Ifluor 488

Islets were isolated from 7–8-week-old animals and cultured ex vivo in RPMI medium (added 10% FBS, 50 IU ml⁻¹ penicillin, 50 μg ml⁻¹ streptomycin, 0.25 μg ml⁻¹ amphotericin B and 50 mg ml⁻¹ gentamicin) for 2 days to reach steady state. Islets were then treated with 500 nM HDACi (TSA) or 500 mM HATi (C646) for 1 day, followed by a 2-d EdU incubation (iFluor 488, Abcam) to track DNA replication.

DNA synthesis was directly measured in live cells with the EdU Staining Proliferation kit iFluor 488 (Abcam, ab219801) following the manufacture protocol. Cells were seeded in 6-well plates with no. 1.5 22×22 mm coverslips (Electron Microscopy Sciences, 72204–01) at 1.5×10⁵ cells/well, cultured for 48 h, then washed with 1x PBS, and treated with complete or serum-free media. After 20–24 h incubation, cells were incubated with 10 μm EdU solution for 4 h, fixed in 4% paraformaldehyde (PFA; ThermoFisher, J19943.K2) for 10 min, permeabilized, and labeled using kit components. Coverslips were mounted using Vectashield with DAPI (Vector, H-1200) microscope slides and sealed with nail polish. Analysis was performed in ImageJ using the Particle Analyzer plugin and proliferation was calculated as the fraction of EdU positive cells.

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.

Primary islets in RPMI medium (10% FBS, 50 IU ml⁻¹ penicillin, 50 μg ml⁻¹ streptomycin, 0.25 μg ml⁻¹ amphotericin B and 50 mg ml⁻¹ gentamicin) were stained with 10 μM EdU using a fluorescence microscopy protocol kit following the manufacturer’s instructions (iFluor 488, ab219801, Abcam). We used an A1 Plus-RSi laser scanning confocal microscope (Nikon) and z-stack function to capture sequential images of the islets and reconstruct their three-dimensional volume. The total volume of EdU-incorporated cells was then calculated with an ImageJ macro (https://visikol.com/wp-content/uploads/2019/02/Visikol-Measure-Volume-Macro.ijm).