A172 and U373, LN18, LN229, T98G and U87MG GBM cell lines and human embryonic kidney 293T cells were obtained from American Tissue Type Culture Collection (Manassas, VA, USA) and cultured in DMEM medium (Gibco, Gaithersburg, MD, USA) with 10% fetal bovine serum and 1% Penicillin-Streptomycin (Gibco, Gaithersburg, MD, USA). GBM8, GBM4 and MGG119 cells [19 (link),34 (link)] were cultured in neurobasal medium (Gibco, Gaithersburg, MD, USA) supplemented with 3 mM L-Glutamine (Mediatech/Sigma-Aldrich, Woburn, MA, USA), B27 (Invitrogen/Gibco, Norcross, GA, USA), 2 μg/mL heparin (StemCell Technologies/Fisher Scientific, Kent, WA, USA), 20 ng/mL human EGF (R&D Systems, Minneapolis, MN, USA), and 20 ng/mL human FGF-2 (PeproTech Hamburg, Germany) (EF media). All cells were grown in 37 °C, 5% CO2 in a humidified incubator. Vitronectin (Gibco, Gaithersburg, MD, USA), Collagen (Gibco, Gaithersburg, MD, USA), recombinant human TGFβ1 (Peprotech 100-21, Hamburg, Germany), Tiplaxtinin (Selleckchem PAI-039, Houston, TX, USA), Repsox (Tocris, Ellisville, MO, USA), and SB431542 (Stemcell Technologies, Kent, WA, USA) were used for dispersal experiments. D-luciferin was used for in vivo imaging (Biotium, Fremont, CA, USA).
Repsox
Manufactured by Bio-Techne
Sourced in United States
RepSox is a small molecule inhibitor of the TGF-beta receptor I kinase. It is often used in cell biology research to modulate TGF-beta signaling.
Automatically generated - may contain errors
Lab products found in correlation
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Vitronectin, by Thermo Fisher Scientific (1 mentions)
Human egf, by R&D Systems (1 mentions)
Sb431542, by STEMCELL (1 mentions)
D luciferin, by Biotium (1 mentions)
Tiplaxtinin, by Selleck Chemicals (1 mentions)
Recombinant human tgf β1, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
U0126, by Merck Group (1 mentions)
Cocl2, by Bio-Techne (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Fugene 6, by Promega (1 mentions)
4 oht, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Amniomax, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 (fgf2), by RnD Systems (1 mentions)
8 protocols using repsox
1
Glioblastoma Cell Culture Protocol
2
Odontogenic Differentiation of SHED
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SHED (1 × 104 cells/well) were plated in 24-well plates in growth medium and incubated overnight, allowing cell attachment. On the following day, the medium was changed to OCM, and the two inhibitors, U0126 (Sigma Aldrich) and RepSox (Tocris, Ellisville, MO, USA), were treated individually. As a control group, DMSO, which was used to dilute the inhibitors, was used as the treatment without inhibitors. The media were refreshed with inhibitors or DMSO every 3 days for 14 days. Odontogenic differentiation-related gene expression levels (DMP1, RUNX2, and OCN) were measured on Days 3 and 7, and ALP activity and mineralization were confirmed by ALP and ARS staining on Days 7 and 14, respectively.
3
RepSox Treatment of M2 Larvae
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M2 larvae were injected with 0.05% dimethyl sulfoxide (DMSO) (vehicle control; n = 40) or RepSox (SJN2511; Tocris Bioscience, Minneapolis, MN; 7.183 ng/g body weight, equivalent to 0.025 µM dissolved in 0.05% DMSO; n = 40) intraperitoneally. After 10 days, animals were euthanized and morphological changes were recorded. Liver samples were snap‐frozen in liquid nitrogen for quantitative RT‐PCR (n = 10 × 1 set and 8 × 3 sets) and UPLC/MS‐MS (n = 6). The animals were treated for 10 days because it took 1 to 2 weeks for the M2 larvae to progress to M3.7
4
Regulation of VHL and ALK5 in Renal Cells
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One day before transfection, ACHN, and A498 cells were seeded into 10 cm plates (1x106 cells/plate). ACHN cells were transfected with pcDNA 3.1(+) or C-terminally hemagglutinin (HA)-tagged ALK5 (ALK5-HA) [21 ] or ALK5-HA and siVHL (Cat#4390824, Ambicon, Thermo Fisher Scientific, Waltham, MA, USA). The transfection was performed using Fugene 6 (Promega, Fitchburg, WI, USA). siVHL was transfected by using RNAimax (Thermo Fisher Scientific). A498 cells were transfected with pcDNA 3.1(+) or ALK5-HA, or ALK5-HA and VHL (NM_000551.2, Origene Technologies, Inc., Rockville, MD, USA) using Fugene 6 (Promega).
The next day, cells were starved for 12h in media containing 2% FBS (starvation media), followed by treatment with 10 ng/ml TGF-β1 (R&D system, Minneapolis, MN, USA) for 6 hours. A selective kinase inhibitor of TβRI; 100 µM RepSox (Tocris, Cat. No. 3742/10) was added to cells 6h before TGF-β1 stimulation to inhibit the kinase activity of TβRI. For the induction of hypoxia, AHCN and A498 cells were treated with 300 µM COCl2 (Tocris, Bristol, UK) for 3h and 6h. Cells without treatment served as control. After the indicated time points, cells were collected for protein extraction.
The next day, cells were starved for 12h in media containing 2% FBS (starvation media), followed by treatment with 10 ng/ml TGF-β1 (R&D system, Minneapolis, MN, USA) for 6 hours. A selective kinase inhibitor of TβRI; 100 µM RepSox (Tocris, Cat. No. 3742/10) was added to cells 6h before TGF-β1 stimulation to inhibit the kinase activity of TβRI. For the induction of hypoxia, AHCN and A498 cells were treated with 300 µM COCl2 (Tocris, Bristol, UK) for 3h and 6h. Cells without treatment served as control. After the indicated time points, cells were collected for protein extraction.
5
Inducible Epidermal β-catenin Activation
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All experimental procedures were carried out under the terms of a UK Home Office project license. All mice (Supplementary Table S1 ) were maintained on a C57BL6/CBA background and male and female mice were used in experiments. K14ΔNβ-cateninER mice were injected with 10 μl tamoxifen (50 μg/g body weight, dissolved in corn oil) (Sigma-Aldrich/Merck, Darmstadt, Germany) intraperitoneally at P0, followed by three topical applications of 4OHT (100 μg in acetone; Sigma-Aldrich). For depilation experiments, four topical applications of 4OHT (100 μg in acetone) were made. To assess proliferation P11 mice were injected intraperitoneally with 500 μg EdU in phosphate buffered saline (Invitrogen/Thermo Fisher Scientific, Waltham, MA) 2 hours before isolation.
Neonatal wound healing was performed as described before (Rognoni et al., 2016 (link)). Skin depilation was performed as described (Sequeira et al., 2014 (link)) with adult (P56) telogen mice. For TGFβ inhibition, skin was treated with RepSox (1 μmol in acetone; Tocris, Minneapolis, MN) daily before isolation.
Neonatal wound healing was performed as described before (Rognoni et al., 2016 (link)). Skin depilation was performed as described (Sequeira et al., 2014 (link)) with adult (P56) telogen mice. For TGFβ inhibition, skin was treated with RepSox (1 μmol in acetone; Tocris, Minneapolis, MN) daily before isolation.
6
Isolation and Culture of Mouse Primary Fibroblasts
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Mouse primary fibroblasts were isolated6 (link) and cultured in DMEM supplemented with 10% FCS or Amniomax (Gibco). Cells were pulsed with 10 μM EdU 3–4 h before fixation and staining with the Click-it EdU Imaging Kit (Invitrogen). To stimulate or inhibit specific signalling pathways in fibroblasts, the following concentrations of GFs and inhibitors were added to the medium for 24 or 48 h: Shh 1 μg ml−1; TGF-β2 10 ng ml−1; FGF2 20 ng ml−1; FGF5 10 ng ml−1 (all from RnD Systems; vehicle: PBS); IPI4182 0.5 μM (Infinity Pharmaceuticals; vehicle: DMSO); RepSox 25 μM (Tocris; vehicle: DMSO); PD173074 2 μM (Tocris; vehicle: DMSO). DED was prepared by floating 5 mm diameter punch biopsies of back skin on 0.8% trypsin (Gibco) dissolved in PBS for 60 min, separating the dermis from the epidermis with forceps and depleting cells from the tissue by at least ten freeze–thaw cycles. Before fibroblasts were seeded onto DEDs, the tissue was placed into 24-well cell culture inserts and equilibrated with medium.
7
Small Molecule Library Procurement and Dilution
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A library of 1,280 biologically active small molecules was procured from Tocris Bioscience. Repsox was procured from Tocris Bioscience and Sigma-Aldrich. Repsox was diluted in PBS to obtain 1–20 mM stock concentration.
The details of the compound and the sources are provided in thesupplemental information (Table S3 ).
The details of the compound and the sources are provided in the
8
Episomal iPSC Induction from Human Fibroblasts and Astrocytes
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Human broblasts and human astrocytes (Catalog #1800, Sciencell) were expanded and maintained with FM medium and AM medium respectively. The following episomal plasmids for iPSCs derivation were used for transfection at a 1:1:1 ratio: pCXLE-hOCT3/4-shp53-F (Addgene, 27077), pCXLE-hSK (Addgene, 27078), pCXLE-hUL (Addgene, 27080), and pCXLE-EGFP (Addgene, 27082). Total 4.5 µg of the plasmid mixture was electroporated into 1-2×10 6 broblast cells or astrocytes according to the manual of Nucleofector kit (NHDF, Lonza) and Nucleofector 2b (Amaxa, Lonza), and selected U-020 and T-020 system respectively. The transfected cells were seeded in Matrigel-coated 35 mm plates and cultured with FM medium containing 15% FBS (NATOCOR) for 2 days. The next day, Cells were re-seeded at a 1-2×10 4 cells/well into 12-well plates with FM medium. Day 0, the medium was changed to E6 and treated with different small molecules for 7 days, and then cultured in E8 medium for 21 days until hiPSCs colonies formed. We tested the compounds including: CHIR99021 (3 µM, Tocris), RepSox (1 µM, Tocris), XAV-939 (10 µM, Tocris), SB431542 (10 µM, MCE), ID-8 (0.5 µM, MCE), VPA (0.5 mM, Cayman), 1-azakenpaullone (0.5 µM, MCE), SAG (100 nM, EMD Millipore), LDN193189 (50 nM, Stemolecule), DAPT (2.5 µM, Sigma), Y27632 (10 µM, Tocris), Forskolin (10 µM, Cayman), JQ1 (50 nM, Sigma) and SB203580 (1 µM, MCE).
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