HEK293T cells were treated with dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA), Z-DEVD-FMK (Apexbio Technology LLC, Houston, TX, USA), Z-IETD-FMK (Apexbio Technology LLC, Houston, TX, USA), and Z-LEHD-FMK (Selleck Chemicals, Houston, TX, USA) (20 mM) for 2 h, followed by co-transfection of various molecules in NF-κB signaling with 3C or empty vectors for 18 h. The samples were harvested and analyzed using western blotting.
Z lehd fmk
Manufactured by Selleck Chemicals
Sourced in United States
Z-LEHD-FMK is a chemical compound that acts as a caspase inhibitor. It functions by inhibiting the activity of caspase-4, an enzyme involved in the regulation of apoptosis, or programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
Z ietd fmk, by Selleck Chemicals (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Z devd fmk, by Apexbio (1 mentions)
Z ietd fmk, by Apexbio (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Caspase glo 3 7 assay, by Promega (1 mentions)
Oxythiamine chloride hydrochloride, by Santa Cruz Biotechnology (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Annexin 5 apoptosis detection kit, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Solarbio (1 mentions)
Z devd fmk, by Selleck Chemicals (1 mentions)
Dimethyl sulfoxide (dmso), by MP Biomedicals (1 mentions)
Ac devd cho, by Selleck Chemicals (1 mentions)
Bx795, by Selleck Chemicals (1 mentions)
Mk 2206, by Selleck Chemicals (1 mentions)
Mab0041, by R&D Systems (1 mentions)
Mab210, by R&D Systems (1 mentions)
Ac yvad cmk, by Merck Group (1 mentions)
Necrostatin 1, by Cayman Chemical (1 mentions)
7 protocols using z lehd fmk
1
Modulation of NF-κB Signaling in HEK293T Cells
2
Chondrogenic Cell Culture and Treatment
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The tissues in Pfirrmann classification grades 2 were washed with cold phosphate-buffered saline solution (PBS). Then the samples were cut into 0.3x0.3 cm2 pieces and incubated with 0.25% type I collagenase at 37.5˚C overnight for digesting. Cell pellets were filtrated with a strainer and re-suspended in Dulbecco's modified Eagle's medium (DMEM) (containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, Gibco; Thermo Fisher Scientific, Inc.). For cell treatment 50 and 100 µM H2O2 was used to induce ROS; siRNA transfection was used to silence ATF4 and CHOP gene expression; 20 µM Z-LEHD-FMK (ZLF, a cell-permeable, irreversible caspase-9 inhibitor; purchased from Selleck) was used to inhibit the expression of caspase-9.
3
Cell Cycle Analysis with Ros Scavengers
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The cell cycle was analyzed as described previously [62 (link)]. Ros scavengers used were: α-tocopherol (100 µM, Vit. E) or Ferrostatin-1 (1 µM). The commercial inhibitors used were: Z-IETD-FMZ (50 µM: Selleckchem, Houston, TX, USA), Z-LEHD-FMK (50 µM: Selleckchem, Houston, TX, USA) and Oxythiamine chloride hydrochloride (OT, 1 and 10 mM: Santa Cruz Biotechnology, Heidelberg, Germany). The cells were seeded in culture plates and pre-treated with commercial inhibitors for 1.5 h before they were exposed to HCA for different times. Caspase-3/7 activity was measured using the Caspase-Glo® 3/7 assay according to the manufacturer’s instructions (Promega, Madison, WI, USA).
4
Apoptosis Induction in Human Hepatoma Cells
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Human HCC cell lines HepG2 and Huh9 were obtained from Shanghai Institutes for Biological Sciences (Shanghai, China). Cells were cultured in RPMI1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific). The cells were cultured in a humidified atmosphere with 5% carbon dioxide at 37°C. IFN-γ was purchased from Solarbio (Beijing, China). FLUD (Fludarabine) and S3I (STAT3 inhibitor) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). z-DEVD-fmk, z-LEHD-fmk, and z-IETD-fmk were purchased from Selleckchem (Houston, TX, USA). Propidium was purchased from Sigma-Aldrich Co. Annexin V apoptosis detection kit was purchased from BD Biosciences (San Jose, NJ, USA).
5
Cisplatin-Induced Apoptosis Pathway
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Cisplatin (DDP) was obtained from Yunnan Phytopharmaceutical Co. Ltd. Specific inhibitors of Akt (MK‐2206), PDK1 (BX795), caspase‐3 (Ac‐DEVD‐CHO), caspase‐8 (Z‐IETD‐FMK) and caspase‐9 (Z‐LEHD‐FMK) were purchased from Selleck Chemicals and dissolved in DMSO (MP Biomedicals). The antibodies against cleaved caspase‐3 (cat. no. 9664), cleaved caspase‐8 (cat. no. 9496), cleaved caspase‐9 (cat. no. 7237), Akt (cat. no. 9272), p‐Akt (S473) (cat. no. 4060), p‐Akt (T308) (cat. no.4056), PDK1 (cat. no. 13037), p‐PDK1 (cat. no. 3438), Ki67 (cat. no. 9027), CD34 (cat. no. 3569), GAPDH (cat. no. 2118), anti‐mouse IgG antibody (cat. no. 7076) and anti‐rabbit IgG antibody (cat. no. 7074) were products of Cell Signaling Technology. Anti‐DFNA5/GSDME (cat. no. ab215191) and anti‐AMIGO2 (cat. no. 821607) antibodies were, respectively, obtained from Abcam and ZENBIO.
6
UVB Irradiation of Cells and Mice
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Mouse dorsal fur was removed via shaving and depilation with Veet (Reckitt Benckiser, UK). Mice were placed in restrainers with facial protection and treated once ±250mJ/cm2 UVB light using the UV-2 ultraviolet irradiation system (Tyler Research). UVB light was provided by cascade-phosphor ultraviolet generators that emit 310nm UVB radiation.
For irradiation of cultured cells at approximately 80% confluence, growth medium was replaced with PBS and cells were treated with 0-50mJ/cm2 UVB light as indicated in specific experiments. PBS was removed immediately following irradiation and replaced with growth medium. Cells were incubated at 37°C for the indicated times.
Inhibitors of cell death (Ac-YVAD-cmk, Sigma Aldrich SML0429; Necrostatin-1, Cayman Chemical 11658; GSK’872, Tocris Bioscience 64-921-0; Z-IETD-FMK, Selleckchem S7314; Z-LEHD-FMK, Selleckchem S7313; Z-VAD-FMK, Selleckchem S7023; UAMC-3203, Selleckchem S8972) were added 30 minutes prior to IFN treatment and again immediately following UVB exposure.
For neutralizing antibody experiments, N/TERTs were treated with isotype control (R&D MAB002, MAB0041) or neutralizing antibodies targeting TRAIL (100ng/ml; R&D MAB375), TNF-α (5µg/ml; R&D MAB210) or FasL (1µg/ml; R&D MAB126) immediately following UVB exposure.
For irradiation of cultured cells at approximately 80% confluence, growth medium was replaced with PBS and cells were treated with 0-50mJ/cm2 UVB light as indicated in specific experiments. PBS was removed immediately following irradiation and replaced with growth medium. Cells were incubated at 37°C for the indicated times.
Inhibitors of cell death (Ac-YVAD-cmk, Sigma Aldrich SML0429; Necrostatin-1, Cayman Chemical 11658; GSK’872, Tocris Bioscience 64-921-0; Z-IETD-FMK, Selleckchem S7314; Z-LEHD-FMK, Selleckchem S7313; Z-VAD-FMK, Selleckchem S7023; UAMC-3203, Selleckchem S8972) were added 30 minutes prior to IFN treatment and again immediately following UVB exposure.
For neutralizing antibody experiments, N/TERTs were treated with isotype control (R&D MAB002, MAB0041) or neutralizing antibodies targeting TRAIL (100ng/ml; R&D MAB375), TNF-α (5µg/ml; R&D MAB210) or FasL (1µg/ml; R&D MAB126) immediately following UVB exposure.
7
Ferroptosis Induction and Regulation
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Z-VAD-FMK, 3-methyladenine (3-MA), deferoxamine and RSL3 were obtained from MedChemExpress (NJ, USA); Z-LEHD-FMK and imidazole ketone erastin (IKE) were purchased from Selleck Chemicals (Houston, USA). Dichlorodihydro-fluorescein diacetate (DCFH-DA) was purchased from Beyotime Biotechnology (Shanghai, China). Antibodies against BCL-2, BAX, caspase 9, caspase 3, poly ADP-ribose polymerase (PARP) and β-actin were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against SLC7A11 and GPX4 were obtained from Abcam (Cambridge, UK). Antibody against GAPDH was acquired from Goodhere Biological Technology (Hangzhou, China).
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