Aspc-1, HepG2, Panc02, and H22 cell lines were obtained from the KeyGEN Biotechnology Company (China). HT1080 and SW480 were obtained from the FuHeng BioLogy Company (China). HT1080 cancer cells were cultured in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum (FBS), glutamine (2 mM), penicillin (100 U/ml), and streptomycin (0.1 mg/ml). SW480, Aspc-1, HepG2, Panc02, and H22 were cultured in high Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, L-glutamine (4 mM), and penicillin (100 U/ml) and streptomycin (0.1 mg/ml). All cell lines were maintained in a humidified atmosphere containing 5% CO2 at 37 °C and tested for mycoplasma prior to the commencement of experiments. Unless otherwise indicated, cell culture medium was changed every 3 days, and cells were passaged using 0.05% trypsin/EDTA. Erastin (#HY-15763), sorafenib (#HY-10201), sulfasalazine (#HY-14655), DON (#HY-108357), RSL3 (#HY-100218A), L-Buthionine-(S,R)-sulfoximine (BSO, #HY-106376A), ferrostatin-1 (#HY-100579), Z-VADFMK (#HY-16658), AICAR (#HY-13417), BafA1 (#HY-100558), N-Acetylcysteine (#HY-B0215), and Necrosulfonamide (#HY-100573) were purchased from MedChemExpress (USA). Compound C (#ab120843) was purchased from Abcam. Deferoxamine mesylate (#D9533) was purchased from Sigma-Aldrich.
Necrosulfonamide
Manufactured by MedChemExpress
Sourced in United States, China
Necrosulfonamide is a chemical compound that functions as a cell-permeable, irreversible inhibitor of the mixed lineage kinase domain-like (MLKL) protein. MLKL is a key effector of necroptosis, a form of programmed cell death. Necrosulfonamide can be used in research applications to study the mechanisms of necroptosis.
Automatically generated - may contain errors
Lab products found in correlation
Ferrostatin 1, by MedChemExpress (7 mentions)
Z vad fmk, by MedChemExpress (6 mentions)
Erastin, by MedChemExpress (5 mentions)
Anti 4 hne ab46545, by Abcam (2 mentions)
Z vad fmk, by Selleck Chemicals (2 mentions)
Deferoxamine, by MedChemExpress (2 mentions)
Ab125066, by Abcam (2 mentions)
Anti slc7a11 nb300 318, by Novus Biologicals (2 mentions)
Anti ftl 10727 1 ap, by Proteintech (2 mentions)
Anti gstz1 gtx106109, by GeneTex (2 mentions)
N acetyl l cysteine nac, by Beyotime (2 mentions)
Ferrostatin 1, by Selleck Chemicals (2 mentions)
Deferoxamine mesylate, by Merck Group (1 mentions)
Sulfasalazine, by MedChemExpress (1 mentions)
Aicar, by MedChemExpress (1 mentions)
N acetylcysteine, by MedChemExpress (1 mentions)
Sorafenib, by MedChemExpress (1 mentions)
Aspc 1, by Keygen Biotech (1 mentions)
Hepg2, by Keygen Biotech (1 mentions)
Compound c, by Abcam (1 mentions)
12 protocols using necrosulfonamide
1
Cell Line Culture and Compound Screening
2
Ferroptosis Regulation Pathway Analysis
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Bafilomycin A1 (S1413) and ZVAD-FMK (S7023) were purchased from Selleckchem (Houston, TX, USA). N-acetyl-L-cysteine (S0077) was from the Beyotime Institute of Biotechnology (Shanghai, China). Sorafenib (HY-10201), Erastin (HY-15763), Ferrostatin-1 (HY-100579), Deferoxamine (HY-B0988), and Necrosulfonamide (HY100573) were obtained from MedChemExpress (MCE; Shanghai, China). Tertiary butylhydroquinone (112941) was purchased from Sigma–Aldrich (Shanghai, China). Brusatol (MB7292) was obtained from Meilunbio (Dalian, China). Carmustine (BCNU) was from Macklin (Shanghai, China). Antibodies against SLC27A5 (NBP1-89267) were brought from Novusbio (Centennial, CO, USA). Anti-NRF2 (ab62352), anti-β-actin (ab6276), and anti-4-HNE (ab46545) were obtained from Abcam (Cambridge, MA, USA). Anti-GSR (sc-133245) was purchased from Santa Cruz Biotechnology (Santa Cruz; CA, USA).
3
Investigating Pancreatic Cancer Cell Line Responses
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Human pancreatic cancer cell lines (PaTU8988 and AsPC-1) were obtained from the Cell Bank of the China Academy of Sciences (Shanghai, China). PaTU8988 cells were cultured in high glucose Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum and antibiotics (100 units/mL penicillin, 100 mg/mL streptomycin). AsPC-1 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) containing 10% fetal bovine serum and antibiotics (100 units/mL penicillin, 100 mg/mL streptomycin). They were maintained at 37°C 5% CO2 and saturated humidity. Artesunate (#HY-N0193), Z-VAD-FMK (#HY-16658), Necrosulfonamide (#HY-100573), Ferrostatin-1 (Fer-1, #HY-100), and Deferoxamine mesylate (DFO, #HY-B0988) were obtained from MedChemExpress (MCE).
4
Ferroptosis Pathway Modulator Protocol
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Erastin (HY-15763), Ferrostatin-1 (HY-100579), Deferoxamine (HY-B0988), and Necrosulfonamide (HY-100573) were purchased from MedChemExpress (MCE; Shanghai, China). Sorafenib (S7397), ZVAD-FMK (S7023), Bafilomycin A1 (S1413), and RSL3 (S8155) were obtained from Selleckchem (Houston, TX, USA). N-acetyl-L-cysteine (NAC, S0077) was from Beyotime (Shanghai, China). Brusatol (Bru, MB7292) was obtained from Meilunbio (Dalian, China). Tertiary butylhydroquinone (tBHQ, 112941) was obtained from Sigma (Shanghai, China). Antibodies raised against GPX4 (ab125066), NRF2 (ab62352), 4-HNE (ab46545), NQO1 (ab34173), and β-actin (ab6276) were obtained from Abcam (Cambridge, MA, USA), anti-SLC7A11 (NB300-318) was from Novusbio (Centennial, CO, USA), anti-FTL (10727-1-AP) was from Proteintech (Shanghai, China), and anti-GSTZ1 (GTX106109) was from GeneTex (San Antonio, CA, USA).
5
Establishing Drug-Resistant Hepatocellular Carcinoma Cell Lines
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A normal hepatocyte line (L02) and HCC cell lines (Hep3B, HepG2, Huh7, and PLC) were purchased from Shanghai Institute of Cell Bank (Shanghai, China). Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, USA) supplemented with NaHCO3 (1.5 g/L). HepG2 and PLC cells were grown in minimum essential medium (Gibco, Grand Island, USA) with NaHCO3 (1.5 g/L) and sodium pyruvate. Cells were cultured with 10% fetal bovine serum (Pansera ES, Pan biotech GmbH, Germany), penicillin (U/ml), and streptomycin (0.1 mg/ml) at 37°C in a humidified atmosphere containing 5% CO2. Cells were exposed to sorafenib (Solarbio, Beijing, China) for the indicated time and at the indicated concentration. Drug-resistant cell lines were established by stepwise selection of cells cultured in growth media with increasing concentrations of the drug over a period of 6 months. Erastin, staurosporine (STS), ferrostatin-1 (Fer-1), deferoxamine (DFO), ZVAD-FMK, and necrosulfonamide (NSA) were purchased from MedChemExpress (New Jersey, USA). sorafenib and pioglitazone (Pg) were purchased from Solarbio Biotechnology Company (Beijing, China).
6
Ferroptosis Regulatory Compound Assay
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Erastin (S7242), ferrostatin-1 (S7243), liproxstatin-1 (S7699), Z-VAD-FMK (S7023), necrosulfonamide (S8251), 3-Methyladenine (S2767), LF3 (S8474), and cisplatin (S1166) were purchased from Selleck Chemicals (Houston, Texas, USA). The stimuli concentrations were as follows: ferrostatin-1, 2 μM; liproxstatin-1, 1 μM; Z-VAD-FMK, 10 μM; necrosulfonamide, 0.5 μM; 3-Methyladenine, 250 μM. Actinomycin D (HY-17559) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). TNF alpha (TNFα, HZ-1014), anti-beta-actin (66009-1-Ig, 1:5000), and anti-GAPDH (60004-1-Ig, 1:5000) were purchased from Proteintech (Wuhan, PR China). Anti-GPX4 (ab125066, 1:2000), anti-beta-catenin (ab32572, 1:1000), and anti-4-HNE (ab46545, 1:100) were purchased from Abcam (Cambridge, UK). Anti-TCF4 (sc-166699, 1:500) for western blotting and anti-CagA (sc-28368, 1:500) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Anti-TCF4 (MA5-32240, 1:100) for IHC was purchased from Thermo Fisher Scientific.
7
Culturing Liver Cancer Cell Lines
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Human liver cancer cells HepG2 were obtained from the American Type Culture Collection (ATCC). HCCC9810 was obtained from Procell (Catalog, CL-0095). Cell cultures were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) (EallBio, Beijing, China; # 03. U16001) at 37°C in a humidified atmosphere of 95% air and 5% CO2. RSL3, dimethyl sulfoxide (DMSO), Z-VAD-FMK, necrosulfonamide, and ferrostatin-1 were purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA).
8
Exploring Ferroptosis Regulation Pathways
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Erastin (#HY-15763), (1S,3R)-RSL3 (RSL3, #HY-100218A), Ferrostatin-1 (#HY-100579), Z-VAD-FMK (#HY-16658B), Necrosulfonamide (#HY-100573), 6-Diazo-5-oxo-L-nor-Leucine (DON, #HY-108357), UDP-GlcNAc Disodium Salt (UDP-GlcNAc, #HY-112174), Cycloheximide (CHX, #HY-12320), MG-132 (#HY-13259) and Piperazine Erastin (#HY-100887) were purchased from Med Chem Express (MCE, USA). Benzyl 2-acetamido-2-deoxy-a-D-galactopyranoside (BADGP, B4894) was purchased from Sigma-Aldrich (USA). Thiamet-G (TMG, S7213) was purchased from Selleck Chemicals (USA). Human OGT, GFPT1 and ZEB1 knockdown plasmids were cloned into the pPLK GFP lentivirus vector (PPL, China) and FV055 lentivirus vector (Fubio, China), respectively. FV026 lentivirus vector (Fubio, China) was used to overexpress ZEB1-WT and ZEB1 mutant (T678A or S555A). The shRNA and overexpression target sequences are listed in Table S1 and 2
9
Oesophageal Cell Lines for Necroptosis
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Oesophageal epithelial cells (HET-1A) (RRID: CVCL_3702) and ESCC cells (KYSE410, KYSE30, KYSE510, KYSE450, and KYSE150) (RRID: CVCL_1352, RRID: CVCL_1351, RRID: CVCL_1354, RRID: CVCL_1353 and RRID: CVCL_1348) were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and Procell Life Science&Technology Co., Ltd (Wuhan, China). Cell lines were cultured in RPMI 1640 medium (PM150110, Procell, China) with 10% fetal bovine serum (164210, Procell, Wuhan, China), penicillin G (100 U/ml, BYT-C0222, Beyotime, China), and streptomycin (100 μg/ml, BYT-C0222, Beyotime, China) and maintained in a humidified incubator with 5% CO2, at 37 °C. All cell lines tested negative for mycoplasma throughout the study. The short tandem repeat (STR) profiling was used to verify the identity of each cell line. Cisplatin (HY-17394, MedChemExpress, Monmouth Junction, NJ, USA), necrostatin-1(Nec-1) (HY-15760, MedChemExpress, Monmouth Junction, NJ, USA), RIPA-56 (HY-101032, MedChemExpress, Monmouth Junction, NJ, USA) and necrosulfonamide (NSA) (HY-100573, MedChemExpress, Monmouth Junction, NJ, USA) were used to treat cells for 24 h to induce and inhibit necroptosis, respectively.
10
Cytotoxicity Evaluation of Compounds
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Cells were plated in 96-well plate at the density of 1 × 104 per well and were allowed to attach for 24 h 5 μM Sorafenib (Cell Signaling Technology, USA), 5 μM ferrostatin-1 (S7243, Selleck, USA), 50 μM deferoxamine (S5742, Selleck, USA), 10 μM Z-VAD(OMe)-FMK (HY-16658, MedChemExpress, USA) or 1 μM Necrosulfonamide (HY-100573, MedChemExpress, USA)were added to the cells as indicated in Figure legends and incubated for 24 h. The cells were harvested and Cytotoxicity LDH assays were performed using the Cytotoxicity LDH assay kit (CK12, Dojindo, Japan) according the manufacturer's instructions as previously described [45 (link)].
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