat anti-rabbit antibody and goat anti-mouse antibody were from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd (China), Lysis buffer and Hoechst33342 Staining Solution for Living Cells were from Beyotime (China), TUNEL Apoptosis Assay Kit was from Roche (Switzerland), Giemsa Stain solution (10× Stock solution) was from Solarbio (China), Matrigel was from BD (America), DMEM medium, Fetal bovine serum, Glutamine, Antibiotics (penicillin 100 units/ml, streptomycin 100 μg/ml), and PBS were from Bioind (Israel), KnockOut™ Serum Replacement was from Gibco (America). ..

    Apoptosis Assay:

    Article Title: Novel agent DMAMCL suppresses osteosarcoma growth and decreases the stemness of osteosarcoma stem cell
    Article Snippet: .. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt assay (MTS; inner salt assay) and Caspase-Glo 3/7 Assay Kit were purchased from Promega (America), RNase A and Propidium iodide (PI) were from Sigma (America), Annexin V-FITC Apoptosis Detection Kit was from Dojindo laboratories (Japan), the primary antibodies against PARP, BAK, BAX, BCL2, and MCL1 were from CST (America), antibodies against CyclinB1, CDC2, CD133, and Nanog were from Wanleibio (China), antibodies againstα-Tubulin was from Biotechnology (China), goat anti-rabbit antibody and goat anti-mouse antibody were from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd (China), Lysis buffer and Hoechst33342 Staining Solution for Living Cells were from Beyotime (China), TUNEL Apoptosis Assay Kit was from Roche (Switzerland), Giemsa Stain solution (10× Stock solution) was from Solarbio (China), Matrigel was from BD (America), DMEM medium, Fetal bovine serum, Glutamine, Antibiotics (penicillin 100 units/ml, streptomycin 100 μg/ml), and PBS were from Bioind (Israel), KnockOut™ Serum Replacement was from Gibco (America). ..

    Giemsa Stain:

    Article Title: Novel agent DMAMCL suppresses osteosarcoma growth and decreases the stemness of osteosarcoma stem cell
    Article Snippet: .. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt assay (MTS; inner salt assay) and Caspase-Glo 3/7 Assay Kit were purchased from Promega (America), RNase A and Propidium iodide (PI) were from Sigma (America), Annexin V-FITC Apoptosis Detection Kit was from Dojindo laboratories (Japan), the primary antibodies against PARP, BAK, BAX, BCL2, and MCL1 were from CST (America), antibodies against CyclinB1, CDC2, CD133, and Nanog were from Wanleibio (China), antibodies againstα-Tubulin was from Biotechnology (China), goat anti-rabbit antibody and goat anti-mouse antibody were from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd (China), Lysis buffer and Hoechst33342 Staining Solution for Living Cells were from Beyotime (China), TUNEL Apoptosis Assay Kit was from Roche (Switzerland), Giemsa Stain solution (10× Stock solution) was from Solarbio (China), Matrigel was from BD (America), DMEM medium, Fetal bovine serum, Glutamine, Antibiotics (penicillin 100 units/ml, streptomycin 100 μg/ml), and PBS were from Bioind (Israel), KnockOut™ Serum Replacement was from Gibco (America). ..

    Knock-Out:

    Article Title: Novel agent DMAMCL suppresses osteosarcoma growth and decreases the stemness of osteosarcoma stem cell
    Article Snippet: .. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt assay (MTS; inner salt assay) and Caspase-Glo 3/7 Assay Kit were purchased from Promega (America), RNase A and Propidium iodide (PI) were from Sigma (America), Annexin V-FITC Apoptosis Detection Kit was from Dojindo laboratories (Japan), the primary antibodies against PARP, BAK, BAX, BCL2, and MCL1 were from CST (America), antibodies against CyclinB1, CDC2, CD133, and Nanog were from Wanleibio (China), antibodies againstα-Tubulin was from Biotechnology (China), goat anti-rabbit antibody and goat anti-mouse antibody were from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd (China), Lysis buffer and Hoechst33342 Staining Solution for Living Cells were from Beyotime (China), TUNEL Apoptosis Assay Kit was from Roche (Switzerland), Giemsa Stain solution (10× Stock solution) was from Solarbio (China), Matrigel was from BD (America), DMEM medium, Fetal bovine serum, Glutamine, Antibiotics (penicillin 100 units/ml, streptomycin 100 μg/ml), and PBS were from Bioind (Israel), KnockOut™ Serum Replacement was from Gibco (America). ..

    Immunoprecipitation:

    Article Title: Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells
    Article Snippet: Proteins using TNT Quick Coupled Transcription/Transcription System (Promega, Charbonnières-les-Bains, France) were produced as follows: 1 μ g of template plasmid DNA was added to the reaction mixture, which was afterwards incubated at 30 °C for 90 min. Twenty microliters of the in vitro translated proteins were used for immunoprecipitation. .. Immunoprecipitation and immunoblotting Cells were lysed in lysis buffer (50 mM Hepes pH 7.4, 140 mM NaCl, 5 mM EDTA, 0.2% NP40, 10 mM sodium fluoride, protease inhibitor cocktail (Roche, Neuilly sur Seine, France)). .. In all, 800 μ g of each lysate was incubated with 3 μ g of CK2α , CK2β (Santa Cruz), Par-4 (Sigma-Aldrich), GFP-tag (Millipore) or HA-tag (Biomol, Hamburg, Germany) antibodies with constant agitation at 4 °C.

    other:

    Article Title: A Novel Cell Lysis Approach Reveals That Caspase-2 Rapidly Translocates from the Nucleus to the Cytoplasm in Response to Apoptotic Stimuli
    Article Snippet: In cells were disrupted in hypotonic buffer without Triton X-100 while in cells were lysed in the presence of 0.5% Triton X-100.

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    Roche caspase lysis buffer
    Cleavage of <t>caspase-7</t> and PARP is inhibited in influenza virus-infected BAD knockdown cells. (A) Nontargeting shRNA control (NSi) and BAD knockdown cells were infected with NY55 at an MOI of 3, and cells were harvested at specific times points. Whole-cell
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    Cleavage of caspase-7 and PARP is inhibited in influenza virus-infected BAD knockdown cells. (A) Nontargeting shRNA control (NSi) and BAD knockdown cells were infected with NY55 at an MOI of 3, and cells were harvested at specific times points. Whole-cell

    Journal: Journal of Virology

    Article Title: Influenza Virus Induces Apoptosis via BAD-Mediated Mitochondrial Dysregulation

    doi: 10.1128/JVI.02017-12

    Figure Lengend Snippet: Cleavage of caspase-7 and PARP is inhibited in influenza virus-infected BAD knockdown cells. (A) Nontargeting shRNA control (NSi) and BAD knockdown cells were infected with NY55 at an MOI of 3, and cells were harvested at specific times points. Whole-cell

    Article Snippet: Cytoplasmic lysate for caspase immunoblotting was obtained by lysing cells in caspase lysis buffer (20 mM Tris-HCl [pH 7.5], 0.5% NP-40, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 100 μM β-glycerol-3-phosphate, protease inhibitor cocktail [Roche]).

    Techniques: Infection, shRNA

    BAD is required for influenza virus-induced caspase-3 and caspase-7 activity. (A) Measurement of caspase-3/7 activity using Caspase-Glo 3/7 assay at 72 hpi in A549 cells infected with NY55 at an MOI of 1 or at 24 h after treatment of cells with 1 μM

    Journal: Journal of Virology

    Article Title: Influenza Virus Induces Apoptosis via BAD-Mediated Mitochondrial Dysregulation

    doi: 10.1128/JVI.02017-12

    Figure Lengend Snippet: BAD is required for influenza virus-induced caspase-3 and caspase-7 activity. (A) Measurement of caspase-3/7 activity using Caspase-Glo 3/7 assay at 72 hpi in A549 cells infected with NY55 at an MOI of 1 or at 24 h after treatment of cells with 1 μM

    Article Snippet: Cytoplasmic lysate for caspase immunoblotting was obtained by lysing cells in caspase lysis buffer (20 mM Tris-HCl [pH 7.5], 0.5% NP-40, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 100 μM β-glycerol-3-phosphate, protease inhibitor cocktail [Roche]).

    Techniques: Activity Assay, Caspase-Glo Assay, Infection

    XIAP downregulation restores TRAIL sensitivity in Bcl-2 overexpressing cells. (A) Downregulation of XIAP by siRNA treatment. Cells were left untreated (UT) or were transiently transfected with non-targeting (siControl) or siRNA specific for XIAP (siXIAP). Protein expression was determined 48 h post transfection from whole cell lysates via immunoblot assay using an XIAP specific antibody. As control c-IAP1 and survivin were included. (B) and (C) NCI-H460 wild type (B) and NCI-H460/Bcl-2 cells (C) had been pretreated as in (A) were treated with serial dilutions of Db-scTRAIL (open symbols). NCI-H460/Bcl-2 cells transfected with siXIAP were incubated with 50 μM z-VAD-fmk and 1 nM Db-scTRAIL (filled triangle). After 24 h viable cells were stained with crystal violet. All values were normalized to those from unstimulated cells. The experiment was performed in triplicates and the data shown are representative of three independent experiments. (D) NCI-H460/Bcl-2 cells had been pretreated as in (A) were treated with Db-scTRAIL (1 nM) for 2 and 4 h and analyzed by immunoblotting using antibodies for cleaved caspase-8, -9 and -3. Tubulin-α was used as loading control. Blot shown is representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps

    doi: 10.1371/journal.pone.0198203

    Figure Lengend Snippet: XIAP downregulation restores TRAIL sensitivity in Bcl-2 overexpressing cells. (A) Downregulation of XIAP by siRNA treatment. Cells were left untreated (UT) or were transiently transfected with non-targeting (siControl) or siRNA specific for XIAP (siXIAP). Protein expression was determined 48 h post transfection from whole cell lysates via immunoblot assay using an XIAP specific antibody. As control c-IAP1 and survivin were included. (B) and (C) NCI-H460 wild type (B) and NCI-H460/Bcl-2 cells (C) had been pretreated as in (A) were treated with serial dilutions of Db-scTRAIL (open symbols). NCI-H460/Bcl-2 cells transfected with siXIAP were incubated with 50 μM z-VAD-fmk and 1 nM Db-scTRAIL (filled triangle). After 24 h viable cells were stained with crystal violet. All values were normalized to those from unstimulated cells. The experiment was performed in triplicates and the data shown are representative of three independent experiments. (D) NCI-H460/Bcl-2 cells had been pretreated as in (A) were treated with Db-scTRAIL (1 nM) for 2 and 4 h and analyzed by immunoblotting using antibodies for cleaved caspase-8, -9 and -3. Tubulin-α was used as loading control. Blot shown is representative of three independent experiments.

    Article Snippet: Cells were then lysed using caspase lysis buffer with freshly added protease inhibitor cocktail (Roche Diagnostics, Germany) incubated on ice and centrifuged at 13000 × g at 4°C for 10 min.

    Techniques: Transfection, Expressing, Incubation, Staining

    Bcl-2 overexpression strongly inhibits caspase activity but affects caspase cleavage preferentially at late activation steps. (A) Cells were stimulated with Db-scTRAIL (1 nM) for the indicated time periods. Whole protein lysates were incubated with fluorogenic caspase substrates Ac-IEPD-AMC (caspase-8), Ac-DMQD-AMC (caspase-3) and Ac-LEHD-AMC (caspase-9), respectively. Increasing fluorescence values were measured every 2 min for 2 h at λ = 460 nm. Data points shown, representing slopes, are mean values ± SD calculated from 3 independent experiments, normalized to the highest value of each experiment. (B) Cells were stimulated with Db-scTRAIL (1 nM) up to 8 h, one group was left untreated as a control. Equal amounts of whole cell lysates were subjected to immunoblot analysis using antibodies specific for cleaved caspase-8, cleaved caspase-3 and caspase-9 followed by HRP-conjugated secondary antibody.

    Journal: PLoS ONE

    Article Title: Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps

    doi: 10.1371/journal.pone.0198203

    Figure Lengend Snippet: Bcl-2 overexpression strongly inhibits caspase activity but affects caspase cleavage preferentially at late activation steps. (A) Cells were stimulated with Db-scTRAIL (1 nM) for the indicated time periods. Whole protein lysates were incubated with fluorogenic caspase substrates Ac-IEPD-AMC (caspase-8), Ac-DMQD-AMC (caspase-3) and Ac-LEHD-AMC (caspase-9), respectively. Increasing fluorescence values were measured every 2 min for 2 h at λ = 460 nm. Data points shown, representing slopes, are mean values ± SD calculated from 3 independent experiments, normalized to the highest value of each experiment. (B) Cells were stimulated with Db-scTRAIL (1 nM) up to 8 h, one group was left untreated as a control. Equal amounts of whole cell lysates were subjected to immunoblot analysis using antibodies specific for cleaved caspase-8, cleaved caspase-3 and caspase-9 followed by HRP-conjugated secondary antibody.

    Article Snippet: Cells were then lysed using caspase lysis buffer with freshly added protease inhibitor cocktail (Roche Diagnostics, Germany) incubated on ice and centrifuged at 13000 × g at 4°C for 10 min.

    Techniques: Over Expression, Activity Assay, Activation Assay, Incubation, Fluorescence

    Caspase-9 plays only a minor role in TRAIL-induced apoptosis in NCI-H460 cells. (A) Cells were preincubated with caspase-9 inhibitor (z-LEHD-fmk, 50 μM) for 1 h, followed by stimulation with serial dilutions of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and absorbance was measured at 550 nm. (B) Western blot showing efficient downregulation of procaspase-9 by siRNA treatment. NCI-H460 cells were transfected with siRNAs (siControl and siCaspase-9) and harvested after 48 h. (C) NCI-H460 cells with downregulated caspase-9 do not show increased TRAIL resistance. Cells transfected with either non-targeting (siControl) or caspase-9 (siCaspase-9) siRNAs were stimulated 48 h post transfection with serial dilutions of Db-scTRAIL for further 24 h. (D) Cells pretreated as in (B) were treated for 2 and 4 h with Db-scTRAIL (1 nM), control cells were left untreated. Whole cell lysates were subjected to western blot analysis using antibodies specific for caspase-9 and caspase-3, α-tubulin was used to confirm equal loading.

    Journal: PLoS ONE

    Article Title: Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps

    doi: 10.1371/journal.pone.0198203

    Figure Lengend Snippet: Caspase-9 plays only a minor role in TRAIL-induced apoptosis in NCI-H460 cells. (A) Cells were preincubated with caspase-9 inhibitor (z-LEHD-fmk, 50 μM) for 1 h, followed by stimulation with serial dilutions of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and absorbance was measured at 550 nm. (B) Western blot showing efficient downregulation of procaspase-9 by siRNA treatment. NCI-H460 cells were transfected with siRNAs (siControl and siCaspase-9) and harvested after 48 h. (C) NCI-H460 cells with downregulated caspase-9 do not show increased TRAIL resistance. Cells transfected with either non-targeting (siControl) or caspase-9 (siCaspase-9) siRNAs were stimulated 48 h post transfection with serial dilutions of Db-scTRAIL for further 24 h. (D) Cells pretreated as in (B) were treated for 2 and 4 h with Db-scTRAIL (1 nM), control cells were left untreated. Whole cell lysates were subjected to western blot analysis using antibodies specific for caspase-9 and caspase-3, α-tubulin was used to confirm equal loading.

    Article Snippet: Cells were then lysed using caspase lysis buffer with freshly added protease inhibitor cocktail (Roche Diagnostics, Germany) incubated on ice and centrifuged at 13000 × g at 4°C for 10 min.

    Techniques: Staining, Western Blot, Transfection

    Smac is a potent regulator of TRAIL-induced apoptosis in NCI-H460 cells. (A) and (B) Smac mimetic SM83 sensitizes NCI-H460/Bcl-2 cells for TRAIL-induced apoptosis. NCI-H460 wild type (A) and NCI-H460/Bcl-2 cells (B) were preincubated with SM83 (1 μM) followed by stimulation with increasing concentrations of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and the absorbance was determined at 550 nm. Values from stimulated cells were normalized to unstimulated cells. Shown are mean values ± SD calculated from triplicates. One representative experiment out of three is shown. (C) Downregulation of Smac protein by siRNA treatment. NCI-H460 cells were transfected with siRNAs directed against Smac (siSmac) or non-targeting (siControl). Expression levels of Smac were determined 48 h later by western blotting. (D) Reduced caspase-3 processing after Smac downregulation is rescued by proteasome inhibition. Cells from (C) were preincubated with MG132 (25 μM) or Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3. (E) and (F) NCI-H460 cells were preincubated with Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3 (E) and apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry (F).

    Journal: PLoS ONE

    Article Title: Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps

    doi: 10.1371/journal.pone.0198203

    Figure Lengend Snippet: Smac is a potent regulator of TRAIL-induced apoptosis in NCI-H460 cells. (A) and (B) Smac mimetic SM83 sensitizes NCI-H460/Bcl-2 cells for TRAIL-induced apoptosis. NCI-H460 wild type (A) and NCI-H460/Bcl-2 cells (B) were preincubated with SM83 (1 μM) followed by stimulation with increasing concentrations of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and the absorbance was determined at 550 nm. Values from stimulated cells were normalized to unstimulated cells. Shown are mean values ± SD calculated from triplicates. One representative experiment out of three is shown. (C) Downregulation of Smac protein by siRNA treatment. NCI-H460 cells were transfected with siRNAs directed against Smac (siSmac) or non-targeting (siControl). Expression levels of Smac were determined 48 h later by western blotting. (D) Reduced caspase-3 processing after Smac downregulation is rescued by proteasome inhibition. Cells from (C) were preincubated with MG132 (25 μM) or Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3. (E) and (F) NCI-H460 cells were preincubated with Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3 (E) and apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry (F).

    Article Snippet: Cells were then lysed using caspase lysis buffer with freshly added protease inhibitor cocktail (Roche Diagnostics, Germany) incubated on ice and centrifuged at 13000 × g at 4°C for 10 min.

    Techniques: Staining, Transfection, Expressing, Western Blot, Inhibition, Flow Cytometry, Cytometry

    Inhibition of TAK1 promotes PANoptosis and PANoptosome formation. (A) Representative cell death images of BMDMs lacking different components of pyroptosis, apoptosis, or necroptosis after LPS priming and inhibition of TAK1. (B) Western blot analysis of PANoptosis activation after LPS priming and TAK1 inhibition. (C) Co-immunoprecipitation of PANoptosome components from primary BMDMs after TAK1 and caspase inhibition. Red asterisks denote a non-specific band.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Identification of the PANoptosome: A Molecular Platform Triggering Pyroptosis, Apoptosis, and Necroptosis (PANoptosis)

    doi: 10.3389/fcimb.2020.00237

    Figure Lengend Snippet: Inhibition of TAK1 promotes PANoptosis and PANoptosome formation. (A) Representative cell death images of BMDMs lacking different components of pyroptosis, apoptosis, or necroptosis after LPS priming and inhibition of TAK1. (B) Western blot analysis of PANoptosis activation after LPS priming and TAK1 inhibition. (C) Co-immunoprecipitation of PANoptosome components from primary BMDMs after TAK1 and caspase inhibition. Red asterisks denote a non-specific band.

    Article Snippet: Immunoblot AnalysisFor caspase-1 analysis, BMDMs were lysed along with the supernatant using 50 μL caspase lysis buffer (1 × protease inhibitors (Roche), 1 × phosphatase inhibitors (Roche), 10% NP-40 and 25 mM DTT) followed by the addition of 100 μL 4 × SDS loading buffer.

    Techniques: Inhibition, Western Blot, Activation Assay, Immunoprecipitation

    XIAP downregulation restores TRAIL sensitivity in Bcl-2 overexpressing cells. (A) Downregulation of XIAP by siRNA treatment. Cells were left untreated (UT) or were transiently transfected with non-targeting (siControl) or siRNA specific for XIAP (siXIAP). Protein expression was determined 48 h post transfection from whole cell lysates via immunoblot assay using an XIAP specific antibody. As control c-IAP1 and survivin were included. (B) and (C) NCI-H460 wild type (B) and NCI-H460/Bcl-2 cells (C) had been pretreated as in (A) were treated with serial dilutions of Db-scTRAIL (open symbols). NCI-H460/Bcl-2 cells transfected with siXIAP were incubated with 50 μM z-VAD-fmk and 1 nM Db-scTRAIL (filled triangle). After 24 h viable cells were stained with crystal violet. All values were normalized to those from unstimulated cells. The experiment was performed in triplicates and the data shown are representative of three independent experiments. (D) NCI-H460/Bcl-2 cells had been pretreated as in (A) were treated with Db-scTRAIL (1 nM) for 2 and 4 h and analyzed by immunoblotting using antibodies for cleaved caspase-8, -9 and -3. Tubulin-α was used as loading control. Blot shown is representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps

    doi: 10.1371/journal.pone.0198203

    Figure Lengend Snippet: XIAP downregulation restores TRAIL sensitivity in Bcl-2 overexpressing cells. (A) Downregulation of XIAP by siRNA treatment. Cells were left untreated (UT) or were transiently transfected with non-targeting (siControl) or siRNA specific for XIAP (siXIAP). Protein expression was determined 48 h post transfection from whole cell lysates via immunoblot assay using an XIAP specific antibody. As control c-IAP1 and survivin were included. (B) and (C) NCI-H460 wild type (B) and NCI-H460/Bcl-2 cells (C) had been pretreated as in (A) were treated with serial dilutions of Db-scTRAIL (open symbols). NCI-H460/Bcl-2 cells transfected with siXIAP were incubated with 50 μM z-VAD-fmk and 1 nM Db-scTRAIL (filled triangle). After 24 h viable cells were stained with crystal violet. All values were normalized to those from unstimulated cells. The experiment was performed in triplicates and the data shown are representative of three independent experiments. (D) NCI-H460/Bcl-2 cells had been pretreated as in (A) were treated with Db-scTRAIL (1 nM) for 2 and 4 h and analyzed by immunoblotting using antibodies for cleaved caspase-8, -9 and -3. Tubulin-α was used as loading control. Blot shown is representative of three independent experiments.

    Article Snippet: Cells were then lysed using caspase lysis buffer with freshly added protease inhibitor cocktail (Roche Diagnostics, Germany) incubated on ice and centrifuged at 13000 × g at 4°C for 10 min.

    Techniques: Transfection, Expressing, Incubation, Staining

    Bcl-2 overexpression strongly inhibits caspase activity but affects caspase cleavage preferentially at late activation steps. (A) Cells were stimulated with Db-scTRAIL (1 nM) for the indicated time periods. Whole protein lysates were incubated with fluorogenic caspase substrates Ac-IEPD-AMC (caspase-8), Ac-DMQD-AMC (caspase-3) and Ac-LEHD-AMC (caspase-9), respectively. Increasing fluorescence values were measured every 2 min for 2 h at λ = 460 nm. Data points shown, representing slopes, are mean values ± SD calculated from 3 independent experiments, normalized to the highest value of each experiment. (B) Cells were stimulated with Db-scTRAIL (1 nM) up to 8 h, one group was left untreated as a control. Equal amounts of whole cell lysates were subjected to immunoblot analysis using antibodies specific for cleaved caspase-8, cleaved caspase-3 and caspase-9 followed by HRP-conjugated secondary antibody.

    Journal: PLoS ONE

    Article Title: Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps

    doi: 10.1371/journal.pone.0198203

    Figure Lengend Snippet: Bcl-2 overexpression strongly inhibits caspase activity but affects caspase cleavage preferentially at late activation steps. (A) Cells were stimulated with Db-scTRAIL (1 nM) for the indicated time periods. Whole protein lysates were incubated with fluorogenic caspase substrates Ac-IEPD-AMC (caspase-8), Ac-DMQD-AMC (caspase-3) and Ac-LEHD-AMC (caspase-9), respectively. Increasing fluorescence values were measured every 2 min for 2 h at λ = 460 nm. Data points shown, representing slopes, are mean values ± SD calculated from 3 independent experiments, normalized to the highest value of each experiment. (B) Cells were stimulated with Db-scTRAIL (1 nM) up to 8 h, one group was left untreated as a control. Equal amounts of whole cell lysates were subjected to immunoblot analysis using antibodies specific for cleaved caspase-8, cleaved caspase-3 and caspase-9 followed by HRP-conjugated secondary antibody.

    Article Snippet: Cells were then lysed using caspase lysis buffer with freshly added protease inhibitor cocktail (Roche Diagnostics, Germany) incubated on ice and centrifuged at 13000 × g at 4°C for 10 min.

    Techniques: Over Expression, Activity Assay, Activation Assay, Incubation, Fluorescence

    Caspase-9 plays only a minor role in TRAIL-induced apoptosis in NCI-H460 cells. (A) Cells were preincubated with caspase-9 inhibitor (z-LEHD-fmk, 50 μM) for 1 h, followed by stimulation with serial dilutions of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and absorbance was measured at 550 nm. (B) Western blot showing efficient downregulation of procaspase-9 by siRNA treatment. NCI-H460 cells were transfected with siRNAs (siControl and siCaspase-9) and harvested after 48 h. (C) NCI-H460 cells with downregulated caspase-9 do not show increased TRAIL resistance. Cells transfected with either non-targeting (siControl) or caspase-9 (siCaspase-9) siRNAs were stimulated 48 h post transfection with serial dilutions of Db-scTRAIL for further 24 h. (D) Cells pretreated as in (B) were treated for 2 and 4 h with Db-scTRAIL (1 nM), control cells were left untreated. Whole cell lysates were subjected to western blot analysis using antibodies specific for caspase-9 and caspase-3, α-tubulin was used to confirm equal loading.

    Journal: PLoS ONE

    Article Title: Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps

    doi: 10.1371/journal.pone.0198203

    Figure Lengend Snippet: Caspase-9 plays only a minor role in TRAIL-induced apoptosis in NCI-H460 cells. (A) Cells were preincubated with caspase-9 inhibitor (z-LEHD-fmk, 50 μM) for 1 h, followed by stimulation with serial dilutions of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and absorbance was measured at 550 nm. (B) Western blot showing efficient downregulation of procaspase-9 by siRNA treatment. NCI-H460 cells were transfected with siRNAs (siControl and siCaspase-9) and harvested after 48 h. (C) NCI-H460 cells with downregulated caspase-9 do not show increased TRAIL resistance. Cells transfected with either non-targeting (siControl) or caspase-9 (siCaspase-9) siRNAs were stimulated 48 h post transfection with serial dilutions of Db-scTRAIL for further 24 h. (D) Cells pretreated as in (B) were treated for 2 and 4 h with Db-scTRAIL (1 nM), control cells were left untreated. Whole cell lysates were subjected to western blot analysis using antibodies specific for caspase-9 and caspase-3, α-tubulin was used to confirm equal loading.

    Article Snippet: Cells were then lysed using caspase lysis buffer with freshly added protease inhibitor cocktail (Roche Diagnostics, Germany) incubated on ice and centrifuged at 13000 × g at 4°C for 10 min.

    Techniques: Staining, Western Blot, Transfection

    Smac is a potent regulator of TRAIL-induced apoptosis in NCI-H460 cells. (A) and (B) Smac mimetic SM83 sensitizes NCI-H460/Bcl-2 cells for TRAIL-induced apoptosis. NCI-H460 wild type (A) and NCI-H460/Bcl-2 cells (B) were preincubated with SM83 (1 μM) followed by stimulation with increasing concentrations of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and the absorbance was determined at 550 nm. Values from stimulated cells were normalized to unstimulated cells. Shown are mean values ± SD calculated from triplicates. One representative experiment out of three is shown. (C) Downregulation of Smac protein by siRNA treatment. NCI-H460 cells were transfected with siRNAs directed against Smac (siSmac) or non-targeting (siControl). Expression levels of Smac were determined 48 h later by western blotting. (D) Reduced caspase-3 processing after Smac downregulation is rescued by proteasome inhibition. Cells from (C) were preincubated with MG132 (25 μM) or Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3. (E) and (F) NCI-H460 cells were preincubated with Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3 (E) and apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry (F).

    Journal: PLoS ONE

    Article Title: Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps

    doi: 10.1371/journal.pone.0198203

    Figure Lengend Snippet: Smac is a potent regulator of TRAIL-induced apoptosis in NCI-H460 cells. (A) and (B) Smac mimetic SM83 sensitizes NCI-H460/Bcl-2 cells for TRAIL-induced apoptosis. NCI-H460 wild type (A) and NCI-H460/Bcl-2 cells (B) were preincubated with SM83 (1 μM) followed by stimulation with increasing concentrations of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and the absorbance was determined at 550 nm. Values from stimulated cells were normalized to unstimulated cells. Shown are mean values ± SD calculated from triplicates. One representative experiment out of three is shown. (C) Downregulation of Smac protein by siRNA treatment. NCI-H460 cells were transfected with siRNAs directed against Smac (siSmac) or non-targeting (siControl). Expression levels of Smac were determined 48 h later by western blotting. (D) Reduced caspase-3 processing after Smac downregulation is rescued by proteasome inhibition. Cells from (C) were preincubated with MG132 (25 μM) or Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3. (E) and (F) NCI-H460 cells were preincubated with Bortezomib (1 μM) followed by stimulation with Db-scTRAIL (1 nM) for 4 h. Immunoblotting was performed to detect cleaved caspase-3 (E) and apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry (F).

    Article Snippet: Cells were then lysed using caspase lysis buffer with freshly added protease inhibitor cocktail (Roche Diagnostics, Germany) incubated on ice and centrifuged at 13000 × g at 4°C for 10 min.

    Techniques: Staining, Transfection, Expressing, Western Blot, Inhibition, Flow Cytometry, Cytometry