To determine the contributions of different caspases to bacteria-induced apoptosis, hatchling animals were exposed to a series of targeted protease inhibitors. The inhibitors used in this study include the pan-caspase inhibitor z-VAD-FMK, the caspase 8 inhibitor Ac-IETD-CHO, and the caspase 9 inhibitor Ac-LEHD-CMK (Santa Cruz Biotechnology, Dallas, TX) as well as the irreversible serine protease inhibitor, Pefabloc (Sigma Aldrich, St. Louis, MO). All inhibitor solutions were 0.22 µm-filter sterilized and stored at – 80 °C until use. Caspase inhibitors were used at final concentrations of 60 and 100 μM for vial and HARV experiments, respectively. For all experiments, the final concentration of the serine protease inhibitor Pefabloc was 25 μM. These concentrations were chosen in part as they did not cause apparent toxicity to the animal and did not impede colonization. Inhibitor treatment occurred for 2 h before the start of the experiment. Dimethyl sulfoxide (DMSO) controls were run in parallel where appropriate.
Z vad fmk
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Germany
Z-VAD-FMK is a broad-spectrum caspase inhibitor that irreversibly binds to the catalytic site of caspase enzymes. It is commonly used in research to prevent apoptosis (programmed cell death) in cellular and animal models.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Z devd fmk, by Santa Cruz Biotechnology (4 mentions)
Caspase 3, by Cell Signaling Technology (3 mentions)
β actin, by Merck Group (3 mentions)
N acetylcysteine nac, by Merck Group (3 mentions)
Abt 737, by Santa Cruz Biotechnology (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Nigericin, by Merck Group (2 mentions)
Nec 1, by Merck Group (2 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
Anti egfr 1005, by Santa Cruz Biotechnology (2 mentions)
Anti fasl nok1, by BD (2 mentions)
Anti tlr2 h 175, by Santa Cruz Biotechnology (2 mentions)
Anti tnfr1 h 271, by Santa Cruz Biotechnology (2 mentions)
Ab9563, by Abcam (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Caspase 8, by Cell Signaling Technology (2 mentions)
56 protocols using z vad fmk
1
Apoptosis Induction by Caspase Inhibitors
2
Antibody Reagents for NF-κB Signaling
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Antibodies against β-actin (clone C4), NF-κB-p65 (clone C-20; C-terminus), NF-κB-p65 (clone F-6), and IκBα (clone H4) were purchased from Santa Cruz Biotechnology. Antibodies against NF-κB-p65 (clone C22B4; N-terminus), IKKα (clone3G12), IKKβ (D30C6), cleaved caspase-3 (Asp175, clone 5A1E), and PARP (clone 46D11) were obtained from Cell Signaling. SVA VP1 and SVA VP2 mouse monoclonals and SVA whole virus antibodies were kindly provided by Dr. Steve Lawson (SDSU). Anti-Flag or anti-HA mouse monoclonal antibodies are commercially available (GenScript and Thermo Scientific, respectively). Anti-rabbit and/or anti-mouse secondary antibodies conjugated with Alexa Fluor® 594 and Alexa Fluor 488® were purchased from Life Technologies. IRDye 800CW-labeled anti mouse IgG and IRDye 680-labeledRD anti rabbit IgG secondary antibodies were purchased from Li-Cor Biosciences. Recombinant TNF-α was obtained from InvivoGen. Staurosporine was purchased from Cell Signaling and Z-VAD-FMK was obtained from Santa Cruz Biotechnology.
3
Dissecting Apoptosis Signaling Pathways
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G1 was purchased from Tocris (Wiesbaden-Nordenstadt, Germany), dissolved (5 mM) in dimethyl sulfoxide (DMSO) (Roth, Karsruhe, Germany) and stored at −20 °C; Thapsigargin, SP600125, GSK2606414 were also purchased from Tocris. Indo-1 AM was from Thermo Fisher Scientific (Waltham, MA, USA). zVAD-fmk was bought from Santa Cruz (Santa Cruz, CA, USA). SB203580 and Kira6 were purchased from MERCK Millipore (Darmstadt, Germany). All substances were dissolved in DMSO. Antibodies were obtained from the following commercial sources: caspase 9 (Ca# 9502), cleaved PARP (Ca# 9541), IRE1α (Cat# 3294), PERK (Cat# 3192), eIF2α (Cat# 5234), phospho-eIF2α (Cat# 3398), BiP GRP78 (Ca# 3177), CHOP (Cat# 2895), p38 MAPK (Ca# 9212), phospho-p38 MAPK (Ca# 4511), phospho-SAPK/JNK (Ca# 4668), caspase 3 (Ca# 9662), BCL-2 (Ca# 2872), Cell Signaling (Danvers, MA, USA); ATF6 (Cat# 73500), BioAcademia (Osaka, Japan); puromycin (Ca# MABE343), cylophilin D (Ca# AP1035), MERCK Millipore (Darmstadt, Germany); phospho-IRE1α (Cat# NBP2-50067), Novus Biologicals (Littleton, CO, USA); cytochrome c (Ca# 556433), BD Biosciences (Franklin Lakes, NJ, USA); β-actin (Cat# A5441, Sigma-Aldrich (Steinheim, Germany)). Secondary, peroxidase-conjugated antibodies were purchased from Dianova (Hamburg, Germany). All other chemicals of analytical grade were obtained from Sigma-Aldrich or Roth.
4
Optimizing Cell Culture for Metabolic Analysis
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L‐aspartic acid (Merck) was added to the growth medium and the pH adjusted to 7.5 with 1 m NaOH before FBS supplementation; hypoxanthine, adenine, and uridine (all from Merck Life Science) were solubilized and added directly to the growth medium. IACS‐010759 [16 (link)], venetoclax/ABT‐199 (Carbosynth, Newbury, UK) [34 (link)], S63845 (Medchemexpress LCC, Monmouth Junction, NJ, USA) [35 (link)], Z‐VAD‐FMK (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) were dissolved in DMSO and added directly to the culture medium; 4‐hydroxytamoxifen (OHT; Merck) was dissolved in ethanol and added directly to the culture medium.
An ADP/ATP Ratio Assay kit (Merck) was used to quantify cellular ADP and ATP, while Caspase‐Glo 3/7 Assay (Promega, Madison, WI, USA) was used to evaluate caspase activity, both according to the manufacturer’s instructions.
An ADP/ATP Ratio Assay kit (Merck) was used to quantify cellular ADP and ATP, while Caspase‐Glo 3/7 Assay (Promega, Madison, WI, USA) was used to evaluate caspase activity, both according to the manufacturer’s instructions.
5
SERPINB1 Regulates NLRP3 Inflammasome Activation
6
Evaluation of Cytotoxic Compounds in Melanocytes
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All chemicals were obtained from Sigma-Aldrich (Darmstadt, Germany) including Dulbecco modified Eagle’s medium (DMEM), unless otherwise stated. 4-hydroxynonenal was purchased from Biomol (Hamburg, Germany), cardamonin and alpinetin (Fig 1 ) from Phytolab (Verstenbergsgreuth, Germany). Penicillin/Streptomycin was from Biochrom and Glutamax from Gibco (Darmstadt, Germany). The fetal bovine serum (FBS) was from Pan-Biotech (Aidenbach, Germany). The melanocyte growth medium and the growth medium supplement mix were purchased from PromoCell (Heidelberg, Germany). Hank’s Balanced Salt Solution (HBSS) was from Gibco (Darmstadt, Germany). DMSO was obtained from Roth (Karlsruhe, Germany). Recombinant human TGFß1 was purchased from R&D Systems (Wiesbaden, Germany) and the pan caspase inhibitor z-VAD-FMK from Santa Cruz Biotechnology (Heidelberg, Germany).
7
Immune Activation Protocol Reagents
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The following chemicals and drugs were utilized in this study: Talabostat/VbP (MCE #HY-13233A), Lipofectamine 2000 (Invitrogen #11668019), double-stranded alternating copolymer poly(dA:dT) (pdAdT) (Sigma #P0883), poly(I:C) (pIC) (Invivogen #tlrl-picw), HT-DNA (Sigma #D6898), double-stranded homopolymer poly(dA):poly(dT) (Sigma #P9764), poly(dG:dC) (Invivogen #tlrl-pgcn), PAM3CSK4 (Invivogen #tlrl-pms), nigericin (Sigma #N7143), diABZI (MCE #HY-112921B), ANS (MCE #HY-18982), H2O2 (Sigma #H1009), MG-132 (MCE #HY-13259), Bortezomib (MCE #HY-10227), MCC950 (Invivogen #inh-mcc), z-VAD-FMK (Santa Cruz #sc-3067), z-DEVD-FMK (Santa Cruz #sc-311558), H-151 (MCE #HY-112693), NAC (Sigma #A9165), KU-44933 (Santa Cruz #sc-202963), NU-7441 (Tocris #3712), Sorafenib (Sigma #SML2633), PLX-4720 (MCE #HY-51424), Doramapimod (MCE #HY-10320), SB-202190 (MCE #HY-10295), and RNA Polymerase III inhibitor (Sigma #557403). gDNA was isolated from the genomic DNA of HEK293T cells. ISD was synthesized from custom oligos as previously described (55 (link)). Recombinant IFNγ was purchased from Peprotech (#300-02). DNase I (Bio-Rad #7326828), S1 Nuclease (Thermofisher #EN0321), and RNase A/T1 Cocktail (Thermofisher #AM2286) were purchased from the indicated vendors. VACV Copenhagen strain WT and ΔF1L were a kind gift of John Bell (56 (link)) and titered by plaque assay.
8
Apoptosis Analysis via PI-Annexin V
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The apoptosis analysis was done using PI-Annexin V double staining method as per the standard procedure (BD Pharmingen, Catalog number: 556547). Briefly, the cells were provided Vehicle, DGLA, Iminodibenzyl, and combination. To validate caspase dependent apoptosis mechanism, cells were pretreated with 30 μM Z-VAD-FMK (Santacruz biotechnology, Catalog number: SC3067) for 2 h, followed by Iminodibenzyl and DGLA treatment. For apoptosis analysis, the cells were collected by trypsinization. The cells were washed twice with ice-cold PBS buffer, followed by mixed with 100 μl 1× binding buffer at a concentration of 1 × 106 cells/ml. The cells were then stained with 5 μl PI and 5 μl Annexin V FITC for 30 min in dark at room temperature. After incubation, the volume was made till 500 μl and the reading was taken by Acuri C6 flow cytometer. The data analysis was done by Acuri C6 software as described elsewhere12 (link),13 (link).
9
Induction of Autophagy in Breast Cell Lines
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Human breast cancer cell lines MCF-7, T47D, SKBR3, BT549, MDA-MB-231, MDA-MB-435S and a human breast non-tumorigenic cell line MCF-10A were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were maintained in a humidified incubator at 37°C and 5% CO2. Cells were treated with Earle's balanced salt solution (EBSS, Sigma) to activate starvation-induced autophagy [27 (link)]. Apoptosis inhibitor Z-VAD-FMK (Santa Cruz), autophagy inhibitor 3-MA (Sigma) and XIAP inhibitor Embelin (Santa Cruz) were treated when necessary.
10
DENV2 Infection Modulation by Necroptosis Inhibitors
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U937-DC-SIGN cells (6 x 105) were plated in a 6 well plate and infected with DENV2 (MOI=6) for 48hrs. Cells were treated with 50µM Nec-1 (Sigma Aldrich), 20µM zVAD-FMK (Santa Cruz Biotechnology), 50ng/ml TNFα (Peprotech), and 10µM Smac mimetic (Fisher Scientific) for 6 hours. Cells were harvested by centrifugation at 1600rpm for 5 minutes and lysed with RIPA buffer supplemented with protease inhibitors. Cell lysates were analyzed by Western blot.
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