No 1.5 borosilicate glass bottom

U2OS cells were cultured on the Lab-Tek II chambered coverglass with a No. 1.5 borosilicate glass bottom (Thermo Fisher Scientific), then treated with 20 nM Cep152 siRNA for 24 h and infected with adenoviruses expressing the indicated mGFP-Cep152 constructs for 14 h. The cells were washed once with phosphate-buffered saline (PBS), replenished with fresh medium, and subjected to time-lapse imaging using a Zeiss LSM880 Airyscan microscope. Centrosome-localized mGFP-Cep152 signals were photobleached with the 75% acousto-optic tunable filter-modulated transmission power of the 488-nm laser at 30 iterations and 3.87 µs pixel dwell time. Fluorescence recovery was monitored by collecting images at three-minute intervals over 42 min. Images were processed using the ZEN 2.3 SP1 software (Carl Zeiss Microscopy LLC), and the processed images were analyzed using the FRAP module of the ZEN software to plot recovery curves.

For the results shown in Fig. 6f, U2OS cells stably expressing EGFP-Plk4 CP were cultured on a Lab-Tek II chambered coverglass with a No. 1.5 borosilicate glass bottom (Thermo Fisher Scientific), and then subjected to live cell imaging using a Zeiss LSM880 Airyscan microscope with a 32-channel GaAsP Airyscan detector, a plan-apochromat ×63 (NA 1.4) oil-immersion objective lens, and a Pecon heated stage incubator (temperature, humidity, and 5% CO₂ control) under the confocal imaging mode. The same area was imaged before and after treating the cells with 6% 1,6-hexanediol for 10 s under the same image acquisition settings. Relative signal intensities were quantified after maximum intensity projection using the ZEN v2.1 software.