Mek inhibitor pd0325901

RiPSCs were derived from embryonic (E16) Fischer 344 rat fibroblasts (Charles Rivers) that were reprogrammed by retroviral expression of Oct4, Sox2, Klf4, CMyc and SV40 LT-Ag, similarly to previous studies (Liao et al., 2009 ; Liskovykh et al., 2011 (link); Makanga et al., 2015 (link)). RiPSCs were then trypsinized and maintained on a feeder layer of irradiated mouse embryonic fibroblasts (MEFs, Globalstem, Life Technologies) in stem cell medium. The riPSC medium consisted of 50% N2 medium (1% N2 supplement, 2.5% BSA, 1% penicillin/streptomycin, 0.3% β-Mercaptoethanol in DMEM/F12), 50% B27 medium (2% B27 supplement, 1% penicillin/streptomycin, 1% L-Glutamine in Neurobasal medium) supplemented with 3 μM GSK3αβ inhibitor CHIR99021 (Cayman chemical), 1 μM MEK inhibitor PD0325901 (Cayman chemical) and 10 μg/mL rat leukemia inhibitory factor (LIF) (Millipore) (Liskovykh et al., 2011 (link); Makanga et al., 2015 (link)). Cells were maintained in a humidified incubator at 37^oC with 5% CO₂ and the medium was changed daily. Colonies with typical iPSC morphology were passaged every 4–7 days by mechanical dissection and subsequent transfer to MEFs. The pluripotent state of riPSCs was assessed by quantitative real time PCR (qRT-PCR) analysis and immunofluorescence detection for the pluripotency markers Oct4, Octalat, Sox2, and Nanog (Supplementary Figure 1).