α-Amanitin, cycloheximide, J-8, and cantharidic acid were purchased from Enzo Life Sciences (USA). Triptolide was purchased from MedChem Express (USA). 3,4-Methylenedioxy-β-nitrostyrene (MNS), 2-aminoethyl diphenylborinate (2-APB), and okadaic acid were purchased from Santa Cruz Biotechnologies (USA). The 81 chemicals used for the screening were provided by the Drug Discovery Initiative, The University of Tokyo (Japan) and are listed in S1 Table . All chemicals were dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chemical, Japan; special grade) as a stock solution and stored at -20°C. Chemical solutions at the appropriate concentrations were prepared by diluting stock solutions in sterilized Milli-Q (stMQ) water just prior to chemical treatment. The final concentration of DMSO in all chemical solutions was adjusted to 1%.
Triptolide
Manufactured by MedChemExpress
Sourced in United States
Triptolide is a naturally occurring compound isolated from the Chinese herb Tripterygium wilfordii Hook F. It is a potent small-molecule inhibitor that has been extensively studied in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Fujifilm (1 mentions)
Cycloheximide (chx), by Enzo Life Sciences (1 mentions)
Cantharidic acid, by Enzo Life Sciences (1 mentions)
Okadaic acid, by Santa Cruz Biotechnology (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Berberine, by MedChemExpress (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Curcumin, by MedChemExpress (1 mentions)
Metformin, by MedChemExpress (1 mentions)
Erastin, by MedChemExpress (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Il 22, by Merck Group (1 mentions)
Mcc950, by MedChemExpress (1 mentions)
Dextran sulfate sodium (dss), by MP Biomedicals (1 mentions)
Male wild type c57bl 6 mice, by Charles River Laboratories (1 mentions)
Cal27, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
7 protocols using triptolide
1
Screening of Small Molecule Compounds
2
Leukemia Cell Culture and Treatment
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Human leukemia chronic myelogenous leukemia K562 and acute promyelocytic leukemia HL-60 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Hyclone; Cytiva). HEK293T (Human embryonic kidney) cells were cultured in DMEM medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Hyclone; Cytiva). All cells were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences and were grown in a medium supplemented with 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin (Gibco; Thermo Fisher Scientific, Inc.). Cells were maintained in a humidified 37°C incubator under a 5% CO2 atmosphere. Cells at 80% confluence were treated with 1 µg/ml DOX (cat. no. HY-15142A; MedChemExpress) for 48 h, 40 nM berberine (cat. no. HY-N0716; MedChemExpress) for 24 h, 40 nM metformin (cat. no. HY-15763; MedChemExpress), 40 nM artemisinin (cat. no. HY-B0094; MedChemExpress) for 24 h, 40 nM curcumin (cat. no. HY-N0005; MedChemExpress) for 24 h, 2 µM erastin (cat. no. HY-B0627; MedChemExpress) for 48 h and 40 nM triptolide (cat. no. HY-32735; MedChemExpress) for 24 or 48 h at 37°C.
3
Triptolide Ameliorates IBS in Rat Model
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A total of 18 adult male Sprague-Dawley (SD) rats, weighing 200–220 g, were obtained from the Experimental Animal Center of the Zhejiang Province (Hangzhou, China). The rats were fed in groups of 3 per cage with a room temperature of 24 ± 1 °C and humidity of 50 ± 10%, and they were maintained on a 12-h light/dark cycle with water and food available ad libitum. After all rats were acclimatized to laboratory conditions for 1 week, the 6 rats per group in each were used to conduct behavior tests. All experiments were approved by Wenzhou Medical University Animal Care and Use Committee (Code of Ethics: wydw2022-0138) and were conducted by the National Institutes of Health Guide for Care and Use of Laboratory Animals. Triptolide was purchased from MedChemExpress, which was dissolved in DMSO and diluted with olive oil. All the rats were randomly divided into 3 groups (n = 6 each): the normal group administered via gavage of the same volume of olive oil, the IBS group and the IBS treated via gavage of Triptolide (100 µg/kg/day) [18 (link), 19 (link)]. 5 weeks later, behavioral tests and visceral sensitivity of the bowel were performed. Then the rats were anesthetized by i.p. injection of pentobarbital sodium, and their ileum and colon were harvested for further experiments. Finally, the rats were sacrificed by injecting excessive pentobarbital sodium.
4
Cell Proliferation Assay with CCK-8
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Cell Counting Kit-8 (CCK-8, Dojindo, Kuma-moto, Japan) was applied to assess the cell proliferation. The HaCaT cells (10,000 cells/well) were plated into 96-well plates, and incubated overnight at 37°C. Later on, cells were transfected with miR-181b-5p antagomir and exposed to 10 μM triptolide (MedChemExpress, Monmouth Junction, NJ, USA) for 24 h, then treated with 100 ng/mL of IL22 (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. After that, 10 μL CCK-8 reagent was added into each well, and the plates were incubated for another 2 h at 37°C. Subsequently, a microplate Reader (Bio-Rad, Hercules, CA, USA) was applied to evaluate the absorbance of each well at 450 nM. triptolide was dissolved with DMSO (20 mM store solution), and then diluted with DMSO medium for cell assays.
5
Modulating Mycobacterial Infection Using Inhibitors
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NF-κB inhibitor Triptolide (TPL) was purchased from MedChemExpress (HY-32735, Monmouth Junction, NJ, USA), and NLRP3 inhibitor MCC950 was purchased from MedChemExpress (HY-12815, Monmouth Junction, NJ, USA). THP-1 macrophages were pr-treated for 2 h using TPL (5 nM) and MCC950 (10 μM), respectively. Followed by infection with BCG for 24 h. Exogenous S100A4 was purchased from Sino Biological (10185-H01H, Beijing, China). The S100A4 inhibitor Niclosamise (Nic) was purchased from MedChemExpress (HY-B0497, Monmouth Junction, NJ, USA). THP-1 macrophages were pr-treated with Exogenous S100A4 at concentrations of 0, 0.25, 0.50, 1.00, and 1.50 μg/mL, Nic at concentrations of 0, 0.5, 1.0, 3.0, and 5.0 μM for 2 h before being infected with BCG for 24 h, respectively. Cellular protein samples were extracted and stored at −20 °C for Western blot analysis.
6
Triptolide Attenuates DSS-Induced Colitis
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Male C57BL/6 wild-type mice were obtained from Beijing Vital River Laboratory Animal Technology (Beijing, China). DSS was purchased from MP Biomedicals (Costa Mesa, CA, USA) and triptolide from MedChemExpress (Shanghai, China). Mice were randomly assigned to three groups, namely, the control group, colitis group and treatment group (n = 6). The control group was provided drinking water without DSS, while the colitis group and the treatment group were given water containing 2.5% DSS; the mice in the treatment group were intraperitoneally injected with 0.02 mg/kg triptolide every other day. The animals’ conditions were monitored through general inspections and weight change. On the fifth day, the colitis group and the treatment group were switched to drinking water without DSS. On the eighth day, the mice were euthanized by neck dislocation under isoflurane anesthesia, and the entire colon was removed, rinsed gently with PBS, and dried on filter paper to measure its length. The colon was then divided into sections for immunological and western blot analysis.
7
Molecular Regulation of OSCC Cell Lines
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The human OSCC cell lines CAL27 was obtained from American Type Culture Collection (ATCC, CRL-2095) and WSU-HN6 was obtained from Central Laboratory of Peking University School and Hospital of Stomatology. The cells were cultured in DMEM (Gibco, USA) containing 10% FBS (Sigma, USA) and 1% penicillin/streptomycin in a 37°C incubator with 5% CO 2 . Triptolide was obtained from MedChemExpress (USA), and added into complete DMEM at 0, 10, 30 and 50nM for further detection.
When the cells reached 50-60% con uence, the siRNA targeting METTL3 (Ruibo Biosciences Inc., China) was transfected using the JetPrimer regent (Polyplus transfection, France) following the manufacturer's instructions. For further functional assays, the transfection medium was aspirated and serum-free DMEM was added to the starve cell for 8h incubation. Lentivirus vectors containing METTL3 shRNA were purchased from Hanbio Biotechnology Co. Ltd. (Shanghai, China). To construct a stable cell line of METTL3 knockdown, WSU-HN6 was transfected with lentivirus in the presence of 5 μg/mL polybrene for 48 h, then selected by 3 μg/mL puromycin. For IGF2BP2 overexpression, the full-length cDNA sequence of human IGF2BP2 was cloned into a PCDNA3.1-3xFlag-C vector. Transfection of IGF2BP2 overexpression plasmid was performed as siRNA transfection. The indicated sequences of siRNA and shRNA are listed in Table S1.
When the cells reached 50-60% con uence, the siRNA targeting METTL3 (Ruibo Biosciences Inc., China) was transfected using the JetPrimer regent (Polyplus transfection, France) following the manufacturer's instructions. For further functional assays, the transfection medium was aspirated and serum-free DMEM was added to the starve cell for 8h incubation. Lentivirus vectors containing METTL3 shRNA were purchased from Hanbio Biotechnology Co. Ltd. (Shanghai, China). To construct a stable cell line of METTL3 knockdown, WSU-HN6 was transfected with lentivirus in the presence of 5 μg/mL polybrene for 48 h, then selected by 3 μg/mL puromycin. For IGF2BP2 overexpression, the full-length cDNA sequence of human IGF2BP2 was cloned into a PCDNA3.1-3xFlag-C vector. Transfection of IGF2BP2 overexpression plasmid was performed as siRNA transfection. The indicated sequences of siRNA and shRNA are listed in Table S1.
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