C apochromat 40 1.2 water objective

The cells were transferred to PLL‐g‐PEG coated fluorodishes (FD35‐100) with CO₂‐independent culture medium (described previously). Cells were stained with Hoechst 33342 solution (PN:62249, Invitrogen) in order to distinguish between mitotic and interphase cells. During imaging they were maintained at 37 °C using ibidi heating stage. Images were recorded using a Zeiss LSM700 confocal microscope of the CMCB light microscopy facility, incorporating a Zeiss C‐Apochromat 40×/1.2 water objective. Images were taken at the equatorial diameter of each cell exhibiting the largest cross‐sectional area.

The suspended (interphase or STC‐arrested mitotic) cells were fixed with 4% formaldehyde/PBS for 1 h, followed by a 30 min permeabilization step in 0.2% Triton X‐100. The cells were then stained for 1 h with 5 µg mL⁻¹ DAPI and 0.2 µg mL⁻¹ Phalloidin‐Alexa Fluor 647 in 2% BSA/PBS solution. Images were recorded using a Zeiss LSM700 confocal microscope of the CMCB light microscopy facility, incorporating a Zeiss C‐Apochromat 40×/1.2 water objective. Images were taken at the equatorial diameter of each cell exhibiting the largest cross‐sectional area.

The spheroid hydrogel constructs were fixed at day 7 with 4% formaldehyde/PBS for 1 h, followed by a 30 min permeabilization step in 0.2% Triton X‐100. The spheroids were then stained for 4 h with 5 µg mL⁻¹ DAPI and 0.2µg mL⁻¹ Phalloidin‐Alexa Fluor 488 in 2% BSA/PBS solution. Then hydrogels were immersed at least for 1 h in PBS. For imaging, hydrogels were cut horizontally with a blade and were placed on a cover slip to image the interior of the hydrogel. Per hydrogel 5–15 spheroids were imaged recording a confocal z‐stack with a Zeiss LSM700 confocal microscope of the CMCB light microscopy facility, using a Zeiss C‐Apochromat 40×/1.2 water objective (z‐step: 0.4–0.9 µm).