Epz004777 idot1l

The DOT1L inhibitor EPZ004777 (iDOT1L) was purchased from AOBIOUS Inc. (Gloucester, MA, USA). WNT inhibitor IWR1 was purchased from Selleckchem (Houston, TX, USA). The constructs pMXs-OCT4, NANOG, LIN28A and GLIS1 were purchased from Addgene (Cambridge, MA, USA). Construction of the polycistronic vector pMXs-KLF4, MYC and SOX2 (KMS) was described in our previous study (Wang et al., 2017 (link)). To clone the pMXs-GNL or NL polycistronic vector, the coding sequences for human NANOG, LIN28A and GLIS1 were PCR-amplified from the above-mentioned Addgene constructs. The amplified DNA sequences for each gene were then inserted into linearized pMXs vectors (Cell Biolabs, San Diego, CA, USA) using an In-Fusion kit (Clontech Inc., Mountain View, CA, USA). 2A sequences (Carey et al., 2009 (link); Ryan and Drew, 1994 (link); Ryan et al., 1991 (link)) were inserted between each gene.

The DOT1L inhibitor EPZ004777 (iDOT1L) was purchased from AOBIOUS Inc. (Gloucester, MA, USA). WNT inhibitor IWR1, GSK-3 inhibitor CHIR-99021, MEK inhibitor PD0325901, ROCK inhibitor Y-27632 (ROCKi), and caspase inhibitor Z-VAD-FMK were purchased from Selleckchem (Houston, TX, USA). Forskolin was purchased from Fisher Scientific (Pittsburg, PA, USA). Human reprogramming factors OCT4, SOX2, KLF4, MYC, NANOG, LIN28A, and KDM4A were used for bovine somatic cell reprogramming. The constructs pMXs-OCT4, FUW-TetO-eGFP, and FUW-m2rtTA were purchased from Addgene (Cambridge, MA, USA). Construction of the polycistronic vector pMXs-KLF4, MYC, and SOX2 (KMS) [68 (link)] and pMXs-LIN28A and NANOG (LN) [30 (link)] were described previously. Human KDM4A was cloned into pMXs-vector similarly to as previously described in [30 (link)]. Primary bovine umbilical cord-derived mesenchymal stem cells (bMSCs) were collected from Wharton’s jelly part of the placenta of a newborn male Holstein calf, and were maintained with low serum MSC medium (ATCC, Manassas, VA, USA).