The human osteoblast-like cells MG-63 (American Type Culture Collection ATCC®, CRL-1427) were cultivated in Dulbecco’s modified eagle medium (DMEM, Life Technologies GmbH, Darmstadt, Germany) with 10% fetal calf serum (FCS) (Biochrom FCS Superior, Merck KGaA, Darmstadt, Germany), as was reported before [8 , 10 ]. For phosphatidylinositol 3-kinase (PI3K) inhibition the cells were treated with 10 µM LY294002 (Cell Signaling Technology Inc., Danvers, MA, USA), and for ROCK inhibition with 20 µM Y27632 (Cell Signaling Technology Inc.) during the 24 h culture time. The inhibitory substances were diluted in dimethylsulfoxide (DMSO, Merck KGAA, Darmstadt, Germany) to a stock solution of 10 mM. The control experiments were also carried out with the appropriate amount of the vehicle DMSO.
Y 27632
Manufactured by Cell Signaling Technology
Sourced in United States
Y-27632 is a ROCK inhibitor that selectively inhibits ROCK1 and ROCK2 kinases. It is commonly used in cell culture studies to modulate cellular processes such as cell morphology, migration, and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (4 mentions)
Ly294002, by Cell Signaling Technology (3 mentions)
Pd98059, by Cell Signaling Technology (2 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (2 mentions)
Stemdiff neural crest differentiation kit, by STEMCELL (2 mentions)
Matrigel, by Cell Signaling Technology (2 mentions)
Accutase, by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
Mtesr medium, by STEMCELL (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Biochrom fcs superior, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ccg 1423, by Selleck Chemicals (1 mentions)
Pf431396, by Selleck Chemicals (1 mentions)
Pf 562271, by Selleck Chemicals (1 mentions)
Verteporfin, by Merck Group (1 mentions)
Sb431542, by Selleck Chemicals (1 mentions)
U0126, by Cell Signaling Technology (1 mentions)
15 protocols using y 27632
1
Osteoblast-like Cell Culture and Inhibition
2
Chemical Reagents for Signaling Pathways
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Chemical reagents U0126 (Cell Signaling Technology, #9903), PD98059 (Cell Signaling Technology, #9900), Y-27632 (Cell Signaling Technology, #13624), SB431542 (Selleckchem, S1067), CCG1423 (Selleckchem, S7719), verteporfin (Sigma, SML0534). VS-6064 (Selleckchem, S7654), PF431396 (Selleckchem, S7644), PF562271 (Selleckchem, S2890), and Vanadate (New England Biolabs, P0758) were used at doses and times indicated in figure legends. All compounds were dissolved in DMSO.
3
Comprehensive Mitophagy Regulation Assay
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The primary antibodies used were RhoA (#2117, for WB), Parkin (#2132), COX-IV (#11967), VDAC (#4661), lamin A/C (#2032), Rho-GDI (#2564), HA (#3724), PKD (#90039), P-PKD S916 (#2051), GAPDH (#2118), α-actinin (#3134) and LC3B (#3868) from Cell Signaling Technology; PINK1 from Novus Biologicals (#NB600-973); RhoA (SC-418, for IP) and ubiquitin (SC-8017) from Santa Cruz Biotechnology; miniSOG from Kerafast (#EFH004). Horseradish peroxidase (HRP)-conjugated secondary antibodies for WB, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), cycloheximide (CHX), MG-132, CID755673, Bafilomycin A1, Evans blue and triphenyltetrazolium chloride (TTC) were purchased from MilliporeSigma. Y-27632 was purchased from Cell Signaling Technology. DharmaFECT-1 and LysoTracker Blue were purchased from Thermo Fisher Scientific. C3 exoenzyme was purchased from Cytoskeletton, Inc.
4
Molecular Mechanisms Regulating MSC Mechanoresponse
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MSCs were cultured in 12-well TCPs at 1 × 105 seeding density per well (for RNA and protein extraction) with α-MEM medium supplemented with 10% MSC-qualified FBS, 1× Glutamax (Gibco, USA), and 1× antibiotic-antimycotic solution. For immunofluorescence staining, the 1 × 104 cells per well were seeded onto sterilized 15-mm coverslips placed at the bottom of each well of a 12-well TCP. The MSCs were serum-starved prior to treatment with inhibitors by removing the growth medium containing 10% MSC-qualified FBS and replacing it with low serum medium containing 1% MSC-qualified FBS overnight. The serum-starved cells were incubated for 4 h in medium containing inhibitors solubilized in DMSO: 100 μg/ml GRGDSP (Sigma, USA), 50 μM PD98059 (Cell Signaling Technology, USA), 30 μM RN1738 (Tocris, USA), and 10 μM Y-27632 (Cell Signaling Technology, USA) separately for the inhibition of integrin, MEK/ERK1/2, and ROCK respectively prior to cLIUS stimulation. Non-cLIUS-treated MSCs incubated in medium containing DMSO (0.125% v/v) served as controls.
5
RTK inhibition and AXL signaling in U2OS cells
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Receptor tyrosine kinases (RTKs) were inhibited in U2OS cells overnight with 1 μM of each of the following inhibitors: Gefitinib (#HY-50895), Lapatinib (#HY-50898), Bemcentinib (#HY-15150), BMS-536924 (#HY-10262) purchased from MedChemExpress; Erdafitinib (#HY-18708) Lenvatinib (#HY-10981), Cabozantinib (#HY-13016), Galunisertib (#HY-13226), Linsitinib (#HY-10191) from Cedarlane Laboratories; and Crizotinib (#PF-02341066) from Selleckchem.com. PI3K was inhibited by treating cells with LY294002 (Millipore Sigma; #440202) at 10 μM for at least 1 h. Gas6-mediated stimulation of AXL in U2OS cells was performed by serum-starving cells for 24 hours prior to the addition of 200 ng/mL recombinant Human Gas6 (R&D Systems; #885-GSB) to the culture medium for 30 min. For AXL receptor blockade, U2OS cells were incubated with 20 μg/mL anti-AXL antibody (R&D Systems; #AF154) or 50 μg/mL anti-GAS6 antibody (R&D Systems; #AB885). Normal goat polyclonal IgG (R&D Systems; #AB-108-C) was used as control (50 μg/mL). Rho-associated kinases (ROCK) were inhibited with Y-27632 (Cell Signaling Technology; #13624) at 1 or 5 μM.
6
Cytoskeletal Regulation and Beta-Catenin Inhibition
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5 × 105 cells were seeded on the PDMS substrate with a total area of 300 mm2 for 1 day, followed by the treatment of Y-27632 (Cell Signaling Technology, MA, USA, 13624) at 10 μM for 2 h or of Blebbistatin (Toronto Research Chemicals, North York, ON, Canada, B592500) at 25 μM for 2 h, to inhibit Rho activity and Myosin II activity, respectively. For beta-catenin inhibition, 5 × 105 cells were seeded on the PDMS substrate with a total area of 300 mm2 and cultured for 1 day, followed by treatment of XAV-939 at 10 μM (Selleckchem, Houston, TX, USA S1180) for 24 h.
7
Engineered MCF-10A cell lines
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MCF-10A cells were bought from ATCC (Manassas, VA, USA). PTEN−/−, 10A-KRAS(G12V) and PTEN−/−KRAS(G12V) cell lines were created from the MCF-10A cell line as previously described [1 (link)]. Cells were cultured at 37 °C and 5% CO2 in DMEM/F-12 media (Invitrogen, Grand Island, NY, USA) with 1% Penicillin-Streptomycin (Gemini Bio-Products, West Sacramento, CA, USA), 0.2 µg/mL recombinant human EGF (Invitrogen), 0.5 µg/mL hydrocortisone (Sigma, St. Louis, MO, USA), 0.2 µg/mL Cholera Toxin (Sigma), and 10 µg/mL Insulin (Sigma). PI3K and ROCK were inhibited by adding 20 mM LY294002 (Cell Signaling Technology, Danvers, MA, USA) or 10 mM Y27632 (Cell Signaling Technology, Danvers, MA, USA) in a 1:1000 volume ratio to cell culture dishes for 24 or 4–6 h before measurement respectively.
8
Differentiation of mESCs to Mesendoderm
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Differentiation to mesendodermal cells was adapted from a published protocol (Thomson et al. 2011 (link)). mESCs were first recovered into 2i media for two days. On day 1 of differentiation, mESCs were seeded on plates coated with 0.1% gelatin (EMD Millipore ES006-B) in N2/B27 differentiation medium, containing a 1:1 mixture of DMEM/F12 (ThermoFisher 11330032) and Neurobasal (ThermoFisher 21103049), supplemented with 0.5% N2 Supplement (ThermoFisher 17502048), 1% B27 Supplement (ThermoFisher 17504044), 1% GlutaMAX (ThermoFisher 35050061), 1% Pen-Strep (ThermoFisher 15140122), 1% MEM nonessential amino acids (ThermoFisher 11140050), 100 μM sodium pyruvate (ThermoFisher 11360070), 0.1 mM 2-mercaptoethanol (Sigma M3148), supplemented with 10 μM ROCK inhibitor Y-27632 (Cell Signaling 13264). On day 2, ROCK inhibitor was removed and differentiation media was supplemented with 3 μM CHIR99021 (R&D Systems 4423) replenished daily for 3 additional days. On day 5, plates were rinsed twice using PBS and cells were collected for further studies.
9
Differentiation of iPSCs into Osteoblasts
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iPSC colonies were maintained in mTesR medium (StemCell Technologies) and cultured on Matrigel (Corning, 354277). Cells were typically passaged once a week using GCDR (StemCell Technologies). iPSCs were differentiated into NCCs with the STEMDiff Neural Crest Differentiation Kit (StemCell Technologies) according to the company instructions. Briefly, one well of iPSCs was detached with Accutase (ThermoFisher) into single cells. Cells were resuspended in provided medium containing 10 µM Y-27632 (Cell Signaling, 13624S) and plated at 8.6×104 cells/cm2 on Matrigel-coated 12-well plates. Cells were cultured with daily medium changes without Y-27632. On day 6, cells were passaged with Accutase into single cells (NCC P1) and maintained in Mesencult-ACF Plus medium (StemCell Technologies) supplemented with 2 mM L-glutamine until confluent. Cells were expanded for three passages before osteoblast differentiation. Briefly, cells were passaged at 5×103 cells/cm2 on uncoated 24-well plates and cultured for 4-12 days in osteogenic medium [DMEM/F-12 with GlutaMAX (Gibco) supplemented with 10% FBS, 0.1 µM dexamethasone, 10 mM β-glycerophosphate and 200 µM ascorbic acid] with medium changes every other day. Cells were fixed with 4% PFA and stained with 2% Alizarin Red (pH 4.8).
10
Trophoblast Stem Cell Differentiation Protocol
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TSCs and TSLCs were differentiated into EVT and ST as described previously (3 (link)). For differentiation of ST, dissociated TSCs and TSLCs were cultured in a 60 mm Petri dish (5 × 105 to 1 × 106 cells/dish) with 4 mL ST differentiation medium (DMEM/F12 supplemented with 1% ITS-X supplement, 0.5% penicillin-streptomycin, 0.3% BSA, 0.1 mM β-mercaptoethanol, 2.5 μM Y27632, 2 μM forskolin [Selleck Chemicals], and 4% SR). ST differentiation medium was replaced on 3 d. ST derived from TSCs and TSLCs were analyzed on 6 d. For differentiation of EVT, dissociated TSCs and TSLCs were plated on 1 μg/mL collagen IV-coated 6-well plate (1 to 1.5 × 104 cells/cm2) with 2 mL of EVT differentiation medium (DMEM/F12 medium supplemented with 1% ITS-X supplement, 0.5% penicillin-streptomycin, 0.3% BSA, 0.1 mM β-mercaptoethanol, 100 ng/mL human neuregulin-1 [NRG1, Cell Signaling Technology], 7.5 μM A83-01, 2.5 μM Y27632, 4% SR, and 2% Matrigel). On 3 d, the EVT medium was replaced with fresh medium supplemented with reduced Matrigel (0.5%) in the absence of NRG1. On 6 d, the EVT medium was replaced again with the medium containing reduced Matrigel (0.5%) but omitting both NRG1 and SR. EVT derived from TSCs and TSLCs were analyzed on 8 d.
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