Ti2e microscope system

To assess the effects of NANOG WT and mutant overexpression on stem cell differentiation, H9 ESCs with lentiviral transfected NANOG WT or W8A were plated on Cultrex-coated 12-well plates. The cells were initially singularized with Accutase (Sigma-Aldrich, A6964–500ML), then counted and centrifuged. The cell pellets were recovered in StemFlex medium with 10 µM Y-27632 (Tocris, 1254) and 2 µg ml⁻¹ puromycin. Finally, each well was seeded with 40,000 cells. On the next day, the medium (without Y-27632) was changed and NANOG expression was induced with 2 µg ml⁻¹ of DOX for 3–7 days. To check for ESC colonies and undifferentiated cells, alkaline phosphatase activity was assessed using a Vector Blue alkaline phosphatase substrate kit (Vector Laboratories, SK-5300) following the manufacturer’s protocol. The plates were also stained with 0.5% crystal violet solution (protein/nucleic acid stain) in Dulbecco’s phosphate buffered saline (DPBS) to detect all cells. The plates were imaged using a Nikon Ti2E microscope system with a Yokogawa W1 spinning disk module. The large six-well images were derived from stitching of smaller images using the NIS software (Nikon).