Validated Gatad2a WT, flox/flox or KO male EpiSCs were treated with ROCKi (10 Y27632) 24 hours before initiation of experiments and were harvested with TrypLE (Thermo Fisher Scientific) as single cells. Cells were either single cells sorted in Gelatin/DR4 MEF coated 96 well plates or bulk plated on 6 well Gelatin/DR4 coated plates. Cells were immediately plated in N2B27 based media with the indicated supplements used at these concentrations: human LIF (20ng/ml), CHIR99021 (CH, 3 µM or 1 µM for titrated version (t2i)- Axon Medchem), PD0325901 (PD, 1 µM - Axon Medchem), PKCi (Go6983, 2 µM – Axon Medchem), SRCi (CGP77675, 1.2 µM – Axon Medchem), ROCK inhibitor (Y27632, 2.5 µM – Axon Medchem), γ-Secretase Inhibitor (DBZ 0.35 µM – Axon Medchem). Following approximately 6 days after plating, reversion efficiency was quantified by microscope or FACS imaging of ΔPE-Oct4-GFP. Constitutive mCherry was used for normalization when found in the cell lines used.
Pd0325901 pd
Manufactured by Axon Medchem
Sourced in Netherlands
PD0325901 (PD) is a lab equipment product. It is a selective and potent inhibitor of MEK1/2, which are key components of the MAPK/ERK signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Tryple, by Thermo Fisher Scientific (1 mentions)
Cgp77675, by Axon Medchem (1 mentions)
Rock inhibitor, by Axon Medchem (1 mentions)
Citral, by Merck Group (1 mentions)
Cd2665, by Bio-Techne (1 mentions)
Cd437, by Bio-Techne (1 mentions)
Pgl3 rare luciferase, by Addgene (1 mentions)
Iwr 1, by Merck Group (1 mentions)
Retinol, by Merck Group (1 mentions)
Prl tk, by Promega (1 mentions)
2 protocols using pd0325901 pd
1
Induced Pluripotent Stem Cell Reprogramming
2
Reprogramming factors and vector systems
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Oct4, c‐Myc, Klf4, Sox2, Rarg, Rara, and Lrh1 cDNAs and the RARA‐DN were cloned into piggyBac (PB) transposons under the control of the CMV early enhancer/chicken β actin (CAG) promoter or the doxycycline (Dox)‐inducible TRE promoter (TRE).
For episomal expression, cDNAs of 4F and 2F were cloned into episomal vectors under the control of CAG promoter to construct two expression vectors: pCEP‐4F and pCEP‐2F.
We also used the PB transposase plasmid, pGL3‐RARE‐Luciferase (Addgene, Cambridge, MA,https://www.addgene.org , plasmid, 13458), pRL‐TK (Renilla luciferase control plasmid) (Promega, Madison, WI, http://www.promega.com ), and TOPflash (T‐cell factor [TCF] reporter plasmid) (Merk Millipore, Darmstadt, Germany, http://www.emdmillipore.com , gift from Dr. Jason Wray and Prof. Austin Smith, University of Cambridge, U.K.).
The diagrams of these constructs and plasmids are listed in Supporting Information Figure S1. ATRA, 9cRA, retinol, citral, and IWR‐1 were purchased from Sigma (Gillingham, UK,https://www.sigmaaldrich.com/united‐kingdom.html ), and CD437 and CD2665 were obtained from Tocris Biosciences (Abingdon, UK, http://www.tocris.com ). PD0325901 (PD) and CHIR99021(CH) were obtained from Axon Medchem, (Groningen, The Netherlands, http://www.axonmedchem.com ).
For episomal expression, cDNAs of 4F and 2F were cloned into episomal vectors under the control of CAG promoter to construct two expression vectors: pCEP‐4F and pCEP‐2F.
We also used the PB transposase plasmid, pGL3‐RARE‐Luciferase (Addgene, Cambridge, MA,
The diagrams of these constructs and plasmids are listed in Supporting Information Figure S1. ATRA, 9cRA, retinol, citral, and IWR‐1 were purchased from Sigma (Gillingham, UK,
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