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To image Polycomb bodies in live cells, PCGF2-HaloTag SCC1^DEG cells were plated on a gelatinised 35 mm Petri dish, 14 mm Microwell 1.5 coverglass dishes (MatTek) at least 5 hours prior to imaging. Cells were labeled with 500 nm Halo-JF549 (Grimm et al., 2015 (link)) for 15 min at 37°C, followed by three washes, changing medium to Fluorobrite DMEM (Thermo Fisher Scientific) for imaging, which was supplemented as described for general ESC culture above. Cells were incubated for a further 30 min in supplemented Fluorobrite DMEM with 10 μg/mL Hoechst 33258 (Thermo Fisher Scientific) at 37°C and washed once more before imaging. Imaging was performed with an IX81 Olympus microscope connected to a Spinning Disk Confocal system (UltraView VoX PerkinElmer) using an EMCCD camera (ImagEM, Hamamatsu Photonics) in a 37°C heated, humidified, CO₂-controlled chamber. Z stacks were acquired using a PlanApo 100x/1.4 N.A. oil-immersion objective heated to 37°C, using Volocity software (PerkinElmer). PCGF2-HaloTag-JF549 was imaged with a 561 nm laser at 1.25 s exposure at 15% laser power, SCC1-AID-GFP with a 488 nm laser at 1 s exposure at 40% laser power, while Hoechst was imaged with a 405 nm laser at 250 ms exposure at 20% laser power. Z stacks were acquired at 150 nm intervals. A total of at least 28 cells were imaged per condition in two biological replicates.

Fluorescence imaging of TS2/4-ATTO647N-labelled LFA-1 was performed on the ventral side of cells using an Olympus IX71 inverted microscope working in total internal reflection fluorescence (TIRF) geometry with a 150×, 1.45NA oil objective. Excitation of ATTO647N was provided by a 640-nm solid-state laser (power density at the focal plane <1 kW/cm²). Fluorescence was collected with the same objective and guided into an EM-CCD camera (Hamamatsu ImagEM) after suitable filtering. Movies were recorded at a frame rate of 10 Hz for a total of typically 200 frames. The sample temperature (34–36 °C) was maintained by a stage heater (Pecon) and an objective heater. Under our experimental conditions, the localization precision on the determination of the centroid positions of individual fluorescent spots resulted 25 nm, as assessed from measurements of individual TS2/4-ATTO647N on fixed cells (Fig. S4).