Lipofectoamine 2000

2TS22C ES cells were cultured in GMEM supplemented with 10 % Knockout Serum Replacement (KSR; Invitrogen), 1 % fetal calf serum (FCS), 1 × non-essential amino acids (Nakarai), 1 mM Sodium pyruvate (Nakarai), 10⁻⁴ M 2-mercaptoethanol and 10³ U/ml of mouse LIF on gelatin coated surface. For the complementation assay, 3 × 10⁴ 2TS22C ES cells were seeded in wells of 48 well plates. The following day, cells were transfected with 1 μg of the PiggyBac Sox expression vector and 1 μg of pCAGGS-PBase [20 (link)] using Lipofectoamine 2000 (Invitrogen), and replated into four wells of a 12-well plate. Cells were selected with 10 μg/ml of Blasticidin S (Invivogen) from one to seven days after transfection. Three wells of cells were stained with Leischman stain to count the numbers of stem cell colonies. One well of cells was dissociated and 3 × 10³ cells seeded into a well of a 12-well plate either in the presence or absence of 1 μg/ml of tetracycline. After six days, one-fifth of the dissociated cells were replated into a well of a 12-well plate followed by culture with tetracycline for 6 days. Stem cell colonies were scored by Leischman staining as well as re-seeding into either 12-well or 48-well plates for RNA preparation and immunostaining, respectively.

H-7 is a protein kinase C inhibitor and has been reported to inhibit the phosphorylation of Rex [45 (link),46 (link)]. HeLa cells were seeded in 48 wells at 2 × 104 cells/250 L/well on 48-well plates After 24 h, the cells were co-transfected with 100 ng each of Renilla-WT and Firefly-PTC, and 200 ng of pME-FLAG (Mock) or pME-FLAG-Rex by Lipofectoamine2000 (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA). After 24 h, H-7 was added to a final concentration of 50 μM (20 h treatment). Similarly, H-7 was added at 40 h (4 h treatment), 42 h (2 h treatment), and at 44 h, and Luc assay was performed simultaneously with H-7-free (0 h treatment) samples.