Mice were randomly allocated to three groups: sham (n = 5 mice), vehicle and treatment (n = 8 mice per group). iCRT3 (EMD Millipore, Billerica, MA) was reconstituted with cell culture grade 100% DMSO as 50 mg/ml stock. 5 and 10 mg/kg body weight (BW) concentrations of iCRT3 were made by diluting stock in sterile normal saline with 5% DMSO. At 5 h after CLP, 5% DMSO in normal saline (vehicle) or iCRT3 at 5 or 10 mg/kg BW doses in 200 μl volume was delivered by intraperitoneal injection using 25 G × 7/8″ hypodermic needle (Becton, Dickinson & Company, Franklin Lakes, NJ). The investigator performing the animal experiments was blinded to the treatment assignment to eliminate any bias.
Icrt3
Manufactured by Merck Group
Sourced in Germany, United States
The ICRT3 is a laboratory equipment designed for measuring the concentration of thyroid hormones, specifically triiodothyronine (T3), in biological samples. It utilizes an immunochemical reaction-based technique to quantify the T3 levels accurately and efficiently.
Automatically generated - may contain errors
Lab products found in correlation
Icrt14, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Lithium chloride (licl), by Merck Group (2 mentions)
Quercetin, by Merck Group (2 mentions)
Bafilomycin, by Merck Group (2 mentions)
Pyrvinium, by Merck Group (2 mentions)
Niclosamide, by Merck Group (2 mentions)
Xav939, by Cayman Chemical (2 mentions)
Icg 001, by R&D Systems (2 mentions)
Pcdna5 to plasmids, by Merck Group (2 mentions)
Nsc668036, by Merck Group (2 mentions)
Cct031374, by R&D Systems (2 mentions)
Icrt5, by R&D Systems (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Turbofect reagent, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Kapa sybr fast qpcr kit master mix 2x abi prism, by Roche (1 mentions)
20 protocols using icrt3
1
Cecal Ligation and Puncture Mice Model
2
Protein Purification and Analysis Protocol
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Protease Inhibitor Cocktail, Aurora-A Inhibitor-I (AAI), nocodazole, Phosphatase Inhibitor Cocktail and Flag-tagged beads were purchased from Sigma-Aldrich. 1,4-dithiothreitol (DTT) and isopropyl β-D-1-thiogalactopyranoside (IPTG) were obtained from Acros Organics. Insect cell-expressed Aurora-A was purchased from Invitrogen. The cDNA reverse transcription kits and TurboFect reagent were purchased from Thermo Fisher Scientific. Hygromycin B and the Midi plasmid extraction kits were obtained from Roche. PVDF membrane, iCRT3 and ECL were purchased from Merck Millipore. The NTA-beads were purchased from QUIAGEN. The protein G beads for immunoprecipitation were obtained from GE. RQ1 (RNase-free DNase) was purchased from Promega. The SYBR Green reagents (the KAPA SYBR FAST qPCR kit Master Mix (2X) ABI Prism) were obtained from KAPA.
3
Wnt/β-Catenin Signaling Pathway Assay
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RAW264.7 cells were seeded the day before transfection at a density of 1.24 × 105 cells per ml. Cells were transiently co-transfected with 250 ng of TOP-TK-Luc or FOP-TK-Luc (Upstate Biotechnology, Lake Placid, NY) and 25 ng pRL-TK (Promega, Madison, WI) reporter plasmids, using the Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. At 24 h after transfection, cells were pre-treated with iCRT3 (EMD Millipore, Billerica, MA) or vehicle for 50 min and then stimulated with LPS (1 ng/ml) for another 24 h. The cells were lysed 48 h post-transfection and luciferase activity was measured with a Dual-Luciferase reporter assay system (Promega) according to the manufacturer’s instructions. TOP-TK-Luc contains optimal and FOP-TK-Luc contains mutated TCF-binding sites placed upstream of a firefly luciferase reporter gene. The TOP and FOP firefly luciferase activity was normalized to Renilla luciferase activity from the cotransfected pRL-TK plasmid used as an internal control for transfection efficiency. All experiments were performed in triplicate at least twice.
4
Wound Healing Assay with FOXC2
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Primary human LECs were plated in 24-well plates and infected with retroviral particles expressing control GFP or human FOXC2. When cells were near confluency (>90% density), scratch wounds were made at the center of the well using a sterile 1000-µL pipette tip. Loose cells were removed by a PBS wash, and DMSO-containing (Sigma) or iCRT3-containing (Sigma) medium was added. Images were recorded at t = 0, t = 24, and t = 48 h using an inverted microscope to measure the wound size.
5
mESC Maintenance and Differentiation
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mESCs used for this study were maintained in feeder-free conditions in standard ESC Complete media containing DMEM high glucose (Sigma-Aldrich) supplemented with 10% serum (ES Cell FBS; Gibco), LIF (Esgro-LIF; Millipore), Glutamax (Life Technologies), and MEM Nonessential Amino Acid Solution (ATCC; protocols were adapted from Faunes et al., 2013 (link)). Healthy cells were maintained with daily media changes and regular passaging every 48–72 h at a proportion of 1:10 on 0.1% gelatin-coated (Millipore) plates after dissociation using TrypLE Express (Life Technologies). For differentiation using RA (Sigma-Aldrich), cells were plated in Complete medium with 0.5 µM RA in the absence of LIF for 48 h. For long-term cultures, cells were plated in limiting dilutions in 6- or 96-well plates for multiple passages (14 d) in stem conditions (serum plus LIF) with DMSO or iCRT3, with daily media changes. AP staining was performed for every passage to monitor the relative pluripotency levels. Small molecules used include 10 µM iCRT3 (ChemDiv) and 1 µM XAV939 (Sigma-Aldrich), which were diluted with DMSO. L-Wnt3a and control L cells were gifts from R.T. Moon (University of Washington, Seattle, WA).
6
Pathway Agonist and Inhibitor Screening
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The signalling pathway agonists/inhibitors Verteporfin (Tocris); iCRT3 (Sigma); DAPT (Merck Millipore) and GANT61(R and D Systems) were applied at the concentrations indicated during a 7-day incubation following 24-hour transduction of GSK3α/β floxed cells with Cre recombinase expressing lentivirus. Cells were stained with Hoechst (Sigma) at 1 µg/ml, imaged, using an IN Cell analyser 2200 (GE Healthcare) and cell number determined using IN Cell analyser workstation software (GE Healthcare).
Live cell imaging was carried out 5–7 days after viral transduction using an IncuCyte ZOOM cell imaging system (Essen Biosciences).
Live cell imaging was carried out 5–7 days after viral transduction using an IncuCyte ZOOM cell imaging system (Essen Biosciences).
7
Investigating DNA Damage and Wnt Signaling Modulation
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The DNA damaging agent methyl methanesulfonate (MMS, Sigma-Aldrich) was used as a positive control in DNA damage studies. Cells were treated with 0.1% (v/v) MMS for 2 hr prior to fixation and processing for imaging. The small molecule inhibitor iCRT3 (Gonsalves et al., 2011 (link)) (SML0211; Sigma-Aldrich, St. Louis, MO) was used to inhibit β-catenin/TCF interactions and their transcriptional activity. iCRT3 (25 µM) and DMSO controls were added to strain array wells 15 min prior to the application of strain. Src Inhibitors PP2 (10 μM; EMD Millipore, Germany) and SU6656 (10 μM, Sigma-Aldrich), CKI inhibitor D4476 (10 μM; Abcam), EGFR inhibitor PD153035 (2.5 μM, Abcam, United Kingdom), and DMSO controls were added to strain device wells 15 min prior to strain application. Fresh inhibitor was added to all experiments every 12 hr. Wnt3A-expressing mouse L cells and parental L cells were grown as indicated (ATCC CRL-2647 and CRL-2648), and conditioned media was obtained as indicated in the comments section of ATCC CRL-2647. Conditioned media from Wnt3A-expressing and parental L cells was added at a 1:2 dilution to MCDK cell culture medium (described above) 15 min prior to strain application.
8
Efficient Stem Cell Culture Protocols
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Gelatine, putrescine, sodium selenite, progesterone, apotransferrin, glucose, insulin, ascorbic acid, valproic acid and ICRT3 were obtained from Sigma (Steinheim, Germany). Accutase was from PAA (Pasching, Austria). FGF-2 (basic fibroblast growth factor), FGF-8b, Sonic hedgehog and noggin and were obtained from R&D Systems (Minneapolis, MN, USA). Y-27632, SB-43154, CHIR99021 and dorsomorphin dihydrochloride were from Tocris Bioscience (Bristol, UK). MatrigelTM was from BD Biosciences (Massachusetts, USA). All cell culture reagents were from Gibco/Invitrogen (Darmstadt, Germany) unless otherwise specified.
9
Canonical Wnt Signaling Modulation
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Short hairpin RNAs (shRNA), cDNAs encoding human WNT7A and FGF1, and pcDNA5/FRT/V5-His, pcDNA6/TR and pcDNA5/TO plasmids were purchased from Sigma-Aldrich, Thermo Scientific (Rockford, IL, USA) and Life Technologies (Life Technologies, Grand Island, NY, USA), respectively. Niclosamide, iCRT3, iCRT14, Pyrvinium, Bafilomycin, Quercetin, NSC668036 and LiCl were purchased from Sigma-Aldrich. XAV939 and IWR were purchased from Cayman Chemical (Ann Arbor, MI, USA). IWP and Box5 were purchased from Thermo Scientific. ICG001, CCT031374 and iCRT5 were obtained from R&D Systems (Minneapolis, MN USA).
10
MOLT4 Cell Wnt Signaling Inhibition
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