Ti u inverted microscope system

Cells were fixed for 10 min at 4°C in 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 10 min at room temperature, and blocked for 1 hour in PBS with 1% horse serum (Sigma-Aldrich, St. Louis, MO). The primary antibodies used were as follows: anti-NANOG (1:400; cat. no. 4903, Cell Signaling), anti-SOX17 (1:100; cat. no. 81778, Cell Signaling), anti-FOXA2 (1:100; cat. no. 685802, BioLegend), anti-SMAD2 (1:100; cat. no. 3122, Cell Signaling), anti-SMAD3 (1:100; cat. no. 9523, Cell Signaling), anti–p-SMAD2 (1:100; cat. no. 3108, Cell Signaling), and anti–p-SMAD3 (1:100; cat. no. 9520, Cell Signaling). The treated cells were subjected to three washes with PBS and further incubation with Alexa Fluor secondary antibodies (1:500; Jackson ImmunoResearch) for 1 hour at room temperature in the dark. The cells were then washed three times with PBS, with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO) added to the first wash to stain the nuclei. Images were acquired using a Confocal Zeiss LSM880. Intensity analysis was performed using ImageJ 1.51j8 (National Institutes of Health, MD, USA). For time-lapse imaging, cells were placed on an inverted microscope (Nikon, Ti-U inverted microscope system) and recorded every 2 hours for 72 hours in an environmental chamber maintained at 37°C with 5% CO₂.