BAD regulates the sensitivity of HCC cells to the cytotoxic effects of sorafenib. 2004). Using gene overexpression and RNA interference, we show that BAD is involved in the cytotoxic effects of sorafenib, a multikinase blocker, which is currently the sole therapeutic drug effective for the treatment of HCC. The DeadEnd™ Assays use this approach, commonly called the TUNEL (TdT-mediated dUTP Nick End Labeling) assay. DeadEnd™ Colorimetric TUNEL System (Cat.# G7360, G7130), 0.3% hydrogen peroxide for blocking endogeneous peroxidases, fixative (e.g., 10% buffered formalin, 4% paraformaldehyde, 4% methanol-free formaldehyde), xylene or xylene substitute [e.g., Hemo-De® Clearing Agent (Fisher Cat.# 15-182-507A)], ethanol (100%, 95%, 85%, 70% and 50%) diluted in deionized water, poly-l-lysine-coated or silanized microscope slides. Sixteen control liver tissue samples were also obtained: the corresponding samples, hereby called “normal,” were obtained from surgical resections done following a diagnosis of hepatic metastasis of colorectal cancer.
Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C. Apoptosis can be induced in response to many external stimuli (extrinsic pathway) including activation of cell surface receptors such as Fas, TNFR1 (tumor necrosis factor receptor 1), TRAIL-R1 (TNF-related apoptosis-inducing ligand receptor 1), TRAIL-R2, p75-NGFR (p75-nerve growth factor receptor) and others (Wajant et al. Total level of Akt is assumed … Rinse 3 times for 5 minutes each time in PBS. To examine this possibility, we analyzed BAD expression levels and phosphorylation on Ser75, an important residue targeted by the RAF-MEK-ERK kinase pathway, a target of sorafenib (17-19), in HCC cells. donkey anti-rabbit biotin conjugate (Jackson Cat.# 711-065-152), peroxidase-labeled streptavidin (eg., KPL Cat.# 14-300-00, diluted 1µg/ml in PBS). Epub 2011 Mar 2. Their expression levels are frequently altered in cancers, enabling tumor cells to survive. (2003) Historical perspectives.
An animated presentation illustrating the death receptor pathway is available.
Schematic of Apo-ONE® Assay protocol. Fluorescently conjugated caspase inhibitors can also be used to label active caspases within cells.
(1999) Apoptosis: The germs of death. Huh7 cells were exposed to ABT-737 or its inactive enantiomer (Ctrl) at a concentration of 5 μmol/L, together with sorafenib at a subtoxic concentration (2 μmol/L). (1994). Mitochondrial apoptotic module.
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Conversely, an 80% reduction in the expression of BAD that was achieved using a siRNA knockdown strategy produced a 40% decrease in the number of cells with condensed chromatin after sorafenib treatment (P < 0.05, Student's t test; Fig. (2000) Quantitation of mitochondrial transmembrane potential in cells and in isolated mitochondria.
This protocol is intended for immunoprecipitation of native proteins utilizing Protein A agarose beads for analysis by western immunoblot or kinase activity. Coverslips were mounted in Mowiol (Calbiochem) and observed with a Nikon Eclipse TE2000U microscope equipped with a plan APO VC 60X/1.40 objective under oil immersion. Subclones derived from Huh7 cells and stably overexpressing green fluorescent protein (GFP) or GFP-BAD were established and characterized by immunoblotting. 2020 Jun 4;87(7):773-82. doi: 10.1002/mrd.23393.
Rinse once in PBS. When Huh7 cells were simultaneously exposed to ABT-737 and sorafenib, we observed a strong synergy between these two compounds in terms of apoptosis induction, estimated by condensed chromatin formation under microscopic examination (Fig. Bleach is frequently used to inactivate the DAB before disposal; however, local requirements for hazardous waste should be followed. 2000 Dec;7(12):1166-73. doi: 10.1038/sj.cdd.4400783. Resuspend the pellet with 20 µl 3X SDS sample buffer. Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® Bad siRNA I (+), using Bad Antibody and p42 MAPK Antibody #9108. Please contact Customer Service to unlock your account. Add 100–200μl of freshly made diaminobenzidine (DAB) solution to each slide. 2003 May 9;278(19):16992-9. doi: 10.1074/jbc.M300039200. Huh-7 cells were treated for 48 h with control or a mixture of two siRNAs targeting BAD, and their BAD protein content was analyzed by immunoblotting. These homogeneous reagents can then be added to the sample in a convenient 1:1 ratio (Figure 3.5) without a separate lysis step. Incubate with Anti-PARP p85 Fragment pAb diluted 1:50 in PBS + 1.0 % donkey serum for 60 minutes. Permeabilize for 10 minutes in PBS + 0.1% Triton® X-100. CytoTox-ONE™ Homogeneous Membrane Integrity Assay (2X concentration; Cat.#. Stable cell lines derived from Huh7 cells overexpressing BAD were found to exhibit a striking 5-fold increase in their sensitivity to sorafenib-induced cytotoxicity measured by determining the mean of the percentage of cells with condensed chromatin (P < 0.05, Student's t test; Fig.
The apoptotic nature of neuron cell death via a chemokine-activated cell-cell communication system involving microglia was characterized. 1995; Gorczyca et al. Label DNA strand breaks with Biotinylated Nucleotide Mix (60 minutes at 37°C). Of the 1,090 somatic cells of the C. elegans adult hermaphrodite, 131 die during normal development (Hengartner, 1997). apply to Products provided by CST, its affiliates or its distributors. Repeat centrifugation and resuspend the cell pellet in PBS to 1.5 × 10, Grow HL-60 cells in RPMI-1640 medium containing 10% fetal bovine serum in a humidified 5% CO, Harvest the cells and resuspend in PBS to 1.5 x 10, Culture cells in a 1:1 mixture of Ham’s F12 nutrients and minimal essential medium supplemented with 10% fetal bovine serum (FBS), 100IU/ml penicillin and 100mg/ml streptomycin in an atmosphere of 95% air and 5% CO.
Pre-equilibrate slides with Equilibration Buffer (5–10 minutes at room temperature). PolyPrep is a registered trademark of Bio-Rad Laboratories, Inc. Prionex is a registered trademark of Pentapharm Ltd. SlowFade is a registered trademark of Molecular Probes, Inc. Superfrost is a registered trademark of Erie Scientific. The corresponding results are presented in Fig. 7). *, P < 0.005 compared with normal tissue; #, P < 0.005 compared with nontumoral tissue. 2008 Dec;27 Suppl 1:S93-104. During the final 1–2 hours of treatment, add 20μl/well CellTiter-Blue® Reagent using diluted 1:4 with Dulbecco's PBS.
Large-scale mutagenesis experiments in the nematode C. elegans identified mutations that disrupted the programmed cell death fates during development, the cell death abnormal (ced) genes (Hedgecock et al. Wash three times in PBS, in Coplin jars, for 5 minutes at room temperature. Schematic diagram of the Caspase-Glo® Assay protocols. and Wyllie, A. Dual staining with the DeadEnd™ Fluorometric TUNEL System and propidium iodide allowed quantification of the TUNEL staining area by analysis of digitized images. Chen PR, Spate LD, Leffeler EC, Benne JA, Cecil RF, Hord TK, Prather RS. One unit of caspase = 0.07ng protein = 1pmol of substrate (Ac-DEVD-pNA) hydrolyzed/minute per the manufacturer's unit definition. EnduRenμ Live Cell Substrate (Cat.# E6481, E6482, E6485), Apo-ONE® Homogeneous Caspase-3/7 Assay Reagent (Cat.# G7790, G7791, G7792), cells transfected with appropriate Renilla luciferase reporter. Vortex gently. Analyze under a fluorescence microscope.
Apoptosis may be induced in experimental systems through a variety of methods, including: Treating cells with the protein synthesis inhibitor, anisomycin, or the DNA topoisomerase I inhibitor, camptothecin, induces apoptosis in the human promyelocytic cell line HL-60 (Del Bino et al.
Caspases constitute a large protein family that is highly conserved among multicellular organisms. Block with hydrogen peroxide (3–5 minutes at room temperature). 2000). Record fluorescence (560.
The natural delay in activation of the caspases after injury allows time for treatment, and molecules that target the caspases have shown therapeutic potential in preclinical animal models (Reed, 2002; Nicholson, 2000).
Posttranscriptional regulation of BAD in HCC. ABT-737 is a recently developed compound that binds to and inhibits the antiapoptotic proteins of the BCL2 family with a specificity mimicking BAD (23, 24). Additional background, optimization and recommended controls for each assay are provided in the technical literature that accompanies each individual assay.
A discussion of every disease caused by modification of the various apoptotic pathways would be impractical, but the concept overlying each on…
Cell viability of Huh7 overexpressing GFP or GFP-BAD was estimated in a clonogenicity assay where different concentrations of sorafenib were tested. These "death receptors" have two distinct signaling motifs: death domains (DD) … Add CaspACE™ FITC-VAD-FMK In Situ Marker to the Jurkat cells at a final concentration of 10µM. Whether the expression of other members of the BCL2 family of proteins is similarly altered in HCC remains unclear. We concluded that the experimental modulation of BCL2 expression and activation afforded by ABT-737 constitutes a promising strategy for the potentiation of apoptosis induction of HCC cells and that BAD is a possible therapeutic target for this type of cancer. The mitochondrial outer membrane (MOM) also undergoes changes that include loss of its electrochemical gradient, possibly by the formation of pores in the MOM, and substances such as cytochrome c leak from the MOM into the cytoplasm. The caspase zymogens contain several domains including an N-terminal prodomain, a large subunit and a small subunit.
TB295 Apo-ONE® Homogeneous Caspase-3/7 Assay. To investigate the possible synergy between ABT-737 and sorafenib, we applied ABT-737 or its inactive enantiomer at subtoxic concentrations (5 μmol/L). 2003).
Molecular Cancer Research Analyze samples immediately using a fluorescence microscope. Bad Antibody detects endogenous levels of total Bad protein. Ther Adv Med Oncol.
Wash twice in PBS for 5 minutes, once in PBS/0.1% Tween® 20 for 5 minutes and once in PBS for 5 minutes, protected from light. The corresponding clones were exposed to sorafenib (10 μmol/L, 18 h).
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Clipboard, Search History, and several other advanced features are temporarily unavailable. *, P < 0.05 compared with GFP cells exposed to sorafenib. After extended incubation, apoptotic cells ultimately shut down metabolism, lose membrane integrity and release their cytoplasmic contents into the culture medium (Riss and Moravec, 2004). Hanafy DM, Burrows GE, Prenzler PD, Hill RA. Caspase-12 in the mouse localizes to the ER and is cleaved in response to ER stress such as the accumulation of unfolded proteins in the ER (Nakagawa et al. Proceed to sample analysis by western immunoblotting or kinase activity (section D).