A cell counting kit-8 (Bimake, Houston, USA) assay was performed to evaluate the viability of hiPSC-RPE cells. Briefly, RPE cells were seeded in a 96-well plate at a same density for 24 h. Then, Y27632 (10 μM) (Med Chem Express), Repsox (10 μM) (Med Chem Express) and a combination of Y27632 (10 μM) + Repsox (10 μM) were added into the culture medium, and the cells were further for additional 24 h. Then, 10 μL CCK-8 reagent was directly added into each well and the plate was incubated for 2 h until the color of medium turned orange. Subsequently, the absorbance value of the medium was read at 450 nm with a microplate reader.
Repsox
Manufactured by MedChemExpress
Sourced in United States
RepSox is a small molecule that functions as a transforming growth factor-beta (TGF-β) receptor inhibitor. It is commonly used in cell culture and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Chir99021, by MedChemExpress (2 mentions)
Cell counting kit 8 (cck8), by Selleck Chemicals (1 mentions)
Y 27632, by MedChemExpress (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
24 well plate, by Corning (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (1 mentions)
A83 01, by MedChemExpress (1 mentions)
Sb431542, by Selleck Chemicals (1 mentions)
F 12 dmem f12, by Thermo Fisher Scientific (1 mentions)
Forskolin, by MedChemExpress (1 mentions)
Ldn 193189, by MedChemExpress (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
Tgf β2, by Thermo Fisher Scientific (1 mentions)
Anti β actin 3700, by Cell Signaling Technology (1 mentions)
Tgf β3, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Avantor (1 mentions)
Ly2109761, by MedChemExpress (1 mentions)
Ab15537, by Abcam (1 mentions)
Ab179695, by Abcam (1 mentions)
6 protocols using repsox
1
Evaluating hiPSC-RPE Cell Viability
2
Directed Differentiation of hESCs into Germ Cells
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hESC were plated on collagen I (Corning)–coated 6-well plates (Corning), 24-well plates (Corning), or glass coverslips (15 mm). Cells were cultured in EpiLife medium until 70 to 80% confluence. The culture medium was replaced by the chemical induction medium consisting of DMEM/F12 (Invitrogen, #10565018), 0.5% N2 (Gibco, #17502048), 0.5% B27 (Gibco, #17504044), and 1% P/S, supplemented with 5 μM Repsox (MedChem Express, #HY-13012), 3 μM CHIR99021 (MedChem Express, #HY-10182), bFGF (10 ng/ml; PeproTech, #100-18B-50), and BMP4 (10 ng/ml; PeproTech, #120-05ET-10). The control group was treated with the basal medium containing 1% dimethyl sulfoxide in the absence of small molecules and growth factors. The chemical induction medium was refreshed every 2 days. After 6 to 8 days of induction, hESC were induced into GCs, and the induced GCs were maintained and passaged in the chemical induction medium for the following use. Other small molecules used in the study included 3 μM Kenpaullone (MedChem Express, #HY-12302), 5 μM A83-01 (MedChem Express, #HY-10432), and 5 μM SB431542 (Selleck, #S1067), and 5 μM DAPT (MedChem Express, HY-13027 ).
3
Screening Stem Cell Signaling Modulators
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A screening of small molecules from the MedChemExpress stem cell signaling library (catalogue No. HY-LO17) was performed using a basic cocktail containing forskolin (5 μM), (MedChemExpress, Monmouth Junction, NJ, USA), CHIR99021 (2 μM), (MedChemExpress, Monmouth Junction, NJ, USA), RepSox (2 μM) (MedChemExpress Monmouth Junction, NJ, USA), and LDN193189 (0.5 μM), (MedChemExpress, Monmouth Junction, NJ, USA) in Dulbecco’s modified Eagle medium: F12 (DMEM: F12) (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing N2 (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) as the only supplement. Each of the small molecules from the library was added to the basic cocktail at a final concentration of 5 μM and investigated for its ability to generate a neuronal-like morphology and Tuj1+ cells.
4
TGF-β Isoforms, Hepatitis C Inhibitors
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TGF‐β1 (100‐21‐10 μg), TGF‐β2 (100‐35B‐10 μg) and TGF‐β3 (100‐36E‐10 μg) proteins were purchased from PeProtech Inc (Peprotech). All TGF‐β isoforms were dissolved according to the manufacturer's protocol and the cell viability after TGF‐β isoforms treatment was determined by a methyl thiazolyl tetrazolium (MTT, Amresco) assay.14 Sofosbuvir (HY‐15005), TβRI/II inhibitor LY2109761 (HY‐12075) and TβRI/ALK5 inhibitor RepSox (HY‐13012) were purchased from MedChem Express (MCE). The following primary antibodies were used: anti‐HCV core (ab2740, Abcam), anti‐NS3 (ab13830, Abcam), anti‐β‐Actin (3700S, Cell Signaling Technology), anti‐TGF‐β1 (ab179695, Abcam), anti‐TGF‐β3 (ab15537, Abcam), anti‐TβRI (ab121024, Abcam), anti‐SMAD2/3 (8685T, Cell Signaling Technology) and corresponding secondary antibodies (ZSGB‐BIO). The siRNAs for TβRI (sc‐40222) and for SMAD2/3 (sc‐37238) and the negative control siRNA‐A (sc‐37007) were purchased from Santa Cruz Biotechnology.
5
Signaling Pathway Inhibitor Protocol
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The PMA, PDBu, dPA, dPPA, LY294002 (PI3K inhibitor) and U0126 (ERK inhibitor) were obtained from Sigma (Saint Louis, USA). Calphostin C was ordered from Cayman (Michigan, USA). RepSox (TGFβ pathway inhibitor) was from MedChemExpress (MCE, New Jersey, USA). Ro318220 were from Santa Cruz (Santa Cruz Biotechnology, California, USA). All chemicals were dissolved in DMSO (sigma) to generate a 50 mM stock solution, with final dilutions of at least 1:2000.
6
Evaluating TGF-β Mediated Tenogenic Differentiation
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Growth factor TGF‐β secreted from TSPCs was detected by TGF‐β ELISA kits according to the manufacturer's protocol. In order to explore whether Mo ions induced tenogenic differentiation of TSPCs through TGF‐β‐related pathways, TSPCs were cultured in medium with TGF‐β inhibitor (RepSox, medchemexpress) (20 µm). Then, the expression of tenogenic differentiation‐related genes was determined by RT‐qPCR. The procedure of RT‐qPCR was the same as mentioned above.
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