NDRG1 or GSK3B genes were inhibited by transient transfection with siRNA (siNDRG1 and siGSK3β) from Santa Cruz (50 ng/ml) and lipofectamine RNAiMAX (Invitrogen) following the manufacturer's instructions for 48h after stimulation with TGFβ1 (10 ng/ml) (PeproTech) for 8h or 14 days. TGFβ1 was replenished every 72h. Scrambled siRNA (SCR) was used as negative control 20 (link). Galunisertib (LY2157299, TGFβR1 inhibitor) (5 μM), CHIR99021 (GSK3α/β inhibitor) (10 μM), LY294002 (PI3K inhibitor) (10 μM), MK2206 (Akt inhibitor) (1 μM), GSK650394 (SGK1/2 inhibitor) (10 μM), and Rapamycin (mTOR inhibitor) (10 μM) were from MedChemExpress. Cells were pre-stimulated with TGFβ1 for 8h and treated with each inhibitor for 24, 48, and 72h.
Galunisertib
Manufactured by MedChemExpress
Sourced in United States
Galunisertib is a small-molecule inhibitor that targets the transforming growth factor-beta (TGF-β) signaling pathway. It functions by selectively inhibiting the TGF-β receptor I kinase activity, thereby modulating the TGF-β signaling cascade.
Automatically generated - may contain errors
Lab products found in correlation
Chir99021, by MedChemExpress (1 mentions)
Mk2206, by MedChemExpress (1 mentions)
Gsk650394, by MedChemExpress (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Ly294002, by MedChemExpress (1 mentions)
Rapamycin, by MedChemExpress (1 mentions)
Ox ldl, by MedChemExpress (1 mentions)
Rhil 2, by Thermo Fisher Scientific (1 mentions)
Easysep human nk cell enrichment kit, by STEMCELL (1 mentions)
Rhtgf β1, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Fujifilm (1 mentions)
8 protocols using galunisertib
1
Modulation of NDRG1 and GSK3B Signaling
2
Oxidized LDL Exposure in HUVECs
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HUVECs were exposed to 200 μg/mL ox-LDL only (Yiyuan Biotechnology, Guangzhou, China), ox-LDL and 10 μM ICA (Weiqi Biotechnology, Sichuan, China), or ox-LDL and 10 μM ICA and galunisertib (lot no. 63191, MedChemExpress, Shanghai, China) for 24 h.
3
Murine Glioblastoma Model Treated with Galunisertib
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Mouse studies were approved by the Animal Experiment Ethical Committee of Hunan University of Medicine (HUM-AE-2018-113). Six-week-old C57BL/6 mice were purchased from Vitalriver. During injection, mice were anesthetized with 2.5% isoflurane. CT-2A cells (5 × 104) in 2 μl Dulbecco’s phosphate-buffered saline (DPBS) were injected into subventrical zone (coordinates: 1 mm anterior to bregma, 1.5 mm from the mid-line, and 2.7 mm below the cranial surface) using the Hamilton microtiter syringe. Pre-warmed pads were employed for mice until fully recovered. Tumor-bearing mice were administered daily with galunisertib (HY-13226, MedChemExpress) by oral gavage (150 mg/kg of galunisertib in 0.5% methylcellulose/Tween 80) starting from 7 days after tumor inoculation. 10 days after first treatment, tumor-bearing mice were sacrificed, and brains were collected for further analysis. The experiment was repeated two times with at least 10 mice per group in each experiment.
4
Gastric Cancer Cell Lines: STAT3 and TGFβ Inhibition
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Human gastric cancer cell MKN45 (RIKEN, Japan), SGC-7901 (Cell Bank, Shanghai), and MKN28 (RIKEN, Japan) were maintained in RPMI-1640. BMFs within 12 generations were used. Napabucasin (STAT3 inhibitor; Cat.No. HY-13,919) was purchased from MedChemExpress, and Galunisertib (TGFβ receptor I inhibitor; Cat.No. S2230) was purchased from Selleck.cn.
5
Fibroproliferative Dermal Fibroblast Induction
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Normal fibroblasts from human neonatal foreskin (referred to as human dermal fibroblasts or HDFs) were used in all experiments. HDFs were purchased from ATCC (PCS-201-010, Manassas, VA, USA). Galunisertib (LY2157299) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). A stock solution of 10 mM Galunisertib was prepared by dissolving in 100% DMSO. Recombinant human transforming growth factor beta-1 (rhTGF-β) was purchased from PeproTech (#100-21, Cranbury, NJ, USA). The treatment of HDFs with rhTGF-β (10 ng/mL) induced a fibroproliferative phenotype, as confirmed by the increased expression of fibrotic markers such as alpha-smooth muscle actin (αSMA) and collagen-1a. This cell population is referred to as fibroproliferative dermal fibroblasts (FPDFs).
6
Isolation and TGF-β Modulation of NK Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque density gradient centrifugation, and NK cells were then purified from the PBMCs of healthy donors by negative selection using the EasySep human NK-cell enrichment kit (Stem cell, Vancouver, Canada). Purified NK cells were resuspended at 2 × 105 cells/well in RPMI 1640 containing 10% fetal calf serum (FCS) and seeded in 96-well plates supplemented with 1000 U/ml recombination human (rh) IL-2 (rhIL-2) to each well in the presence or absence of 10 ng/ml rhTGF-β1 (Peprotech, Rocky Hill, NJ). In some cases, NK cells were treated with 5 μM of the TGF-β receptor I inhibitor galunisertib (MedChem Express, Monmouth Junction, NJ) for 1 hour followed by stimulation with 10 ng/ml rhTGF-β1. After 48 hours, plated cells were harvested and stained with antibodies (listed below) for flow cytometry analysis.
7
THP-1 Macrophage Differentiation Protocol
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To differentiate the THP-1 cells into macrophages, they were stimulated by 20 nM phorbol 12-myristate 13-acetate (PMA; Fujifilm Wako) for 72 h, as reported previously [44 (link)]. Differentiation was confirmed by the adherence of the cells to the bottom of dishes or culture plates. Galunisertib was purchased from MedChemExpress (Middlesex, NJ, USA).
8
Galunisertib Cytotoxicity Assay
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Cells were seeded at a density of 1 × 10 3 cells/well in 96-well plates and treated with 0.2% FBS, 10–100 µM galunisertib (MedChemExpress, Monmouth Junction, NJ, USA), or vehicle (10% FBS and 0.2% DMSO (Dimethyl sulfoxide), as a control) for 48, 72, or 96 h. Cell viability was evaluated using the WST-1 assay (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt, Roche Diagnostics, Basel, Switzerland) for mitochondrial dehydrogenase activity, as previously described [12 (link)].
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