Ix3 svr stage top incubator

U2OS knock-in cells were seeded at 15,000 cells/well in an 18-well ibidi glass-bottom μ-slide and allowed to adhere o/n. After treatment with the indicated amounts of drugs for 4 h, the cells were imaged with an Olympus IX83 epifluorescence microscope equipped with an Orca Fusion scMOS camera, a 100× oil objective (NA 1.45), an X3-ZDC2 TruFocus drift compensator and an Okolab IX3-SVR stage top incubator set to 37°C and 5% CO2. 100 consecutive frames in the FITC channel were taken per field of view at 250 ms intervals and analyzed as previously described^{26 (link)} using custom Mathematica scripts. In short, the surface fluctuation u of individual nucleoli was quantified by averaging the changes in nucleolar contour over both time and polar angle using the Interpolation and Fourier transformation functions of Mathematica. The average surface fluctuation $〈u〉$ was then used to estimate nucleolar surface tension γ based on the relation $\gamma ={\text{k}}_{\text{B}}\text{T}/〈u^2〉$ .

1 × 10⁶ U2OS knock-in cells were seeded in a 10cm culture dish and incubated for 24 h. The culture medium was then replaced with fresh DMEM containing 2mM thymidine. After 18 h, cells were washed twice with PBS, allowed to recover for 6 h in fresh DMEM, and subjected to a second block with 2mM thymidine for 18 h.
Synchronized cells were then trypsinized, seeded into 8-well glass-bottom μ-slides (ibidi) at a density of 30,000 cells/well, and allowed to adhere for three hours. Culture medium was then exchanged with L-15 medium (Gibco) supplemented with 10% FBS and the indicated amounts of drugs. Cells were imaged every 15 min for a total of 24 h in a 3 × 3 grid per well using an Olympus IX83 epifluorescence microscope equipped with an Orca Fusion scMOS camera, a 403 air objective (NA 0.95), an X3-ZDC2 TruFocus drift compensator and an Okolab IX3-SVR stage top incubator set to 37°C. Images were quantified using custom Mathematica scripts.
To determine cell cycle stage, samples were taken immediately after synchronization and at the time point of drug addition, fixed and permeabilized with ice-cold 70% ethanol at 4°C overnight, stained with 1 μg/mL propidium iodine in PBS containing 0.1% Triton X-100 and 10 μg/mL RNase, and analyzed using a Sony SH800 flow cytometer.