Journal: Journal of Toxicology

Article Title: Caspase-10 Is the Key Initiator Caspase Involved in Tributyltin-Mediated Apoptosis in Human Immune Cells

doi: 10.1155/2012/395482

Figure Lengend Snippet: TBT induces recruitment of caspase-8 and caspase-10 by TRAIL-R2. Jurkat A3 wt cells were treated with or without TBT for 3 h before cells were lysed and immunoprecipitation (IP) was carried out with anti-TRAIL-R2 antibody. Initiator caspase-8 and caspase-10 were detected by western blotting (WB) in the precipitates (a). Both antibodies recognise full length procaspases and the processed subunits. Asterisk indicates an unspecific band. Caspase-10 was further investigated in all three cell lines after IP with anti-TRAIL-R2 antibody (b). After 2 or 3 h of incubation with TBT, cells were analysed by IP/WB. In all three cell lines, the amount of procaspase-10 and/or its cleavage products increase over time in the precipitate. Loading control for TRAIL-R2 is shown below. Arrows and numbers give the molecular weight of procaspases and cleaved subunits. Both parts of the western blot (upper part containing the procaspase-10, lower part with the cleavage products) are differentially exposed to visualize the weak bands of the cleavage products.

Article Snippet: The primary antibodies anti-caspase-6 (Cat no. 9762), anti-caspase-7 (Cat no. 9492) and anti-caspase-9 (Cat no. 9502) were purchased from Cell Signaling Technology (Frankfurt, Germany), anti-caspase-3 (Cat no. C31720), and anti-caspase-8 (Cat no. 551242 clone 3-1-9) from BD Pharmingen (Heidelberg, Germany), anti-caspase-10/a (Cat no. M059-3) from MoBiTec (MBL) (Göttingen, Germany), anti-TRAIL-R2 (DR5) (Cat no. PC392) from Calbiochem (Darmstadt, Germany), anti-BID (Cat no. AF846) from R&D Systems (Wiesbaden, Germany), and anti-PARP (Cat no. 1835238) from Roche Biochemica (Mannheim, Germany).

Techniques: Immunoprecipitation, Western Blot, Incubation, Molecular Weight

Journal: Journal of Toxicology

Article Title: Caspase-10 Is the Key Initiator Caspase Involved in Tributyltin-Mediated Apoptosis in Human Immune Cells

doi: 10.1155/2012/395482

Figure Lengend Snippet: Proposed scheme of TBT-induced apoptosis in Jurkat cells. TBT activates caspase-10 upstream of mitochondria which leads to BID cleavage, and activation of the mitochondria. Downstream caspases were then cleaved and the caspase-cascade is provoked. Caspase-8 deficiency cannot prevent apoptosis, but inhibition of caspase-10 (AEVD) or inhibition of the two important caspases downstream of the mitochondria, caspase-9 and caspase-3 (LEHD and DEVD), totally suppress cell death.

Article Snippet: The primary antibodies anti-caspase-6 (Cat no. 9762), anti-caspase-7 (Cat no. 9492) and anti-caspase-9 (Cat no. 9502) were purchased from Cell Signaling Technology (Frankfurt, Germany), anti-caspase-3 (Cat no. C31720), and anti-caspase-8 (Cat no. 551242 clone 3-1-9) from BD Pharmingen (Heidelberg, Germany), anti-caspase-10/a (Cat no. M059-3) from MoBiTec (MBL) (Göttingen, Germany), anti-TRAIL-R2 (DR5) (Cat no. PC392) from Calbiochem (Darmstadt, Germany), anti-BID (Cat no. AF846) from R&D Systems (Wiesbaden, Germany), and anti-PARP (Cat no. 1835238) from Roche Biochemica (Mannheim, Germany).

Techniques: Activation Assay, Inhibition

Journal: Cell Death & Disease

Article Title: The E3 ubiquitin ligase Itch regulates death receptor and cholesterol trafficking to affect TRAIL-mediated apoptosis

doi: 10.1038/s41419-023-06417-4

Figure Lengend Snippet: A Western blot analysis of basal expression of FLIP(L), FLIP(S), FADD, Procaspase-8, TRAIL-R1, TRAIL-R2 in OE33 Ctrl (shCTRL) and Itch knockdown (KD) cells stably expressing shRNA targeting ITCH. B Bar graph showing the densitometry of Western blot analysis of Itch expression normalised to the actin loading control in five independent experiments. C Bar graph showing the quantification for FLIP-Long and -Short expression in control shRNA and Itch knockdown (KD) cell lines. N = 5 independent experiments. D OE33 Ctrl and Itch KD cell lines were subjected to treatment with increasing doses of izTRAIL for 24 h (0–50 ng/mL). Cell viability was measured using the CellTiterGlo Assay. Data was normalised to an untreated control. E OE33 Ctrl and Itch KD cells were treated with 0–20 ng/mL TRAIL for 24 h and then stained with FITC-conjugated Annexin V and propidium iodide to assess the number of apoptotic cells under each treatment condition. Bar graph showing the percentage of apoptotic cells (Annexin V-positive cells) of the population. F Caspase -8 and ( G ) Caspase -3/-7 activity in OE33 Ctrl and Itch KD cell lines was measured following treatment with increasing concentrations of izTRAIL (0–50 ng/mL). Activity was measured 6 h post treatment using the Caspase -3/-7 or the Caspase -8 Glo assays. H Western blot analysis of cleaved Caspase-8, Bid and PARP in OE33 Ctrl and Itch KD cell lines following treatment for 6 or 24 h with 5 ng/ml izTRAIL. I Densitometry of the experiment in ( C ) in four independent repeats. Error bars represent the standard error of the mean. Statistical significance was measured by Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following commercial antibodies were used: TRAIL-R2/DR5 (rabbit, Cat# 3696 S, Cell Signalling Technology); BID (rabbit; Cat# 2002S, Cell Signalling Technology); PARP (rabbit, Cat# 9542 S, Cell Signalling Technology); Caspase 3 (rabbit, Cat# 9662 S, Cell Signalling Technology); FLIP NF6 (mouse, Cat# AG-20B-0056-C100, Adipogen); Itch (mouse, Cat# 611198, BD Biosciences); FADD (mouse, Cat# 556402, BD Pharmigen); Caspase-8 (mouse, Cat# ALX-804-242-C100, Enzo); TRAIL-R1/DR4 (rabbit, Cat# AB16955, Calbiochem); SREBP2 (rabbit, Cat# ab30682, Abcam); beta-Actin (mouse, Cat# A5316, Sigma), cytochrome C (BD Biosciences, # 556433), BAX 6A7 (Thermo #MA5-14003), StAR (#8449; Cell Signalling Technology), VDAC2 (ab155803; AbCam), Bax (#2772; Cell Signalling Technology) and GAPDH (mouse, Cat# sc-47724, Santa Cruz).

Techniques: Western Blot, Expressing, Stable Transfection, shRNA, Staining, Activity Assay

Journal: Cell Death & Disease

Article Title: The E3 ubiquitin ligase Itch regulates death receptor and cholesterol trafficking to affect TRAIL-mediated apoptosis

doi: 10.1038/s41419-023-06417-4

Figure Lengend Snippet: A Immunoprecipitation of the DISC using an activating TRAIL-R2 antibody from OE33 cells with a stable Itch knockdown (KD) or a control. Cells were treated for 1 h to induce formation of the DISC. Proteins were separated by size on a SDS-PAGE gel prior to Western blot analysis. B Densitometry of cleaved caspase-8 normalised to the pro-caspase-8 in the DISC IP from OE33 cells from three independent experiments. C Immunoprecipitation of the DISC in HT29 cells treated with control or ITCH siRNA using the TRAIL-R2 antibody. D Immunoprecipitation of the DISC in HCT116 cells treated with control or ITCH siRNA using the TRAIL-R2 antibody. Statistical significance was measured by Student’s t test, ns represents a non-significant difference.

Article Snippet: The following commercial antibodies were used: TRAIL-R2/DR5 (rabbit, Cat# 3696 S, Cell Signalling Technology); BID (rabbit; Cat# 2002S, Cell Signalling Technology); PARP (rabbit, Cat# 9542 S, Cell Signalling Technology); Caspase 3 (rabbit, Cat# 9662 S, Cell Signalling Technology); FLIP NF6 (mouse, Cat# AG-20B-0056-C100, Adipogen); Itch (mouse, Cat# 611198, BD Biosciences); FADD (mouse, Cat# 556402, BD Pharmigen); Caspase-8 (mouse, Cat# ALX-804-242-C100, Enzo); TRAIL-R1/DR4 (rabbit, Cat# AB16955, Calbiochem); SREBP2 (rabbit, Cat# ab30682, Abcam); beta-Actin (mouse, Cat# A5316, Sigma), cytochrome C (BD Biosciences, # 556433), BAX 6A7 (Thermo #MA5-14003), StAR (#8449; Cell Signalling Technology), VDAC2 (ab155803; AbCam), Bax (#2772; Cell Signalling Technology) and GAPDH (mouse, Cat# sc-47724, Santa Cruz).

Techniques: Immunoprecipitation, SDS Page, Western Blot

Journal: Cell Death & Disease

Article Title: The E3 ubiquitin ligase Itch regulates death receptor and cholesterol trafficking to affect TRAIL-mediated apoptosis

doi: 10.1038/s41419-023-06417-4

Figure Lengend Snippet: A Caspase-8 activity and ( B ) Caspase-3/-7 activity was measured following treatment with increasing concentrations of izTRAIL (0–50 ng/mL). Activity was measured 6 h post treatment using the Caspase-3/-7 or the Caspase-8 Glo assays. Cells were treated with U18666A (1 ng/ml) for 24 h. C Western blot analysis of cell lysates after treatment with U18666A and izTRAIL for key apoptotic proteins; caspase-8, -3, Bid and PARP. D–G Quantification of an inhibition of apoptosis in OE33 cells treated with U18666A (1 ng/ml) in combination with izTRAIL, showing a significant loss of cleavage of caspase-8, -3, Bid, and PARP in three independent experiments. H Bar graph showing the mean fluorescence of cell surface expression of TRAIL-R2 stained control and cells treated with U18666A in three independent experiments. I Bar graph showing the mean fluorescence intensity of cell surface expression of TRAIL-R1 in control and U18666A-treated (1 ng/ml) OE33 cells in three independent experiments ( n = 10,000 cells per experiment). J Fluorescence images of OE33 cells showing the accumulation of cholesterol, stained with Filipin-III, in mitochondria after U18666A treatment. Scale bar, 5 µm. Statistical significance was measured by Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following commercial antibodies were used: TRAIL-R2/DR5 (rabbit, Cat# 3696 S, Cell Signalling Technology); BID (rabbit; Cat# 2002S, Cell Signalling Technology); PARP (rabbit, Cat# 9542 S, Cell Signalling Technology); Caspase 3 (rabbit, Cat# 9662 S, Cell Signalling Technology); FLIP NF6 (mouse, Cat# AG-20B-0056-C100, Adipogen); Itch (mouse, Cat# 611198, BD Biosciences); FADD (mouse, Cat# 556402, BD Pharmigen); Caspase-8 (mouse, Cat# ALX-804-242-C100, Enzo); TRAIL-R1/DR4 (rabbit, Cat# AB16955, Calbiochem); SREBP2 (rabbit, Cat# ab30682, Abcam); beta-Actin (mouse, Cat# A5316, Sigma), cytochrome C (BD Biosciences, # 556433), BAX 6A7 (Thermo #MA5-14003), StAR (#8449; Cell Signalling Technology), VDAC2 (ab155803; AbCam), Bax (#2772; Cell Signalling Technology) and GAPDH (mouse, Cat# sc-47724, Santa Cruz).

Techniques: Activity Assay, Western Blot, Inhibition, Fluorescence, Expressing, Staining

Journal: Cell Death & Disease

Article Title: The E3 ubiquitin ligase Itch regulates death receptor and cholesterol trafficking to affect TRAIL-mediated apoptosis

doi: 10.1038/s41419-023-06417-4

Figure Lengend Snippet: A Cell viability assay of the Itch KD cell line in response to Cisplatin treatment for 72 h at 0–6 µg/ml. B Apoptosis assay using the Itch KD cell line treated with 0–6 µg/ml Cisplatin for 72 h. Data was expressed as a percentage of control. C Analysis of apoptotic markers in whole cell lysates from cells treated with Cisplatin in U18666A. D Western blot analysis of total expression levels of STARD1 and VDAC2 (dimers) in control and Itch KD OE33 cell lines. Error bars represent the standard error of the mean. Statistical significance was calculated by Student’s t test. E Quantification of STARD1 band intensity normalised to the loading control in three independent experiments. Statistical analysis was performed using Student’s t test, *** p < 0.001. F Immunoprecipitation of VDAC2 from OE33 detergent extract. The samples were incubated overnight with a control IgG and the VDAC2 antibody and washed four times. Co-immunoprecipitated proteins were separated by SDS-PAGE electrophoresis and STARD1 and Itch were detected by Western blotting. G A schematic image illustrating the suggested model for Itch in regulation of extrinsic and intrinsic apoptosis. A reduction in TRAIL-R at the cell surface impairs downstream apoptotic signalling, which moreover is not further amplified at the mitochondria. Loss of Itch expression results in an increase in mitochondrial cholesterol which impairs membrane fluidity, binding of caspase-8, subsequent activation of Bid and Bax. Together, this results in reduced pore formation in the outer mitochondrial membrane and reduced cytochrome c release. Mechanistically loss of Itch expression results in reduced ubiquitination and degradation of the STARD1/VDAC2 lipid transfer complex that mediates cholesterol import to mitochondria. A stabilisation of STARD1/VDAC2 promotes import of mitochondrial cholesterol which is anti-apoptotic. Statistical significance; * p < 0.05, *** p < 0.001.

Article Snippet: The following commercial antibodies were used: TRAIL-R2/DR5 (rabbit, Cat# 3696 S, Cell Signalling Technology); BID (rabbit; Cat# 2002S, Cell Signalling Technology); PARP (rabbit, Cat# 9542 S, Cell Signalling Technology); Caspase 3 (rabbit, Cat# 9662 S, Cell Signalling Technology); FLIP NF6 (mouse, Cat# AG-20B-0056-C100, Adipogen); Itch (mouse, Cat# 611198, BD Biosciences); FADD (mouse, Cat# 556402, BD Pharmigen); Caspase-8 (mouse, Cat# ALX-804-242-C100, Enzo); TRAIL-R1/DR4 (rabbit, Cat# AB16955, Calbiochem); SREBP2 (rabbit, Cat# ab30682, Abcam); beta-Actin (mouse, Cat# A5316, Sigma), cytochrome C (BD Biosciences, # 556433), BAX 6A7 (Thermo #MA5-14003), StAR (#8449; Cell Signalling Technology), VDAC2 (ab155803; AbCam), Bax (#2772; Cell Signalling Technology) and GAPDH (mouse, Cat# sc-47724, Santa Cruz).

Techniques: Viability Assay, Apoptosis Assay, Western Blot, Expressing, Immunoprecipitation, Incubation, SDS Page, Electrophoresis, Amplification, Membrane, Binding Assay, Activation Assay

Journal: American Journal of Cancer Research

Article Title: miR-3132 upregulates surface TRAIL to induce apoptotic cell death in cancer cells

doi:

Figure Lengend Snippet: TRAIL is required for miR-3132 dependent apoptosis in cancer cells. (A) Expression of TRAIL at the cell surface post-transfection with 50 nM miR-3132 was tested at the indicated times by flow cytometry. Graphs indicate geometric mean (arbitrary units) of TRAIL expression from duplicate samples. (B, C) Sub-G1 analysis in HCT-116 and HT29 cells, to measure cell death post-treatment with miR-3132, in the presence/absence of RIK2 or CC8 inhibitor. (D) Using same experimental conditions as (B, C), markers of apoptosis were assessed by western blot. 48 hrs co-treatment samples for HCT-116 and 72 hrs co-treatment samples for HT-29 for results in (B-D).

Article Snippet: The following antibodies were used: BCL XL (CST, 2764S), PARP (CST, 9542), p53-DO1 (Santa Cruz Biotechnology, sc-126), p21 (Calbiochem, OP64), β-actin (Sigma, A5441), XIAP (CST, 242S), FOXM1 (CST, D12D5), p-FOXM1 (CST, 14170S), Cyclin B1 (Santa Cruz Biotechnology, sc-245), CC3 (CST Asp175, cat. no. 9661), CC8 (CST Asp391, cat. no. 9496) and CC9 (CST Asp330, cat. no. 7237).

Techniques: Expressing, Transfection, Flow Cytometry, Western Blot

Journal: Cells

Article Title: The Proapoptotic Gene Bad Regulates Brain Development via p53-Mediated Stress Signals in Zebrafish

doi: 10.3390/cells10112820

Figure Lengend Snippet: p53/caspase-8 death signaling involvement in Bad knockdown embryos at 24 hpf. ( A ) A network signaling death pathway was proposed based on accumulated data. The p53/caspase-8/tBid-mediated death pathway was induced. It also required upstream signaling from loss of Bad, leading to enhanced environmental stress with increased ROS and enhanced apoptotic cell death via the stress/death gene p53 and its correlated downstream gene expression caspase-8 or activation of tBid by cleavage from Bid. ( B ) Clear upregulation of p53 and caspase-8 demonstrated via qRT-PCR. All data were analyzed using either paired or unpaired Student’s t -tests, as appropriate. * p < 0.01. ( C ) Identification of apoptotic-related protein expression of p53/caspase-8/tBid as key molecules by Western blotting analysis at 24 hpf. Both p53 and caspase-8 showed stronger expression compared to the control group, correlated with downstream Bid cleavage and anti-apoptotic number Bcl-2 and Bcl-xL expression levels. ( D ) qRT-PCR evaluation of apoptosis-related gene expression with Bad knockdown in p53 mutant lines at 24 hpf. ( E ) Western blot analysis of apoptosis-related protein expression with Bad knockdown in p53 mutant fish lines correlated with downstream protein Bid cleavage and anti-apoptotic number Bcl-2 and Bcl-xL expression levels.

Article Snippet: The gels were immunoblotted with the following antibodies: Anti-bad antibodies (AnaSpec; Cat No:55478), anti-p53 antibodies (GeneTex; Cat No:GTX102965), anti-caspase 8 monoclonal antibodies (BD Bioscience; Cat No:551244), anti-tBid antibodies (Cell Signaling; Cat No:#20025), anti-caspase 3 monoclonal antibodies (Calbiochem; PC66T), anti-Bcl-2 polyclonal antibodies (Cell Signaling; Cat No:#5071), anti-Bcl-xL polyclonal antibodies (AnaSpec; Cat No:554265), and anti-ß-actin monoclonal antibodies (Calbiochem; Cat No:MAB1501), followed by peroxidase-labeled goat anti-mouse secondary antibodies (1:15,000 dilution; Amersham Biosciences, Piscataway, NJ, USA) or peroxidase-labeled goat anti-rabbit secondary antibodies (1:7,500 dilution; Amersham Biosciences, Piscataway, NJ, USA).

Techniques: Knockdown, Gene Expression, Activation Assay, Quantitative RT-PCR, Expressing, Western Blot, Control, Mutagenesis