Kinetics of GFP expression in the iSmad1iresEGFP subclone 5. (A) Phase and (B) GFP fluorescence images of untreated and 1-μg/mL doxycycline-treated iSmad1iresEGFP sc5 ES cells, respectively. (C) Phase and (D-F) GFP fluorescence images of day 5.75 iSmad1iresEGFP sc5 EBs that were continuously induced starting on day 0 (D), day 2.75 (E), and day 4 (F). In panels G-J, the iSmad1iresEGFP EBs were induced from day 2 to day 2.25 according to the washout method described in “Materials and methods,” and GFP expression was monitored. (G) Day 2.25 EBs, (H) day 2.5 EBs, (I) day 3 EBs, (J) day 4 EBs. Images were visualized using an AxioCam MRm camera (Zeiss, Thornwood, NY) attached to a Zeiss Axiovert 200M microscope that was equipped with a 10×/0.30 numerical aperture Plan-Neofluar objective. Images were processed using Zeiss Axiovision software version 4.5.