Journal: PLoS ONE

Article Title: Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

doi: 10.1371/journal.pone.0002844

Figure Lengend Snippet: A) Cells were stably transfected with plasmids encoding one of three different bid -specific shRNAs or a scrambled control shRNA as described in . For analysis of the bid knockdown, cells were subjected to Western blotting with a polyclonal Bid antibody. α-Tubulin served as a loading control. B) Control cells expressing the scrambled (Ctrl 1, Ctrl 2), or the bid specific shRNA (Bid kd 4) and clone (Bid kd 1) were incubated with Cycloheximide (CHX) (1 µg/ml) and an agonistic Fas antibody or vehicle (clone CH11) at the indicated concentrations or vehicle for 4 h. Caspase-3 like activity was measured by cleavage of the fluorigenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05: difference from control cells (Ctrl). C) Cell lysates were subjected to Western blotting with antibodies for the indicated proteins, as described in . D) HeLa Bid kd cells were stably transfected with a vector coding for YFP- bid -CFP (GFP-Bid); cell lysates were subjected to Western blotting with GFP and Bid antibodies. E) HeLa Bid kd cells expressing GFP-Bid were treated with CHX (1 µg/ml) and an activating Fas antibody (100 ng/ml). Controls were treated with vehicle for 4 h; caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl).

Article Snippet: The following primary antibodies were used: a rabbit polyclonal APAF-1 (Chemicon, Carrigtwohill, Ireland), a rabbit polyclonal Bad or caspase-3 (Cell Signaling, Bray, Ireland), a mouse monoclonal Bak or Bcl-2 (Santa Cruz, Heidelberg, Germany), a rabbit polyclonal Bax (Upstate, Carrigtwohill, Ireland), a rabbit polyclonal Bcl-X (BD Biosciences, Erembodgem, Belgium), a mouse monoclonal Bim or caspase-8 (Alexis Biochemicals, Blessington, Ireland), a goat polyclonal Bid (R&D Systems, Abingdon, UK), a rabbit polyclonal caspase-9 (Calbiochem, Darmstadt, Germany), a rabbit polyclonal DR4 or DR5 (Abcam, Cambridge, UK), a rabbit poyclonal GFP (Clontech, Oxford, UK), a mouse monoclonal KDEL (Stressgen, York, UK), a rabbit polyclonal cleaved PARP (NEB, Bray, Ireland), a rabbit polyclonal smac/DIABLO (R&D Systems), a mouse monoclonal XIAP (BD Biosciences), a mouse monoclonal β-actin or α-tubulin (Sigma Aldrich, Dublin, Ireland).

Techniques: Stable Transfection, Transfection, Control, shRNA, Knockdown, Western Blot, Expressing, Incubation, Activity Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

doi: 10.1371/journal.pone.0002844

Figure Lengend Snippet: Control and HeLa Bid kd were pre-incubated with the pan-caspase inhibitor zVAD-fmk (100 µM) for 1 h followed by treatment for the specified times with CHX (1 µg/ml) in combination with an agonistic Fas antibody or recombinant TRAIL at the indicated concentrations. Controls were treated with vehicle. A, B) Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl). C, D) Cells were treated as indicated and lysates were subjected to Western blotting with a polyclonal caspase-3, a monoclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. E, F) Cells were treated as indicated and apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl).

Article Snippet: The following primary antibodies were used: a rabbit polyclonal APAF-1 (Chemicon, Carrigtwohill, Ireland), a rabbit polyclonal Bad or caspase-3 (Cell Signaling, Bray, Ireland), a mouse monoclonal Bak or Bcl-2 (Santa Cruz, Heidelberg, Germany), a rabbit polyclonal Bax (Upstate, Carrigtwohill, Ireland), a rabbit polyclonal Bcl-X (BD Biosciences, Erembodgem, Belgium), a mouse monoclonal Bim or caspase-8 (Alexis Biochemicals, Blessington, Ireland), a goat polyclonal Bid (R&D Systems, Abingdon, UK), a rabbit polyclonal caspase-9 (Calbiochem, Darmstadt, Germany), a rabbit polyclonal DR4 or DR5 (Abcam, Cambridge, UK), a rabbit poyclonal GFP (Clontech, Oxford, UK), a mouse monoclonal KDEL (Stressgen, York, UK), a rabbit polyclonal cleaved PARP (NEB, Bray, Ireland), a rabbit polyclonal smac/DIABLO (R&D Systems), a mouse monoclonal XIAP (BD Biosciences), a mouse monoclonal β-actin or α-tubulin (Sigma Aldrich, Dublin, Ireland).

Techniques: Control, Incubation, Recombinant, Activity Assay, Western Blot, Binding Assay

Journal: PLoS ONE

Article Title: Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

doi: 10.1371/journal.pone.0002844

Figure Lengend Snippet: Control and HeLa Bid kd cells were pre-incubated with the pan-caspase inhibitor zVAD-fmk (100 µM) for 1 h before treatment with the indicated concentrations of Staurosporine for the indicated times. A) Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. n.s. = not significant versus control (Ctrl). B) Cells were treated as indicated and lysates were subjected to Western blotting with a polyclonal caspase-3, a monoclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. C, D) The small molecule Bid inhibitor BI6C9 mimics the effects of the knockdown of Bid in HeLa cells. Parental HeLa cells were pre-incubated with the indicated concentrations of BI6C9 for 16 h prior to treatment with an agonistic Fas antibody, Staurosporine (STS) or vehicle, for 6 h. Apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from control cultures treated with Fas antibody or Staurosporine in the absence of BI6C9.

Article Snippet: The following primary antibodies were used: a rabbit polyclonal APAF-1 (Chemicon, Carrigtwohill, Ireland), a rabbit polyclonal Bad or caspase-3 (Cell Signaling, Bray, Ireland), a mouse monoclonal Bak or Bcl-2 (Santa Cruz, Heidelberg, Germany), a rabbit polyclonal Bax (Upstate, Carrigtwohill, Ireland), a rabbit polyclonal Bcl-X (BD Biosciences, Erembodgem, Belgium), a mouse monoclonal Bim or caspase-8 (Alexis Biochemicals, Blessington, Ireland), a goat polyclonal Bid (R&D Systems, Abingdon, UK), a rabbit polyclonal caspase-9 (Calbiochem, Darmstadt, Germany), a rabbit polyclonal DR4 or DR5 (Abcam, Cambridge, UK), a rabbit poyclonal GFP (Clontech, Oxford, UK), a mouse monoclonal KDEL (Stressgen, York, UK), a rabbit polyclonal cleaved PARP (NEB, Bray, Ireland), a rabbit polyclonal smac/DIABLO (R&D Systems), a mouse monoclonal XIAP (BD Biosciences), a mouse monoclonal β-actin or α-tubulin (Sigma Aldrich, Dublin, Ireland).

Techniques: Control, Incubation, Activity Assay, Western Blot, Knockdown, Binding Assay

Journal: PLoS ONE

Article Title: Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

doi: 10.1371/journal.pone.0002844

Figure Lengend Snippet: Control cells and HeLa Bid kd cells were pre-incubated with the pan-caspase inhibitor zVAD-fmk (100 µM) where specified for 1 h before treatment with the ER stressors Tunicamycin, Brefeldin A, and Thapsigargin at the indicated concentrations for the indicated times. A) Cell lysates were subjected to Western blotting with a monoclonal KDEL antibody, which detects Grp94 and Grp78, and a monoclonal α-tubulin antibody. B, C, D) Caspase-3 like activity was measured by cleavage of the fluorigenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. n.s. = not significant versus control (Ctrl). E) Cells were treated as indicated and lysates were subjected to Western blotting with a polyclonal caspase-3 antibody, a monoclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. F) Apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. n.s. = not significant versus control (Ctrl).

Article Snippet: The following primary antibodies were used: a rabbit polyclonal APAF-1 (Chemicon, Carrigtwohill, Ireland), a rabbit polyclonal Bad or caspase-3 (Cell Signaling, Bray, Ireland), a mouse monoclonal Bak or Bcl-2 (Santa Cruz, Heidelberg, Germany), a rabbit polyclonal Bax (Upstate, Carrigtwohill, Ireland), a rabbit polyclonal Bcl-X (BD Biosciences, Erembodgem, Belgium), a mouse monoclonal Bim or caspase-8 (Alexis Biochemicals, Blessington, Ireland), a goat polyclonal Bid (R&D Systems, Abingdon, UK), a rabbit polyclonal caspase-9 (Calbiochem, Darmstadt, Germany), a rabbit polyclonal DR4 or DR5 (Abcam, Cambridge, UK), a rabbit poyclonal GFP (Clontech, Oxford, UK), a mouse monoclonal KDEL (Stressgen, York, UK), a rabbit polyclonal cleaved PARP (NEB, Bray, Ireland), a rabbit polyclonal smac/DIABLO (R&D Systems), a mouse monoclonal XIAP (BD Biosciences), a mouse monoclonal β-actin or α-tubulin (Sigma Aldrich, Dublin, Ireland).

Techniques: Control, Incubation, Western Blot, Activity Assay, Binding Assay

Journal: PLoS ONE

Article Title: Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

doi: 10.1371/journal.pone.0002844

Figure Lengend Snippet: A) HeLa Control and HeLa Bid kd cells were pre-incubated with the pan-caspase inhibitor zVAD-fmk for 1 h where specified; cells were subsequently treated with Tunicamycin or vehicle for the indicated times. Cell lysates were subjected to Western blotting with a polyclonal DR4, a polyclonal DR5, and a monoclonal β-actin antibody. B, C) Control cells and HeLa Bid kd cells were pre-incubated for 16 h with the indicated concentrations of Tunicamycin, Thapsigargin or vehicle before treatment with TRAIL (10 ng/ml). Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from Tunicamycin, Thapsigargin or TRAIL individual treatments. # p<0.05: difference from control cells (Ctrl). D) Control cells and HeLa Bid kd cells were pre-incubated with Tunicamycin (0.3 µM) or vehicle for the indicated times followed by treatment with TRAIL (10 ng/ml) for the indicated times. Cell lysates were subjected to Western blotting with a polyclonal Bid, a polyclonal caspase-3, a polyclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. E) Control cells and HeLa Bid kd cells were pre-incubated with Tunicamycin (0.3 µM) for 16 h followed by treatment with recombinant TRAIL (10 ng/ml) for the indicated times. Apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from Tunicamycin or TRAIL individual treatments. # p<0.05: difference from control cells (Ctrl). F) Control cells and HeLa Bid kd cells were pre-incubated with a DR5 blocking peptide (50 ng/ml) for 1 h followed by pre-treatment with Tunicamycin (0.3 µM) or vehicle for 16 h. Subsequently, cells were treated with recombinant TRAIL for 3 h. Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from Tunicamycin or TRAIL individual treatments. # p<0.05: difference from control cells (Ctrl).

Article Snippet: The following primary antibodies were used: a rabbit polyclonal APAF-1 (Chemicon, Carrigtwohill, Ireland), a rabbit polyclonal Bad or caspase-3 (Cell Signaling, Bray, Ireland), a mouse monoclonal Bak or Bcl-2 (Santa Cruz, Heidelberg, Germany), a rabbit polyclonal Bax (Upstate, Carrigtwohill, Ireland), a rabbit polyclonal Bcl-X (BD Biosciences, Erembodgem, Belgium), a mouse monoclonal Bim or caspase-8 (Alexis Biochemicals, Blessington, Ireland), a goat polyclonal Bid (R&D Systems, Abingdon, UK), a rabbit polyclonal caspase-9 (Calbiochem, Darmstadt, Germany), a rabbit polyclonal DR4 or DR5 (Abcam, Cambridge, UK), a rabbit poyclonal GFP (Clontech, Oxford, UK), a mouse monoclonal KDEL (Stressgen, York, UK), a rabbit polyclonal cleaved PARP (NEB, Bray, Ireland), a rabbit polyclonal smac/DIABLO (R&D Systems), a mouse monoclonal XIAP (BD Biosciences), a mouse monoclonal β-actin or α-tubulin (Sigma Aldrich, Dublin, Ireland).

Techniques: Control, Incubation, Western Blot, Activity Assay, Recombinant, Binding Assay, Blocking Assay

Journal: PLoS ONE

Article Title: Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

doi: 10.1371/journal.pone.0002844

Figure Lengend Snippet: HeLa control and HeLa Bid kd cells were treated with the indicated concentrations of Etoposide, Oxaliplatin and Doxorubicin, or vehicle, for 24 h; cells were pre-incubated with the pan-caspase inhibitor zVAD-fmk (100 µM) for 1 h where specified. A, B, C) Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl). D) Cells were treated as indicated and lysates were subjected to Western blotting with a polyclonal Bid, a polyclonal caspase-3, a polyclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. E) Apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl).

Article Snippet: The following primary antibodies were used: a rabbit polyclonal APAF-1 (Chemicon, Carrigtwohill, Ireland), a rabbit polyclonal Bad or caspase-3 (Cell Signaling, Bray, Ireland), a mouse monoclonal Bak or Bcl-2 (Santa Cruz, Heidelberg, Germany), a rabbit polyclonal Bax (Upstate, Carrigtwohill, Ireland), a rabbit polyclonal Bcl-X (BD Biosciences, Erembodgem, Belgium), a mouse monoclonal Bim or caspase-8 (Alexis Biochemicals, Blessington, Ireland), a goat polyclonal Bid (R&D Systems, Abingdon, UK), a rabbit polyclonal caspase-9 (Calbiochem, Darmstadt, Germany), a rabbit polyclonal DR4 or DR5 (Abcam, Cambridge, UK), a rabbit poyclonal GFP (Clontech, Oxford, UK), a mouse monoclonal KDEL (Stressgen, York, UK), a rabbit polyclonal cleaved PARP (NEB, Bray, Ireland), a rabbit polyclonal smac/DIABLO (R&D Systems), a mouse monoclonal XIAP (BD Biosciences), a mouse monoclonal β-actin or α-tubulin (Sigma Aldrich, Dublin, Ireland).

Techniques: Control, Incubation, Activity Assay, Western Blot, Binding Assay

Journal: PLoS ONE

Article Title: Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

doi: 10.1371/journal.pone.0002844

Figure Lengend Snippet: A, B) HeLa Control and HeLa Bid kd cells were treated with Etoposide (10 µM) or Oxaliplatin (30 µg/ml) for the indicated times; the pan-caspase inhibitor zVAD (100 µM) was added to the cells 1 h prior to treatment where specified; cell lysates were subjected to Western blotting with a polyclonal DR4, a polyclonal DR5, and a monoclonal β-actin antibody. C, D) HeLa control and HeLa Bid kd cells were pre-incubated with Etoposide (10 µM), Oxaliplatin (30 µg/ml), or vehicle for 16 h followed by treatment with TRAIL (10 ng/ml) for 3 h. Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from Etoposide, Oxaliplatin or TRAIL individual treatments. # p<0.05 difference from control cells (Ctrl).

Article Snippet: The following primary antibodies were used: a rabbit polyclonal APAF-1 (Chemicon, Carrigtwohill, Ireland), a rabbit polyclonal Bad or caspase-3 (Cell Signaling, Bray, Ireland), a mouse monoclonal Bak or Bcl-2 (Santa Cruz, Heidelberg, Germany), a rabbit polyclonal Bax (Upstate, Carrigtwohill, Ireland), a rabbit polyclonal Bcl-X (BD Biosciences, Erembodgem, Belgium), a mouse monoclonal Bim or caspase-8 (Alexis Biochemicals, Blessington, Ireland), a goat polyclonal Bid (R&D Systems, Abingdon, UK), a rabbit polyclonal caspase-9 (Calbiochem, Darmstadt, Germany), a rabbit polyclonal DR4 or DR5 (Abcam, Cambridge, UK), a rabbit poyclonal GFP (Clontech, Oxford, UK), a mouse monoclonal KDEL (Stressgen, York, UK), a rabbit polyclonal cleaved PARP (NEB, Bray, Ireland), a rabbit polyclonal smac/DIABLO (R&D Systems), a mouse monoclonal XIAP (BD Biosciences), a mouse monoclonal β-actin or α-tubulin (Sigma Aldrich, Dublin, Ireland).

Techniques: Control, Western Blot, Incubation, Activity Assay