Caspase 3 Antibody Search Results


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    Cell Signaling Technology Inc cleaved caspase 3
    REV-ERB agonist SR9011 inhibit autophagy a–b, SR90011 treatment reduces the number of autophagosomes both in MCF7 and T47D, n= biological independent samples MCF7 20µM 24h n=9 (mock), n=4 (SR9011) ** P =0.0056, T47D 48h 20µM n=5 (mock), n=4 (SR9011) ** P =0.0079; c–d, SR9011 induces accumulation of p62 as shown by immunofluorescence both in MCF7 and T47D n= biological independent samples 48h MCF7 p62 n=3 (mock), n=4 (SR9011) * P =0.0286; 48h T47D n=5 (mock, SR9011) ** P =0.004; e, Accumulation of p62 is confirmed by immunoblot (48h, 20µM A375); f–g, Inhibition of autophagy precedes apoptosis induction as shown by immunofluorescence of p62, cleaved <t>Caspase</t> 3 and TUNEL assay (n= biological independent samples n=4 mock 48h, n=5 SR9011 p62, n=3 48h SR9011; n=6 (mock, SR9011 72h p62), n=10 (mock 72h), n=8 (SR9011 72h) A375 20µM, Cl. Casp. 3 48h * P =0.0286; Cl. Casp. 3 72h **** P
    Cleaved Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cleaved caspase 3/product/Cell Signaling Technology Inc
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    99
    Cell Signaling Technology Inc caspase 3
    Evaluation of V-9302  in vivo  in HCC1806 cell line xenograft-bearing mice ( A ) Tumor volumetric analysis of mice treated with vehicle or V-9302 (75 mg/kg per day) for 10 days.  n  = 5 mice per group. Treatment started 9 days post injection. P value at day 10 determined by Student’s  t  test. ( B ) Immunofluorescence analysis of tumor tissues harvested from vehicle- or V-9302-treated mice; LC3B and pAKT (Ser473) shown (pink). CD-31 positive vessels shown in green and nuclei in blue (DAPI). 20× magnification. P values determined by Student’s  t  test. ( C ) Effects of V-9302 treatment on pS6-positive cells and caspase 3-positive cells by immunohistochemistry. Quantitative analysis consisted of the mean counts of at least three representative fields from three vehicle and three V-9302-treated mice. For box plots, center line is plotted at the median; the box spans from the first quartile to the third quartile; whiskers represent min to max. Error bars represent ± std. dev.
    Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase 3/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    caspase 3 - by Bioz Stars, 2021-05
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    99
    Cell Signaling Technology Inc anti caspase 3
    MLKL encoding mRNA induces cell death in vitro and in vivo. a – f In vitro cell death characterization. B16-OVA cells were transfected with PBS or with Fluc-, tBid-, or MLKL-mRNA. a At different time points after transfection, cells were collected and analyzed by flow cytometry. Graph showing the percentages of sytox + cells (left). Representative flow cytometric plots 24 h after transfection (right). b Impact of the pan-caspase inhibitor zVAD-fmk on cell death induction (top) and caspase activity (bottom) upon transfection of B16-OVA cells with tBID- or MLKL-mRNA. c Western blot analysis of the expression of MLKL, <t>caspase-3,</t> and cleaved caspase-3 in cell lysates prepared 24 h after mRNA transfection. Tubulin served as a loading control. d B16 cells were co-transfected with luciferase NF-κB reporter plasmid and a plasmid expressing β-galactosidase. Twenty four hours later, the cells were transfected with PBS, GFP-, tBid-, or MLKL-mRNA or, as a positive control for NF-κB activation, with TRAF6 expression vector or they were stimulated with TNF. The normalized luciferase activity in the lysates determined at different time points after mRNA transfection is depicted. e B16 cells were transfected with a GFP expression plasmid. Twenty four hours later, the cells were transfected with PBS, Fluc-, tBid-, or MLKL-mRNA and the cells were treated or not with actinomycin D as indicated. Twenty four hours after mRNA transfection, cell death and GFP expression were quantified using flow cytometry. Horizontal lines indicate the mean. f Still images of B16-OVA cells at 0 and 24 h after transfection with tBid- or MLKL-mRNA visualized by time-lapse microscopy (scale bar = 10 µm). g In vivo cell death characterization. Subcutaneously growing B16-OVA tumors were injected with PBS or with 10 µg mRNA encoding Fluc, tBid, or MLKL followed by electroporation. Twenty four hours later, the tumor cells were isolated and analyzed by flow cytometry for sytox uptake. Graph showing the percentages of sytox + cells (left) and representative flow cytometry plots (right). Horizontal lines indicate the mean
    Anti Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti caspase 3/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti caspase 3 - by Bioz Stars, 2021-05
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    99
    Cell Signaling Technology Inc caspase 9
    MLKL encoding mRNA induces cell death in vitro and in vivo. a – f In vitro cell death characterization. B16-OVA cells were transfected with PBS or with Fluc-, tBid-, or MLKL-mRNA. a At different time points after transfection, cells were collected and analyzed by flow cytometry. Graph showing the percentages of sytox + cells (left). Representative flow cytometric plots 24 h after transfection (right). b Impact of the pan-caspase inhibitor zVAD-fmk on cell death induction (top) and caspase activity (bottom) upon transfection of B16-OVA cells with tBID- or MLKL-mRNA. c Western blot analysis of the expression of MLKL, <t>caspase-3,</t> and cleaved caspase-3 in cell lysates prepared 24 h after mRNA transfection. Tubulin served as a loading control. d B16 cells were co-transfected with luciferase NF-κB reporter plasmid and a plasmid expressing β-galactosidase. Twenty four hours later, the cells were transfected with PBS, GFP-, tBid-, or MLKL-mRNA or, as a positive control for NF-κB activation, with TRAF6 expression vector or they were stimulated with TNF. The normalized luciferase activity in the lysates determined at different time points after mRNA transfection is depicted. e B16 cells were transfected with a GFP expression plasmid. Twenty four hours later, the cells were transfected with PBS, Fluc-, tBid-, or MLKL-mRNA and the cells were treated or not with actinomycin D as indicated. Twenty four hours after mRNA transfection, cell death and GFP expression were quantified using flow cytometry. Horizontal lines indicate the mean. f Still images of B16-OVA cells at 0 and 24 h after transfection with tBid- or MLKL-mRNA visualized by time-lapse microscopy (scale bar = 10 µm). g In vivo cell death characterization. Subcutaneously growing B16-OVA tumors were injected with PBS or with 10 µg mRNA encoding Fluc, tBid, or MLKL followed by electroporation. Twenty four hours later, the tumor cells were isolated and analyzed by flow cytometry for sytox uptake. Graph showing the percentages of sytox + cells (left) and representative flow cytometry plots (right). Horizontal lines indicate the mean
    Caspase 9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase 9/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    caspase 9 - by Bioz Stars, 2021-05
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    N/A
    Rabbit Anti Human Caspase 3 cleaved Polyclonal
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    The Caspase 3 Antibody from Novus Biologicals is a rabbit polyclonal antibody to Caspase 3 This antibody reacts with human The Caspase 3 Antibody has been validated for the following
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    Mouse monoclonal Caspase 3 antibody
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    Image Search Results


    REV-ERB agonist SR9011 inhibit autophagy a–b, SR90011 treatment reduces the number of autophagosomes both in MCF7 and T47D, n= biological independent samples MCF7 20µM 24h n=9 (mock), n=4 (SR9011) ** P =0.0056, T47D 48h 20µM n=5 (mock), n=4 (SR9011) ** P =0.0079; c–d, SR9011 induces accumulation of p62 as shown by immunofluorescence both in MCF7 and T47D n= biological independent samples 48h MCF7 p62 n=3 (mock), n=4 (SR9011) * P =0.0286; 48h T47D n=5 (mock, SR9011) ** P =0.004; e, Accumulation of p62 is confirmed by immunoblot (48h, 20µM A375); f–g, Inhibition of autophagy precedes apoptosis induction as shown by immunofluorescence of p62, cleaved Caspase 3 and TUNEL assay (n= biological independent samples n=4 mock 48h, n=5 SR9011 p62, n=3 48h SR9011; n=6 (mock, SR9011 72h p62), n=10 (mock 72h), n=8 (SR9011 72h) A375 20µM, Cl. Casp. 3 48h * P =0.0286; Cl. Casp. 3 72h **** P

    Journal: Nature

    Article Title: Pharmacological activation of REV-ERBs is lethal in cancer and oncogene induced senescence

    doi: 10.1038/nature25170

    Figure Lengend Snippet: REV-ERB agonist SR9011 inhibit autophagy a–b, SR90011 treatment reduces the number of autophagosomes both in MCF7 and T47D, n= biological independent samples MCF7 20µM 24h n=9 (mock), n=4 (SR9011) ** P =0.0056, T47D 48h 20µM n=5 (mock), n=4 (SR9011) ** P =0.0079; c–d, SR9011 induces accumulation of p62 as shown by immunofluorescence both in MCF7 and T47D n= biological independent samples 48h MCF7 p62 n=3 (mock), n=4 (SR9011) * P =0.0286; 48h T47D n=5 (mock, SR9011) ** P =0.004; e, Accumulation of p62 is confirmed by immunoblot (48h, 20µM A375); f–g, Inhibition of autophagy precedes apoptosis induction as shown by immunofluorescence of p62, cleaved Caspase 3 and TUNEL assay (n= biological independent samples n=4 mock 48h, n=5 SR9011 p62, n=3 48h SR9011; n=6 (mock, SR9011 72h p62), n=10 (mock 72h), n=8 (SR9011 72h) A375 20µM, Cl. Casp. 3 48h * P =0.0286; Cl. Casp. 3 72h **** P

    Article Snippet: Apoptosis was evaluated by immunostaining of cleaved caspase 3 (Cell signaling #9664 1:200) and by TUNEL assay using In Situ Cell Death Detection Kit, Fluoresceine or TMR red (Roche).

    Techniques: Immunofluorescence, Inhibition, TUNEL Assay

    SR9009 and SR9011 treatment evokes an apoptotic response and induces inhibition of autophagy in OIS cells a, Proliferation assay shows that REV-ERBs agonists impair viability of OIS cells (6 days, 20µM). b–c, Immunofluorescence assay for cleaved Caspase 3 and TUNEL assay shows apoptosis induction specifically in OIS (n=biological independent samples, n=7 mock, n=9 SR9009, n=14 SR9011, 72h, 20µM; one-way ANOVA, Cl. Casp 3 **** P

    Journal: Nature

    Article Title: Pharmacological activation of REV-ERBs is lethal in cancer and oncogene induced senescence

    doi: 10.1038/nature25170

    Figure Lengend Snippet: SR9009 and SR9011 treatment evokes an apoptotic response and induces inhibition of autophagy in OIS cells a, Proliferation assay shows that REV-ERBs agonists impair viability of OIS cells (6 days, 20µM). b–c, Immunofluorescence assay for cleaved Caspase 3 and TUNEL assay shows apoptosis induction specifically in OIS (n=biological independent samples, n=7 mock, n=9 SR9009, n=14 SR9011, 72h, 20µM; one-way ANOVA, Cl. Casp 3 **** P

    Article Snippet: Apoptosis was evaluated by immunostaining of cleaved caspase 3 (Cell signaling #9664 1:200) and by TUNEL assay using In Situ Cell Death Detection Kit, Fluoresceine or TMR red (Roche).

    Techniques: Inhibition, Proliferation Assay, Immunofluorescence, TUNEL Assay

    REV-ERBs agonist SR9009 inhibits autophagy a–b , SR9009 treatment reduces the number of autophagosomes, as shown by immunofluorescence of LC3B, (n=biological independent samples MCF7 (n=6 mock), (n=5 SR9009) and T47D (n=5 mock) (n=4 SR9009) Mann–Whitney test one-tailed MCF7 20µM 24h * P =0.0152, T47D 20µM 48h ** P =0.0079; c–d SR9009 induces accumulation of p62 as shown by immunofluorescence; n= biological independent samples MCF7 (n=3 mock), (n=8 SR9009) and T47D (n=5 mock), (n=4 SR9009) Mann–Whitney test one-tailed 48h MCF7 p62 ** P =0.0061; 48h T47D ** P =0.0079; e, Inhibition of autophagy is confirmed by the immunoblot for p62 (20µM 48h, A375); f–g, Inhibition of autophagy precedes apoptosis induction as shown by immunofluorescence of p62, cleaved Caspase 3 and TUNEL assay; n=biological independent samples, Mann–Whitney test one-tailed, A375 20µM Cl. Casp. 3 48h (n=3) * P =0.0179; Cl. Casp. 3 72h (n=7) **** P

    Journal: Nature

    Article Title: Pharmacological activation of REV-ERBs is lethal in cancer and oncogene induced senescence

    doi: 10.1038/nature25170

    Figure Lengend Snippet: REV-ERBs agonist SR9009 inhibits autophagy a–b , SR9009 treatment reduces the number of autophagosomes, as shown by immunofluorescence of LC3B, (n=biological independent samples MCF7 (n=6 mock), (n=5 SR9009) and T47D (n=5 mock) (n=4 SR9009) Mann–Whitney test one-tailed MCF7 20µM 24h * P =0.0152, T47D 20µM 48h ** P =0.0079; c–d SR9009 induces accumulation of p62 as shown by immunofluorescence; n= biological independent samples MCF7 (n=3 mock), (n=8 SR9009) and T47D (n=5 mock), (n=4 SR9009) Mann–Whitney test one-tailed 48h MCF7 p62 ** P =0.0061; 48h T47D ** P =0.0079; e, Inhibition of autophagy is confirmed by the immunoblot for p62 (20µM 48h, A375); f–g, Inhibition of autophagy precedes apoptosis induction as shown by immunofluorescence of p62, cleaved Caspase 3 and TUNEL assay; n=biological independent samples, Mann–Whitney test one-tailed, A375 20µM Cl. Casp. 3 48h (n=3) * P =0.0179; Cl. Casp. 3 72h (n=7) **** P

    Article Snippet: Apoptosis was evaluated by immunostaining of cleaved caspase 3 (Cell signaling #9664 1:200) and by TUNEL assay using In Situ Cell Death Detection Kit, Fluoresceine or TMR red (Roche).

    Techniques: Immunofluorescence, MANN-WHITNEY, One-tailed Test, Inhibition, TUNEL Assay

    Evaluation of V-9302  in vivo  in HCC1806 cell line xenograft-bearing mice ( A ) Tumor volumetric analysis of mice treated with vehicle or V-9302 (75 mg/kg per day) for 10 days.  n  = 5 mice per group. Treatment started 9 days post injection. P value at day 10 determined by Student’s  t  test. ( B ) Immunofluorescence analysis of tumor tissues harvested from vehicle- or V-9302-treated mice; LC3B and pAKT (Ser473) shown (pink). CD-31 positive vessels shown in green and nuclei in blue (DAPI). 20× magnification. P values determined by Student’s  t  test. ( C ) Effects of V-9302 treatment on pS6-positive cells and caspase 3-positive cells by immunohistochemistry. Quantitative analysis consisted of the mean counts of at least three representative fields from three vehicle and three V-9302-treated mice. For box plots, center line is plotted at the median; the box spans from the first quartile to the third quartile; whiskers represent min to max. Error bars represent ± std. dev.

    Journal: Nature medicine

    Article Title: Pharmacological Blockade of ASCT2-dependent Glutamine Transport Leads To Anti-tumor Efficacy in Preclinical Models

    doi: 10.1038/nm.4464

    Figure Lengend Snippet: Evaluation of V-9302 in vivo in HCC1806 cell line xenograft-bearing mice ( A ) Tumor volumetric analysis of mice treated with vehicle or V-9302 (75 mg/kg per day) for 10 days. n = 5 mice per group. Treatment started 9 days post injection. P value at day 10 determined by Student’s t test. ( B ) Immunofluorescence analysis of tumor tissues harvested from vehicle- or V-9302-treated mice; LC3B and pAKT (Ser473) shown (pink). CD-31 positive vessels shown in green and nuclei in blue (DAPI). 20× magnification. P values determined by Student’s t test. ( C ) Effects of V-9302 treatment on pS6-positive cells and caspase 3-positive cells by immunohistochemistry. Quantitative analysis consisted of the mean counts of at least three representative fields from three vehicle and three V-9302-treated mice. For box plots, center line is plotted at the median; the box spans from the first quartile to the third quartile; whiskers represent min to max. Error bars represent ± std. dev.

    Article Snippet: Tissues were sectioned (5 µm thickness) and stained for pS6 (Cell Signaling, #4858) and caspase 3 (Cell Signaling #9661).

    Techniques: In Vivo, Mouse Assay, Injection, Immunofluorescence, Immunohistochemistry

    Evaluation of V-9302 in vivo ( A ) Pharmacodynamic [ 18 F]-4F-Gln PET imaging prior to and 4 h following a single administration of V-9302 (75 mg/kg) in HCC1806 cell line xenograft-bearing mice (arrows indicate xenograft tumor on right flank*). ( B ) Mean time activity curves (TACs) from tumor regions of interest ( n = 4 measurements per condition); data prior to and 4 hrs following V-9302 administration. ( C ) P values determined by Student’s t test. Quantified tracer accumulation in xenograft tumors, muscle, and liver ( n = 4 measurements per condition). Volumetric analysis over 21 day treatment regimen (Vehicle or V-9302; 75 mg/kg, daily) of HCT-116 ( D ) and HT29 ( F ) cell line xenografts propagated in athymic nude mice ( n = 10 mice per group). Treatment started 12 days post tumor injection for HCT-116 and 4 days post injection for HT29. P values on day 21 determined by Student’s t test. Immunohistochemistry for pS6 and caspase 3 in vehicle-treated or V-9302-treated HCT-116 ( E ) and HT29 ( G ) xenografts. Representative photomicrographs and quantitation shown; magnification 20×. P values determined by Student’s t test. ( H ) Volumetric analysis over 31 day treatment regimen (Vehicle or V-9302; 75 mg/kg, daily) on athymic nude mice bearing patient-derived xenograft tumors (PDX A 008, F3 generation, treatment started 28 days post implantation, KRAS G12V ;p53 R248Q ;PTEN L140Y ; n = 10 mice per group) P value on day 31 determined by Student’s t test. ( I ) Photographs of A 008 PDX-bearing mice treated with V-9302 or vehicle; day 16 of 31. (Error bars represent ± std. dev. *Central photopenia observed.

    Journal: Nature medicine

    Article Title: Pharmacological Blockade of ASCT2-dependent Glutamine Transport Leads To Anti-tumor Efficacy in Preclinical Models

    doi: 10.1038/nm.4464

    Figure Lengend Snippet: Evaluation of V-9302 in vivo ( A ) Pharmacodynamic [ 18 F]-4F-Gln PET imaging prior to and 4 h following a single administration of V-9302 (75 mg/kg) in HCC1806 cell line xenograft-bearing mice (arrows indicate xenograft tumor on right flank*). ( B ) Mean time activity curves (TACs) from tumor regions of interest ( n = 4 measurements per condition); data prior to and 4 hrs following V-9302 administration. ( C ) P values determined by Student’s t test. Quantified tracer accumulation in xenograft tumors, muscle, and liver ( n = 4 measurements per condition). Volumetric analysis over 21 day treatment regimen (Vehicle or V-9302; 75 mg/kg, daily) of HCT-116 ( D ) and HT29 ( F ) cell line xenografts propagated in athymic nude mice ( n = 10 mice per group). Treatment started 12 days post tumor injection for HCT-116 and 4 days post injection for HT29. P values on day 21 determined by Student’s t test. Immunohistochemistry for pS6 and caspase 3 in vehicle-treated or V-9302-treated HCT-116 ( E ) and HT29 ( G ) xenografts. Representative photomicrographs and quantitation shown; magnification 20×. P values determined by Student’s t test. ( H ) Volumetric analysis over 31 day treatment regimen (Vehicle or V-9302; 75 mg/kg, daily) on athymic nude mice bearing patient-derived xenograft tumors (PDX A 008, F3 generation, treatment started 28 days post implantation, KRAS G12V ;p53 R248Q ;PTEN L140Y ; n = 10 mice per group) P value on day 31 determined by Student’s t test. ( I ) Photographs of A 008 PDX-bearing mice treated with V-9302 or vehicle; day 16 of 31. (Error bars represent ± std. dev. *Central photopenia observed.

    Article Snippet: Tissues were sectioned (5 µm thickness) and stained for pS6 (Cell Signaling, #4858) and caspase 3 (Cell Signaling #9661).

    Techniques: In Vivo, Positron Emission Tomography, Imaging, Mouse Assay, Activity Assay, Injection, Immunohistochemistry, Quantitation Assay, Derivative Assay

    MLKL encoding mRNA induces cell death in vitro and in vivo. a – f In vitro cell death characterization. B16-OVA cells were transfected with PBS or with Fluc-, tBid-, or MLKL-mRNA. a At different time points after transfection, cells were collected and analyzed by flow cytometry. Graph showing the percentages of sytox + cells (left). Representative flow cytometric plots 24 h after transfection (right). b Impact of the pan-caspase inhibitor zVAD-fmk on cell death induction (top) and caspase activity (bottom) upon transfection of B16-OVA cells with tBID- or MLKL-mRNA. c Western blot analysis of the expression of MLKL, caspase-3, and cleaved caspase-3 in cell lysates prepared 24 h after mRNA transfection. Tubulin served as a loading control. d B16 cells were co-transfected with luciferase NF-κB reporter plasmid and a plasmid expressing β-galactosidase. Twenty four hours later, the cells were transfected with PBS, GFP-, tBid-, or MLKL-mRNA or, as a positive control for NF-κB activation, with TRAF6 expression vector or they were stimulated with TNF. The normalized luciferase activity in the lysates determined at different time points after mRNA transfection is depicted. e B16 cells were transfected with a GFP expression plasmid. Twenty four hours later, the cells were transfected with PBS, Fluc-, tBid-, or MLKL-mRNA and the cells were treated or not with actinomycin D as indicated. Twenty four hours after mRNA transfection, cell death and GFP expression were quantified using flow cytometry. Horizontal lines indicate the mean. f Still images of B16-OVA cells at 0 and 24 h after transfection with tBid- or MLKL-mRNA visualized by time-lapse microscopy (scale bar = 10 µm). g In vivo cell death characterization. Subcutaneously growing B16-OVA tumors were injected with PBS or with 10 µg mRNA encoding Fluc, tBid, or MLKL followed by electroporation. Twenty four hours later, the tumor cells were isolated and analyzed by flow cytometry for sytox uptake. Graph showing the percentages of sytox + cells (left) and representative flow cytometry plots (right). Horizontal lines indicate the mean

    Journal: Nature Communications

    Article Title: Treatment with mRNA coding for the necroptosis mediator MLKL induces antitumor immunity directed against neo-epitopes

    doi: 10.1038/s41467-018-05979-8

    Figure Lengend Snippet: MLKL encoding mRNA induces cell death in vitro and in vivo. a – f In vitro cell death characterization. B16-OVA cells were transfected with PBS or with Fluc-, tBid-, or MLKL-mRNA. a At different time points after transfection, cells were collected and analyzed by flow cytometry. Graph showing the percentages of sytox + cells (left). Representative flow cytometric plots 24 h after transfection (right). b Impact of the pan-caspase inhibitor zVAD-fmk on cell death induction (top) and caspase activity (bottom) upon transfection of B16-OVA cells with tBID- or MLKL-mRNA. c Western blot analysis of the expression of MLKL, caspase-3, and cleaved caspase-3 in cell lysates prepared 24 h after mRNA transfection. Tubulin served as a loading control. d B16 cells were co-transfected with luciferase NF-κB reporter plasmid and a plasmid expressing β-galactosidase. Twenty four hours later, the cells were transfected with PBS, GFP-, tBid-, or MLKL-mRNA or, as a positive control for NF-κB activation, with TRAF6 expression vector or they were stimulated with TNF. The normalized luciferase activity in the lysates determined at different time points after mRNA transfection is depicted. e B16 cells were transfected with a GFP expression plasmid. Twenty four hours later, the cells were transfected with PBS, Fluc-, tBid-, or MLKL-mRNA and the cells were treated or not with actinomycin D as indicated. Twenty four hours after mRNA transfection, cell death and GFP expression were quantified using flow cytometry. Horizontal lines indicate the mean. f Still images of B16-OVA cells at 0 and 24 h after transfection with tBid- or MLKL-mRNA visualized by time-lapse microscopy (scale bar = 10 µm). g In vivo cell death characterization. Subcutaneously growing B16-OVA tumors were injected with PBS or with 10 µg mRNA encoding Fluc, tBid, or MLKL followed by electroporation. Twenty four hours later, the tumor cells were isolated and analyzed by flow cytometry for sytox uptake. Graph showing the percentages of sytox + cells (left) and representative flow cytometry plots (right). Horizontal lines indicate the mean

    Article Snippet: Twenty four hours after transfection, the lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% acrylamide) and MLKL and caspase-3 were visualized by western blotting with, respectively, anti-MLKL (1000× dilution) (Millipore, Cat. No. MABC604) and anti-caspase 3 (1000× dilution) (Cell Signaling Technology, Cat. No. 9662S) antibodies.

    Techniques: In Vitro, In Vivo, Transfection, Flow Cytometry, Cytometry, Activity Assay, Western Blot, Expressing, Luciferase, Plasmid Preparation, Positive Control, Activation Assay, Time-lapse Microscopy, Injection, Electroporation, Isolation