Uplanfl n 10x 0.30 n a objective

mESCs were seeded onto the micropatterns, ensuring complete coverage, and cultured for 16 h. Subsequently, the medium was changed to a LIF-free medium. To assess cell proliferation at different time points, the culture medium was supplemented with 10 µM of EdU two hours before fixation at each time point. The detection of EdU incorporation was performed according to the protocol provided by the EdU proliferation kit (Abcam, ab219801, iFluor 488). In brief, the cells were washed twice with Wash Buffer and then fixed with 4% PFA for 10 min at room temperature, while avoiding exposure to light. After the fixation step, the cells were washed twice and permeabilized with 1x Permeabilization Buffer for 20 min at room temperature. Subsequently, the cells were washed twice and incubated in the dark for 30 min at room temperature in a Reaction mix containing TBS, 4 mM Copper Sulfate, 1.2 µM iFluor 488 azide dye (500 µM in DMSO), and 1x EdU additive solution. Following another round of washing, the cells were stained with Hoechst 33342 (5 µg/mL). Finally, the cells were imaged using a spinning disk confocal microscope (OLYMPUS UPlanFL N 10x/0.30 N.A. objective and Andor iXon camera). The Hoechst 33342 and EdU signals were excited at wavelengths of 405 and 475 nm, respectively.

To detect apoptosis of mESCs, Cleaved Caspase-3 (Cell Signaling, 94530) and 7-AAD (Thermo Fisher, 00-6993-50) were measured to assess the middle and late stage of apoptosis respectively. mESCs were seeded onto the micropatterns, ensuring complete coverage, and cultured for 16 h. Subsequently, the medium was changed to a LIF-free medium. To assess the middle stage of apoptosis after 48 h differentiation, fixed cells were stained with Caspase-3 (cleaved) as mentioned in the section of “ Immunostaining”. To assess the late stage of apoptosis after 48 h differentiation, live cells were stained with 7-AAD and Hoechst 33342 (Sigma-Aldrich, B2261-25MG) for 30 min at a 37 °C incubator. Finally, the cells were imaged using a spinning disk confocal microscope (OLYMPUS UPlanFL N 10x/0.30 N.A. objective and Andor iXon camera). Quantification of apoptosis from nuclear morphology was performed on DAPI images acquired by ARTseqFISH as previously described^{62 (link)}.