86r 1kt

Immunofluorescence analysis was performed on 1% Matrigel-coated glass coverslip in wells or in situ in microfluidic channels with the same protocol. Cells were fixed in 4% formaldehyde (Sigma-Aldrich 78775) in PBS for 10 min at RT, washed in PBS, permeabilized for 1 h in PBS + 0.3% Triton X-100 (PBST) at RT, and blocked in PBST + 5% of HS (ThermoFisher 16050-122) for 5 h at RT. Cells were incubated overnight at 4 °C with primary antibodies (see Supplementary Table 2) in PBST + 3% of HS. After washing with PBS, cells were incubated with secondary antibodies (Alexa, Life Technologies) (Supplementary Table 2) for 45 min at RT. Nuclei were stained with either DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich F6057) or Hoechst 33342 (ThermoFisher 62249). In the case of Phalloidin staining (see Fig. 8f), Alexa Fluor 488 Phalloidin and Hoechst were added with secondary antibodies. Images were acquired with a Zeiss LSN700 or a Leica SP5 confocal microscope using ZEN 2012 or Leica TCS SP5 LAS AF (v2.7.3.9723) software, respectively.
For alkaline phosphatase staining, cells were fixed with a citrate–acetone–formaldehyde solution and stained using an alkaline phosphatase detection kit (Sigma-Aldrich 86R-1KT). Plates were scanned using an Epson scanner and scored manually.

ZHBTc4 YPET-MD cells expressing SNAP-MD-OCT4 or SNAP-MD*-OCT4 or ZHBTc4 cells expressing mCherry-OCT4-AID from the EF1α promoter were plated at 400 cells per well in a 6-well plate with ES cell medium (see above) with 0 or 1 µg/ml dox and medium was refreshed every other day. At day 7, flat and dome-shaped colonies were scored according to morphology and counted (SNAP-MD-OCT4 and SNAP-MD*-OCT4) or total colonies were counted (mCherry-OCT4-AID) followed by alkaline phosphatase staining (Sigma #86R-1KT). For RT-qPCR experiments, cells were collected at day 7 (Figure 3—figure supplement 1G) or after 26 hr of dox treatment and 2 hr with or without IAA treatment (Figure 5—figure supplement 1F–G) and RNA was extracted using GenElute Mammalian Total RNA MiniPrep Kit (Sigma #RTN350). cDNA was synthesized using oligodT primers (Figure 3—figure supplement 1G) or random hexamers (Figure 5—figure supplement 1F–G) using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific #18064014). qPCR was performed on a 7900HT (Applied Biosystems). The 2^(-Ct) value of each primer pair was normalized to that of Rps9 in the same sample. Primers are listed in Supplementary file 4.