Glass bottomed microwell dishes

For imaging, samples were mounted in 100% BABB in glass-bottomed micro-well dishes (MatTek, Ashland, Massachusetts) and covered with a #1 cover glass (Menzel-Glaser, Braunschweig, Germany) to avoid direct contact of the microscope objective and immersion water with the BABB. Imaging was performed with an upright Leica SP5 confocal microscope (Leica Microsystems, Eindhoven, The Netherlands) equipped with a long (1.95 mm) working distance $20\times$ APO water-immersion objective (NA 1.0). Samples were excited by the 514- and 633-nm laser lines from argon and HeNe lasers, respectively. Alexa 514 and Alexa 647 emission light was detected at 525 to 600 nm and 643 to 700-nm, respectively. Z-stacks were recorded with a $3\text{-}\mu \mathrm{m}$ z-step size and a $0.72\text{-}\mu \mathrm{m}$ pixel size. To compensate for loss of signal and optimize the collection of structural information, laser intensity and detector sensitivity were (semi-) automatically adjusted within a preset range. Images were projected in 3D using FIJI (ImageJ 1.49 s³¹ (link)).

For all biological experiments, HeLa cells were cultured at 37 °C in 5% CO₂ in DMEM medium supplemented with penicillin-streptomycin (1×), and 10% fetal bovine serum. 15 × 10⁴ HeLa cells were seeded the day before treatment on glass-bottomed microwell dishes (MatTek Corp.). HeLa cells were treated with BTI, T1, T2, or T3 (stock solutions: 2.5 mM in DMSO) dissolved in complete medium at 1 µM and incubated at 37 °C in 5% CO₂ for 24 h. CellROX^TM green reagent (Invitrogen, Ref No: C10444) (5 µM) was added to the cells for 30 min at 37 °C in 5% CO₂. When required, the cells were photo-irradiated by using an EVOS® FL cell imaging system equipped with an adjustable-intensity LED cube (excitation = 470/22 nm) operating at 23 mW cm⁻² for 10 min. Then, cells were washed twice with 1× PBS. Finally, the cells were kept into live cell imaging solution (Molecular Probes^TM, Ref No: A14291DJ) and imaged by using a Leica SP8 FALCON confocal microscope. Maximum intensity projection of Z-stack images was used for data presentation. All data were processed by using ImageJ software.

Embryos were fixed in 4% PFA overnight at 4 °C and then washed three times in PBT (PBS, 0.1% Triton X-100). Embryos were preincubated in blocking solution (PBT, 10% FBS) during 1 hour and then incubated overnight at 4 °C. In this study, anti-GATA6 (1/100, AF1700, R&D Systems), anti-CDX2 (1/100, MU392A-UC, Biogenex), anti-NANOG (1/100, 8822, Cell Signaling; 1/100, 14–5761, eBioscience), and anti-SOX17 (1/100, AF1924, R&D systems) were used. After several washes in PBT, embryos were incubated with secondary antibodies coupled with Alexa 488 nm, 546 nm, 647 nm (1/300, Invitrogen) and Hoechst (1.6 µM, Sigma Aldrich) 2 hours at room temperature and then washed in PBT. We manually counted the number of cells positive only for GATA6/SOX17/NANOG or double positive (GATA6 or SOX17/NANOG). Embryos were and analyzed using a SP5 (Leica) or LSM800 (Zeiss) confocal microscope. Images were acquired using glass bottomed microwell dishes (MatTek Corporation) using the same objective (Plan-apochromat 20 × /NA 0.75), speed (700 Hz), pinhole 1 airy unit, and laser intensities with optical section thickness of 1.2–1.4 μm. 16-bit resulting images were analyzed and eventually processed using Icy, Fiji and Photoshop CS5 softwares.