B0556

All cells were were fixed with 4% PFA for 30 min, followed by permeabilizing with 0.5% Triton X-100/PBS for 20 min at RT. The cells were blocked in a blocking buffer (3% BSA, 2%donkey serum in PBS) for 10 min. Subsequently, the cells were stained with primary antibody against FBL (Abcam, ab4566;1:200), NPM (Sigam, B0556; 1:200 and Abcam, ab183340,1:100), LIN28A (CST, 3978 S; 1:200) overnight at 4 °C. The next day, they were washed three times for 10 min with PBS, and the cells were stained with a secondary antibody (Anti-mouse IgG Alexa Fluor® 647 Conjugate, Jackson ImmunoResearch, 115-605-003,1:400; atto 488-goat anti-rabbit IgG, Sigma, 18772, 1:150) for 1 h at 37 °C. Following washing three times with PBS, DAPI was used for nucleus staining. STED images were acquired using Abberior Instruments with z-stack module. The x, y, and z axis resolution was 30 nm. STED Resol. was 5%. The images were analyzed using Fiji/ImageJ. In order to quantify nucleolus regularity, Boyce-Clark index was used^{37 (link)}: $sbc=\sum _{i=1}^n\mid \left(\frac{\mathrm{ri}}{{\sum }_{i=1}^n\mathrm{ri}}\right)\mathrm{\times }100-\frac{100}{n}\mid$ r_i is the length from the vantage center to the boundary (semidiameter) of the nucleolus, n is the number of the semidiameter. FBL regularity data were up to the nearest integer in Fig. 7g.

Related ESCs were fixed with 4% PFA for 30 min, followed by permeabilizing with 0.5% Triton X-100/PBS for 20 min at RT. The cells were blocked in a blocking buffer (3% BSA, 2%donkey serum in PBS) for 10 min. Subsequently, the cells were stained with primary antibody (Rabbit polyclonal anti-LIN28A, CST, #3978, 1:200; Mouse monoclonal anti-FBL, Abcam, ab4566, 1:200; Mouse monoclonal anti-NPM, Sigma-Aldrich, B0556, 1:200; Rabbit monoclonal anti-NPM, Abcam, ab183340,1:100; Rabbit monoclonal anti-NANOG, Abcam, ab214549, 1:200) overnight at 4 °C. The next day, they were washed three times for 10 min with PBS, and the cells were stained with the appropriate Alexa Fluor conjugated secondary antibody for 1 h at 37 °C. Following washing three times with PBS, DAPI was used for nucleus staining. The cells were detected using the Zeiss LSM800 fluorescence microscope at a 63× oil objective. For high resolution microscopy imaging, LSM800 with the Airyscan module was used.