Celllight actin rfp

Cells were grown in plastic 96-well plates (Corning) and stained with Hoechst for 10 min, followed by three PBS washes before imaging with fresh media. Cells in the 96-well plates were placed in a 37 °C environmental chamber supplied with 5% CO₂ (ImageXpress Micro XL). The plates were imaged with the ImageXpress Micro XL epifluorescence microscope (Molecular Devices Incorporated) controlled by MetaXpress software (version 5.0.2.0. Molecular Devices Incorporated). Images were captured through a 40 × 0.75 NA dry objective with 2 × 2 binned resolution. For blebbing analysis, images were taken every 1 min. For F-actin-labeled live cell imaging of parental HeLa and Tet-On HeLa, the cells were incubated with CellLight Actin-RFP, BacMam 2.0 (Thermo Fisher Scientific, catalog C10583) 48 h before imaging, and Tet-On HeLa cells were treated with doxycycline 24 h before imaging. Images were captured every 5 min.

U2OS cells were grown in 96‐well plates (Corning) and imaged for up to 24 h at 37°C in a 5% CO₂ environmental chamber using a 40× 0.75 NA dry objective with the MetaXpress 5.0.2.0 software (Molecular Devices Inc.) on the ImageXpress Micro XL epifluorescence microscope (Molecular Devices Inc.). For the analysis of cell division kinetics, cells were stained with Hoechst (1 μg/ml) to label DNA and images were taken every 5 min, and movies were made in the MetaXpress 5.0.2.0 software (Molecular Devices Inc.). For actin localisation, U2OS cells were seeded at 20% confluency in 24‐well plates, and 1 μl CellLight Actin‐RFP (Thermo Fisher Scientific) was added per 5,000 cells and incubated at 37°C for 16 h. Following the incubation, U2OS cells were seeded on L‐shaped micropatterns (CYTOO) at 30,000 per ml. For the analysis of actin localisation, images were taken every 2 min, and movies were made in the MetaXpress 5.0.2.0 software (Molecular Devices Inc.). Images of actin localisation were projected from prophase to metaphase (ImageJ, z‐projection standard deviation) for analysis. For the analysis of cortical membrane elongation, images were taken every 1 min, and movies were made in the MetaXpress 5.0.2.0 software (Molecular Devices Inc.).