Sphingosine‐1‐phosphate (S1P) was purchased from Enzo Life Science, (USA Cat# BML‐SL140‐000). Sphingosine kinase inhibitor (SK1‐II) was purchased from Tocris (Cat# 2097). LY2109761 was purchased from Sigma Aldrich (Italy, Cat# SML2051). Periodic acid‐Schiff was purchased from Sigma Aldrich (St Louis, USA Cat # 395B‐1KT). TGF‐β was purchase by R&D (USA, Cat #DB100B). Anti‐actin alpha SMA from Sigma Aldrich (Cat # A5228). Anti‐IL33 was purchased from ThermoFisher (Italy Car# AF3626). Anti‐FGF2 was purchased from Santa Cruz Technology (USA Cat# sc‐74412). Diamino‐benzidinic acid system was purchased from Sigma Aldrich (Italy Cat# D3939). OCT medium was purchased from Tissue‐Tek OCT (Pella Inc. Italy, Cat # 27050). High‐capacity cDNA kit and SYBR Green Real‐Time PCR Master Mix were purchased from ThermoFisher Scientific (Monza, Italy Cat # 4368814 and 4385612). CD90 PE mouse/rat and CD326 APC mouse were purchase from a Milteny iBiotec (Bologna, Italy Cat# 130‐094‐528 and #130‐096‐417). TGF‐β1 protein (Cat #ab50036), anti‐wide spectrum Cytokeratin (Cat #ab9377), rabbit anti‐mouse, Vimentin (Cat # ab92547), goat anti‐mouse IgG H&L FITC GtxMu‐003‐D (Cat # ab96885594) and goat anti‐rabbit IgG H&L DyLight (Cat #ab96886) were purchased from AbCAM (Cambridge, UK). Other compounds (e.g., ovalbumin, DAPI, collagenase) were purchased from Sigma Aldrich (Italy).
Ly2109761
Manufactured by Merck Group
Sourced in United States
LY2109761 is a laboratory equipment product manufactured by Merck Group. It is designed for use in scientific research and analysis. The core function of LY2109761 is to provide a reliable and precise measurement or analysis tool for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Collagenase, by Merck Group (1 mentions)
High capacity cdna kit, by Thermo Fisher Scientific (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Sphingosine 1 phosphate (s1p), by Enzo Life Sciences (1 mentions)
Anti fgf 2, by Santa Cruz Biotechnology (1 mentions)
Tgf β1 protein, by Abcam (1 mentions)
Sybr green real time pcr master mix, by Thermo Fisher Scientific (1 mentions)
Periodic acid schiff, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Vimentin, by Abcam (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Collagen, by BD (1 mentions)
Pitavastatin, by Selleck Chemicals (1 mentions)
Gemcitabine, by Merck Group (1 mentions)
M30 apoptosense ck18 kit, by Diapharma (1 mentions)
Paclitaxel, by Merck Group (1 mentions)
Valsartan, by Merck Group (1 mentions)
Bosentan, by Merck Group (1 mentions)
11 protocols using ly2109761
1
Molecular Regulation of Tissue Fibrosis
2
Fibroblast Migration Assay with LY2109761
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A scratch wound assay was used to evaluate cell migration as previously described [16 (link)]. Briefly, fibroblasts were seeded in 6-well plates coated with collagen (BD Biosciences, San Diego, CA, USA) and cultured until they reached 80% confluence. Cells were then treated with LY2109761 (dissolved in DMSO; Sigma-Aldrich, St. Louis, MO, USA) at 5 or 10 μM for 36 h. Control fibroblasts were treated with DMSO only. A pipette tip was used to make a “wound” of approximately 0.45–0.50 mm in width at a 90° angle to the bottom of the dish. Cell migration into the wound area was assessed immediately after wound generation and at 24 and 48 h postwound generation using a light microscope (Nikon, Tokyo, Japan). The percentage of cells migrating into the wound area was calculated. Three independent fields were analyzed in each well, and each experiment was performed in triplicate.
3
Spheroid Viability Assay with Drugs
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Monospheroids and heterospheroids were seeded as described before [16 (link),19 (link)], and after one day, treated with 5 µM TGF-β receptor type I/II (TGFBR1/2) kinase inhibitor (LY2109761, Sigma-Aldrich, #SML2051) or different therapeutic compounds, including 50 µM gemcitabine (Sigma-Aldrich, #G6423), 1 µM paclitaxel (Sigma-Aldrich, #580555), 1 µM SN38 (active metabolite of irinotecan; Sigma-Aldrich, #H0165) or 5 µM pitavastatin (a HMG-CoA reductase inhibitor; Selleckchem, #S1759). Three days later, spheroids were collected for RNA extraction or apoptosis detection. M30 Apoptosense® CK18 Kit (Diapharma #P10011) was used according to the manufacturer's instructions to quantitatively detect apoptosis in epithelial cells and differences between treatment groups were assessed by paired t-test.
4
Fibroblast Growth Signaling Inhibitors
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Ang II, ET-1, PD123319, gallein (Gβγ inhibitor), and BQ788 were obtained from Tocris Bioscience (Ellisville, MO, USA). Recombinant human TGF-β1, valsartan, bosentan, ambrisentan, LY2109761, FR180204, and SB203580 were obtained from Sigma Aldrich (Saint Louis, MO, USA). SIS3 (Smad3 inhibitor) and FR900359 (Gαq inhibitor) were obtained from Cayman Chemical (Ann Arbor, MI, USA). Fibroblast growth medium and related cell culture reagents were obtained from Promocell (Heidelberg, Germany).
5
Culturing Nasopharyngeal Carcinoma Cells
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Two nasopharyngeal carcinoma cell lines 13–9B and C666-1 NP69 were purchased from American Type Culture Collection (Manassas, VA, USA). A normal nasopharyngeal cell line NP69 was purchased from Xiang Ya Central Experiment Laboratory (Changsha, China; http://gdyjzx.csu.edu.cn/zxjs/sysjs/xbs.htm ). Cells were cultured in RPMI-1640 medium (cat. no. 30-2001; American Type Culture Collection) contaning 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C and 5% CO2. Serum was not added in case of drug treatment. Cells were also treated with a TGF-β inhibitor, LY2109761 (100 nM; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), for 24 h at 37°C and 5% CO2.
6
Modulation of TGFβ Signaling Pathway
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LY2109761 is a selective TGFβ receptor type I/II (TβRI/II) dual kinase inhibitor and was purchased from Sigma. A TGFβ antibody (1D11) neutralizing all three TGFβ isoforms was purchased from BioXCell. The trivalent TGFβ receptor trap RER was described by us previously and is based on the human TβRII‐rat BG endoglin (orphan) domain and human TβRII (Qin et al., 2016 (link); Zhu et al., 2018 (link)). In this study, we used mRER, which is based on an all‐murine sequence, except for linkers, which are non‐natural.
7
Modulating Collagen Production via TGF-β Signaling
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Immediately post transfection with pcDNA3.1 or pcDNA3.1Grem1, cells were treated with antibodies to TGF-β1 at 1 μg/ml (Mab1835 clone; R&D systems, United Kingdom) or a matched isotype control antibody (1 μg/ml) in 24 well plates to neutralize TGF-β1 levels in the media of the cells. After 48h the media was measured for collagen by Sicrol assay as described above. As described above cells were transfected with SBE luciferase plasmid plus and minus TGF-β1 and neutralizing antibody. To inhibit the TGF-β receptor post transfection we immediately treated the cells with LY2109761 (Sigma Aldrich, United Kingdom) at 70 nM and post 48 h the media was collected and collagen quantified. To determine the role of the TGF-β non-canonical pathway we incubated the dermal fibroblasts with U0126, an MEK inhibitor (Cell Signaling, United Kingdom) 10 μM in DMSO, transfection or not and after 48 h the cell media was harvested and collagen measured by the Sircol assay.
8
Modulating DNA-PKcs in Keratinocytes
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Immortalized human epidermal keratinocytes (HaCaT cells) and the cSCC cell line SCL-1 were supplied by the Department of Dermatology at the First Hospital of China Medical University, and was approved by our institutional ethics committee. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/L streptomycin in a 5% CO2 incubator at 37°C. For the inhibitor and small interfering RNA (siRNA) studies, the cells were seeded in different plates and incubated until they attached firmly to the flask, and then the cells were treated with the DNA-PK inhibitor NU7026 (Selleckchem, Shanghai, China) or transfected with a specific siRNA targeting DNA-PKcs (RiboBio, Guangdong, China). The sequences of the three DNA-PKcs-specific siRNA (si-h-PRKDC) oligonucleotides were as follows: si-h-PRKDC_001, 5ʹ-GCCAGTTTATCAATCTGAT-3ʹ; si-h-PRKDC_002, 5ʹ-GGCCTTATGTACAGCATCA-3ʹ; and si-h-PRKDC_003, 5ʹ-GATCGCACCTTACTCTGTT-3ʹ. The si-h-PRKDC transfection efficiency was determined by RT-PCR. For the TGF-β1 or TGF-β receptor I/II (TGF-βR I/II) dual inhibitor LY2109761 study, the cells were treated with 5 ng/mL TGF-β1 (Sigma-Aldrich) or 300 nmol/L LY2109761 (Selleckchem, Shanghai, China).
9
GDF-15 Recombinant Protein Synthesis
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Recombinant human GDF-15 was purchased from Pepro Tech. TTX, 4-AP, rapamycin, SB431542, PP1, LY2109761 and poly-L -lysine were purchased from Sigma. U0126 was purchased from Selleckchem. FBS, DMEM and antibiotic/antimycotic solution were purchased from Gibco Life Technologies.
10
Inhibition of TGF-β1 and PI3K in GSCs
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