OCA, GW4064, cycloheximide (CHX) and IDN-6556 were from MedChem Express (NJ, USA). Cholic acid (CA), chenodeoxycholic acid (CDCA), dehydrocholic acid (dhCA), lipopolysaccharide (LPS, from Escherichis coli 0111:B4), d-galactosamine (GalN), carbon tetrachloride (CCl4) and mineral oil were from Sigma–Aldrich (St. Louis, MO, USA). Recombinant human tumor necrosis factor α (TNFα) was obtained from Peprotech (Rocky Hill, USA). α-Smooth muscle action (α-SMA) antibody was purchased from Abcam (Cambridge, UK).
Recombinant human tumor necrosis factor α
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant human tumor necrosis factor α (TNFα) is a cytokine protein produced through recombinant DNA technology. It is a key mediator of the inflammatory response and plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Mineral oil, by Merck Group (1 mentions)
Cycloheximide (chx), by MedChemExpress (1 mentions)
Idn 6556, by MedChemExpress (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Celltrace far red, by Thermo Fisher Scientific (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
Rhodamine 6g, by Thermo Fisher Scientific (1 mentions)
Dihydrorhodamine 123, by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (1 mentions)
Recombinant human ifn γ, by Thermo Fisher Scientific (1 mentions)
Recombinant human interleukin 6 il 6, by Thermo Fisher Scientific (1 mentions)
Recombinant human ifn α2, by BioLegend (1 mentions)
Ibet151 gsk1210151a, by Selleck Chemicals (1 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Erk1 erk2, by Cell Signaling Technology (1 mentions)
13 protocols using recombinant human tumor necrosis factor α
1
Hepatoprotective Compound Screening Protocol
2
In Vitro Tendinopathy Model with TNF-α
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Recombinant human tumor necrosis factor-α (TNF-α) was purchased from PeproTech (No. 300-01A; Rocky Hill, NJ, USA). TNF-α was dissolved with pure ethanol to prepare a 20 µg/mL stock stored at 4 °C. The test concentrations used were 10, 50, 100, and 500 ng/mL in a growth medium. Tenocytes were finally treated with TNF-α (10 ng/mL) for 24 h to establish an in vitro tendinopathy model (Supplementary Figure S1 ).
3
Prebiotic Effects of 2′-FL, 3′-SL, GOS, and Lactose
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2′-fucosyllactose (2′-FL, cat# GY1141, ≥98%) and 3′-Sialyllactose sodium salt (3′-SL, cat# GY1143, ≥98%) were purchased from HuicH Biotech Co., Ltd. (Shanghai, China). Galacto-oligosaccharide (GOS, cat#G9150, ≥70%) and lactose (Lac, cat#SL8740, ≥98%) were purchased from Solarbio Life Sciences (Beijing, China). LS174T cells (cat#CL-0145) were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). Recombinant human tumor necrosis factor α (TNF-α, cat#200-13-2) was obtained from Peprotech (Rocky Hill, NJ, USA). Anti-MUC2 Polyclonal Antibody (cat#K106881P) and FITC Goat Anti-Rabbit IgG (cat#A22120) were purchased from Bioss (Beijing, China). Anti-NLRP6 Antibody (cat#ABF29) was obtained from Merch (Shanghai, China). The siRNA of NLRP6 was designed and produced from GenePharma (Shanghai, China). Lipofectamine™ 3000 transfection reagent (cat#L3000008) was purchased from ThermoFisher Scientific (Shanghai, China).
4
Purification and Preparation of Bilirubin
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Unconjugated bilirubin (bilirubin IXα) was obtained from Porphyrin Products (Logan, UT) and further purified according to the method of McDonagh and Assisi30 to eliminate potential lipid contaminants. Unless otherwise indicated, bilirubin was freshly prepared in 0.1 mol/L of potassium phosphate (pH 12), as previously described by our group.19 The addition of a small aliquot (≤0.4% vol/vol) of this vehicle solution had no effect on the pH of the culture medium or on cell viability.31 Recombinant human tumor necrosis factor α (TNF‐α) was purchased from PeproTech (Rocky Hill, NJ) and solubilized in DMSO. Allopurinol (AP) was purchased from MP Biomedicals (Santa Ana, CA). ML171 (2‐acetylphenothiazine) and mouse immunoglobulin G (IgG) were purchased from Calbiochem (San Diego, CA). Mouse anti‐human CD18 (β2; ab8220) and mouse anti‐human CD49d (α4; clone 2B4) were purchased from Abcam (Paris, France) and R&D Systems (Minneapolis, MN), respectively. Mouse anti‐human VCAM‐1 (clone P3C4) and mouse anti‐human ICAM‐1 (clone P2A4) were purchased from Millipore (Temecula, CA). CellTrace Far Red, dihydrorhodamine 123, Texas Red‐dextran 10 000 molecular weight, and rhodamine 6G were obtained from Molecular Probes (Eugene, OR). Human serum albumin was purchased from Sigma‐Aldrich (St. Louis, MO).
5
Investigating MAGEC2 Regulation in Melanoma
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Human melanoma cell line A375 were purchased from ATCC (Rockville, MA, USA) and maintained in DMEM (ATCC) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA), and human melanoma cell line Hs 695T cells were purchased from Cobioer Biosciences Company (Nanjing, China) and maintained in MEM (Gibco, Gaithersburg, MD, USA) supplemented with non‐essential amino acids, 1 mM sodium pyruvate, and 10% FBS (Invitrogen). MAGEC2‐knockdown Hs 695T cells were established by transfecting pGPU6/Neo‐shMAGEC2 or pGPU6/Neo‐shNC vectors and selected using 1.5 mg/mL G418 for 2 weeks. Target sequences are as follows: MAGEC2 shRNA, 5′‐CAATTGATACCGCAGATGA‐3′; and control shRNA, 5′‐TTCTCCGAACGTGTCACGT‐3′. Recombinant human tumor necrosis factor‐α (TNF‐α) was purchased from PeproTech (Rocky Hill, NJ, USA) and used at 20 ng/mL, and cycloheximide (CHX) was from Sigma‐Aldrich (St. Louis, MO, USA) and used at 10 μg/mL.
6
Cytokine and Inhibitor Treatment Protocol for Cellular Responses
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For cytokine treatment, Recombinant Human IFNα2 (Biolegend, San Diego, CA, USA), Recombinant Human soluble TRAIL (PeproTech, Cranbury, NJ, USA), Recombinant Human IFNγ (Gibco), Recombinant Human Tumor Necrosis Factor α (TNFα, PeproTech) or Recombinant Human Interleukin-6 (IL-6, PeproTech) dissolved in PBS was added to the cell media at the concentrations indicated in the figures.
For BET inhibition, cells were treated with 1 µM JQ1 (Sigma) or 1 µM I-BET151 (GSK1210151A; Selleckchem). For the inhibition of pan-caspases, Z-VAD-FMK (A1902-25; Apexbio) was used at a final concentration of 20 µM. For sensitization to TRAIL-induced cell death, cells were treated with 1µM BV6 (S7597; Selleckchem). All drugs were dissolved in DMSO; DMSO was used as vehicle control.
For BET inhibition, cells were treated with 1 µM JQ1 (Sigma) or 1 µM I-BET151 (GSK1210151A; Selleckchem). For the inhibition of pan-caspases, Z-VAD-FMK (A1902-25; Apexbio) was used at a final concentration of 20 µM. For sensitization to TRAIL-induced cell death, cells were treated with 1µM BV6 (S7597; Selleckchem). All drugs were dissolved in DMSO; DMSO was used as vehicle control.
7
Inhibiting Oridonin-Induced Apoptosis
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Oridonin (purity >99.5%) was purchased from Xi’an Haoxuan Biotechnique, China. It was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM and stored at −20°C. Both N-acetyl-l-cysteine (NAC) and ATRA were purchased from Sigma-Aldrich. Recombinant human tumor necrosis factor (TNFα) was obtained from Peprotech (Rocky Hill, NJ, USA). Cycloheximide was purchased from Sigma-Aldrich. ERK inhibitor PD98059, p38 inhibitor SB203580, JNK inhibitor SP600125, and NF-κB inhibitor Bay 11–7082 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). When cells were treated with these reagents, matching concentrations of vehicle were used as the control and the final concentration of DMSO was kept at or below 0.1% in all experiments.
Antibodies recognizing p65, IκBα, and RARα were purchased from Santa Cruz Biotechnology. Antibodies recognizing phospho-IκBα (Ser32/Ser36), phospho-p65, IκB kinase beta (IKKβ), phospho-IKKα/β, phospho-ERK1/ERK2, ERK1/ERK2, phospho-p38, p38, phospho-JNK, and JNK were purchased from Cell Signaling Technology (Beverly, MA, USA).
Antibodies recognizing p65, IκBα, and RARα were purchased from Santa Cruz Biotechnology. Antibodies recognizing phospho-IκBα (Ser32/Ser36), phospho-p65, IκB kinase beta (IKKβ), phospho-IKKα/β, phospho-ERK1/ERK2, ERK1/ERK2, phospho-p38, p38, phospho-JNK, and JNK were purchased from Cell Signaling Technology (Beverly, MA, USA).
8
Endothelial Cell Signaling Pathway Analysis
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Antibodies against VEGF (A-20), MMP-9 (H-129), and β-actin (I-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). In addition, antibodies against PI3K Class III (D4E2), phospho-PI3K Class III (Ser 249), AKT (5G3), phospho-AKT (Ser473), p38 (D13E1), phospho-p38 Thr180/Tyr182 (D3F9), ERK (p44/42), phospho-ERK (Thr202/Tyr204), phospho-p65 (Ser536) (93H1), p65 (C22B4), phospho-JNK (Thr183/Tyr185), JNK, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). EGMTM-2 Endothelial Cell Growth Medium-2 BulletKitTM was purchased from Lonza (Walkersville, MD, USA). The EZ-Cytox cell viability assay kit and EZ-LDH cytotoxicity assay kit were purchased from DoGenbio (Seoul, Korea). Recombinant human tumor necrosis factor (TNF)-α was purchased from Peprotech (Rocky Hill, NJ, USA). Mayer’s hematoxylin solution was purchased from Muto Pure Chemicals (Tokyo, Japan).
9
Quantifying Fibrosis-related Protein Expression
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Anti-fibronectin antibody and horseradish peroxidase-conjugated rabbit anti-goat IgG antibody were purchased from Abcam (Cambridge, MA, USA), FITC-conjugated anti-α-SMA antibody was purchased from Sigma (St. Louis, MO, USA), and all other antibodies were obtained from Cell Signaling (Beverly, MA, USA). Recombinant human tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β2 were purchased from PeproTech (Rocky Hill, NJ, USA) and R&D Systems (Minneapolis, MN, USA), respectively. 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) was obtained from Toronto Research Chemicals (North York, ON Canada). Dipyridamole (DPY) and 5’-amino-5’-deoxyadenosine (AMDA) were obtained from Sigma (St. Louis, MO, USA).
10
Investigating TP Treatment on LPS or TNF-α-Stimulated Cells
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PBMCs or MH7A were placed into 6-well plates at a concentration of 1.0 × 106 cells/ml and 1 × 105 cells/ml, respectively, followed by 2 h of pretreatment with or without 100 ng/ml of lipopolysaccharides (LPS) (Sigma, St. Louis, MO, United States) or 20 ng/ml recombinant human tumor necrosis factor (TNF)-α (PeproTech, New Jersey, United States). Then, PBMCs were incubated with 6.25 nM or 12.5 nM TP (purity: 99.8%, National Institutes for Food and Drug Control, Beijing, China) for another 24-h, MH7A was incubated with 12.5 nM or 25 nM TP for another 24-h. Finally, the cells were harvested for real-time PCR analysis.
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