EC culture was performed according to a previously described protocol (Chapman et al., 2001 (link)), with some modifications. An HH 9 chick embryo was placed on the culture gel (0.15% glucose, 0.3% agarose, 62.5 mM NaCl, ovalbumin) with the dorsal side down. Then, 100 μL of the chemical such as activators and inhibitors diluted in Tyrode’s solution (8 g NaCl, 0.2 g KCl, 0.2 g CaCl2, 0.1 g MgCl2, 0.05 g NaH2PO4, 1 g glucose, 1 g NaHCO3, up to 1 L H2O) was added dropwise. The lid of the dish was closed and left at room temperature for approximately 1 h, and the dish was incubated at 38.6°C. The following chemical solutions were used: 1% DMSO (control), 1 μM PD173074 (Santa Cruz Biotechnology, TX, USA; sc-202610), 10 μM XAV939 (Cayman Chemical, MI, USA; 13,596), 20 μM CHIR-99021 (Focus Biomolecules, PA, USA; 10–1,279), 300 μM iCRT3 (Selleck, TX, USA; S8647), and 500 μM LF3 (Selleck, TX, USA; S8474), 10 μM U73122 (Cayman Chemical, MI, USA; 70,740), 100 μM LY294002 (FUJIFILM Wako, Japan; 129–04861), 50 μM Ruxolitinib (Selleck, TX, USA; S1378), 100 μM U0126 (Chemscene, NJ, USA; CS-0173).
Chir 99021
Manufactured by Focus Biomolecules
Sourced in United States
CHIR-99021 is a small molecule that functions as a highly selective and potent glycogen synthase kinase 3 (GSK3) inhibitor. It has an IC50 value of approximately 10 nM for inhibiting GSK3 activity. CHIR-99021 is often used in cell culture and research applications to modulate GSK3-related signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000 transfection kit, by Thermo Fisher Scientific (3 mentions)
U73122, by Cayman Chemical (1 mentions)
Xav939, by Cayman Chemical (1 mentions)
Icrt3, by Selleck Chemicals (1 mentions)
Pd173074, by Santa Cruz Biotechnology (1 mentions)
Ly294002, by Fujifilm (1 mentions)
Ruxolitinib, by Selleck Chemicals (1 mentions)
Il 34, by Thermo Fisher Scientific (1 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
Genejuice, by Merck Group (1 mentions)
Cx3cl1, by Thermo Fisher Scientific (1 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
5 aza 2 deoxycytidine 5 aza dc, by Merck Group (1 mentions)
6 protocols using chir 99021
1
Chick Embryo Explant Culture Assay
2
Differentiation of Microglia from hiPSCs
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Details of microglial differentiation from hiPSCs are summarized in Sonn et al., 2022 (link). Briefly, GeneJuice (Sigma–Aldrich, 70,967) and Opti-MEM medium (Gibco™, 31,985,070) were used to establish a doxycycline-inducible PU.1-overexpression clone of hiPSCs. Then, the generation of microglial cells from iPSCs was divided into two stages. First, hematopoietic progenitor cells were generated from hiPSCs (hiHPCs; ∼day 18) in modified StemPro™-34 SFM (1×) (Gibco™, 10,639,011) medium supplied with multiple cytokines and chemicals (BMP4 (PEPROTECH), CHIR99021 (Focus Biomolecules), VEGF (Thermo Fisher Scientific), FGF2 (PEPROTECH), SCF (PEPROTECH), IL-3 (PEPROTECH), IL-6 (PEPROTECH), and IWR-1e (Thermo Fisher Scientific). Doxycycline (1 μg/mL) was added to the medium during days 6 ∼ 18.
Second, microglia-like cells were differentiated from hiHPCs (day 19∼) in modified DMEM/F12 (1:1, Gibco™) supplied with a cocktail of five cytokines (100 ng/mL IL-34, 50 ng/mL TGFβ1, 25 ng/mL M-CSF, 100 ng/mL CD200, and 100 ng/mL CX3CL1, all from PEPROTECH). Induced microglia were used for analysis on day 21.
Second, microglia-like cells were differentiated from hiHPCs (day 19∼) in modified DMEM/F12 (1:1, Gibco™) supplied with a cocktail of five cytokines (100 ng/mL IL-34, 50 ng/mL TGFβ1, 25 ng/mL M-CSF, 100 ng/mL CD200, and 100 ng/mL CX3CL1, all from PEPROTECH). Induced microglia were used for analysis on day 21.
3
Immortalized Liver Cell Line Generation
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Hepatocytes were isolated using the two‐step collagenase perfusion technique from 12‐week‐old male C57BL/6J mice, plated on collagen‐coated dishes, and cultured in Williams’ E medium supplemented with epidermal growth factor (10 ng/mL), insulin (10–7 M), and 10% fetal bovine serum. After 24 hours, the hepatocytes were transfected with the SB13 transposase‐expression plasmid and the transposon cassette plasmids using the Lipofectamine 3000 Transfection Kit (Thermo Fischer Scientific, Waltham, MA). Transformed hepatocytes were cloned using a limiting dilution technique. In some experiments, cloned transformed hepatocytes were treated with a DNA methyltransferase (DNMT) inhibitor (5‐aza‐2′‐deoxycytidine [5‐azadC]; Sigma‐Aldrich, Darmstadt, Germany; 3 µM for 3 days); an MEK inhibitor (PD98059; Cell Signaling Technology; 40 µM for 2 days); a Myc inhibitor (10058‐F4; Abcam; 50 µM for 2 days); and a GSK3β inhibitor (CHIR99021; Focus Biomolecules, Plymouth Meeting, PA; 10 µM for 2 days).
4
Wnt/β-Catenin Signaling Pathway Modulation
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DMSO, anti‐β‐catenin antibody, RPMI 1640, and pyrvinium pamoate were from Sigma‐Aldrich (St. Louis, MO). Complete protease inhibitor cocktail (Complete) was from Roche Diagnostics (Tokyo, Japan). CHIR‐99021 was from Focus Biomolecules (Plymouth Meeting, PA). PPKF115‐584 was from BioVision, Inc (Milpitas, CA). Immobilon membrane was from Millipore (Bedford, MA). The Kras inhibitor SAH‐SOS1A was from Merck (Darmstadt, Germany). Anti‐Lamin B1, anti‐C/EBPα, anti‐C/EBPɛ, anti‐calcium pump pan PMCA ATPase (PMCA), anti‐G‐CSF receptor antibodies, and AKT inhibitor (AKTi‐1/2) were from Abcam (San Francisco, CA). Anti‐GSK3β, anti‐p‐GSK3β (Ser9), and anti‐Tcf4/Tcf7L2 antibodies were from Cell Signaling Technology (Danvers, MA). Anti‐G‐CSF antibody was from R&D Systems (Minneapolis, MN). Human Kras and control siRNAs were from Dharmacon (Lafayette, CO). PE‐labeled mouse IgG1k isotype control and PE‐labeled anti‐human CD11b were from eBioscience, Inc (San Diego, CA). Nuclear Extract Kits were from Active Motif (Carlsbad, CA), and Kras Activation Assay Kits were from Cell Biolabs, Inc (San Diego, CA). The luciferase assay system was from Promega Corporation (Madison, WI). Plasmids of Super8xTOPFlash (M50) and Super8xFOPFlash (M51) were kind gifts from Dr. Craig C. Malbon (State university of New York at Stony Brook, Stony Brook, NY).
5
Expansion of Murine Embryonic Stem Cells
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Every time a new batch of EBRTcPTbx6 cells was thawed, 1.5 μg/mL of puromycin was added for one week before propagation. EBRTcPTbx6 cells were maintained with Glasgow minimum essential medium (GMEM, Sigma–Aldrich), 10% FBS (MP Biomedicals), 0.1 mM NEAA (Wako), 1 mM sodium pyruvate (Sigma–Aldrich), 0.1 mM 2-mercaptoethanol (Sigma–Aldrich), 10 ng/mL doxycycline (Wako), 100 U/mL penicillin (Meiji Seika Pharma) and 100 μg/mL streptomycin (Meiji Seika Pharma), supplemented with 1000 U/mL mouse LIF (ORF genetics), 3 μM CHIR-99021 (FOCUS Biomolecules), and 0.4 μM PD-0325901 (Adooq Bio Science). 1 × 105 EBRTcPTbx6 cells were inoculated to a 60 mm cell culture dish (TrueLine, Nippon Genetics) that had been plated with MMC (Wako)-treated SNL 76/7 feeder cells (obtained from Riken Bioresource Center) on the previous day. The cells were passaged every other day, and subjected to isolation from feeder cells using differential adhesive properties to a gelatin-coated cell culture dish before performing differentiation assays as described below.
6
Immunoblotting and Immunocytochemistry Antibody Protocols
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The following primary and secondary antibodies were used for immunoblotting analysis and immunocytochemistry: anti‐TAK1 rabbit polyclonal antibody, anti‐phospho‐SAPK/JNK (Thr183/Tyr185) rabbit polyclonal antibody, and anti‐phospho‐p38 MAPK (Thr180/Tyr182) antibody (Cell Signaling Technology, Danvers, MA, USA); anti‐TNF Receptor I rabbit polyclonal antibody (Abcam, Cambridge, UK); anti‐IL‐1 mouse mAb (Santa Cruz Biotechnology, Dallas, TX, USA); anti‐α‐tubulin (DM1A) mouse mAb (Merck Millipore, Billerica, MA, USA); IRDye 680 goat anti‐mouse IgG and IRDye 800CW goat anti‐rabbit (LI‐COR Biosciences, Lincoln, NE, USA); and Alexa Fluor anti‐mouse F4/80 antibody (BioLegend, San Diego, CA, USA). Mouse IL‐1β and human TNF‐α were from Wako (Osaka, Japan).
Blasticidin S and puromycin were purchased from InvivoGen (San Diego, CA). (5z)‐7‐oxozeaenol, JNK inhibitor VIII, anisomycin, dibutyryl cyclic AMP, and TPA were purchased from Merck Millipore. Epidermal growth factor, SB203580, IMD‐0354, and PolyI:C were purchased from Sigma‐Aldrich (St. Louis, MO, USA). The GSK‐3β inhibitor, CHIR‐99021, was purchased from Focus Biomolecules (Plymouth Meeting, PA, USA).
Blasticidin S and puromycin were purchased from InvivoGen (San Diego, CA). (5z)‐7‐oxozeaenol, JNK inhibitor VIII, anisomycin, dibutyryl cyclic AMP, and TPA were purchased from Merck Millipore. Epidermal growth factor, SB203580, IMD‐0354, and PolyI:C were purchased from Sigma‐Aldrich (St. Louis, MO, USA). The GSK‐3β inhibitor, CHIR‐99021, was purchased from Focus Biomolecules (Plymouth Meeting, PA, USA).
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