Axioimager inverted z1

HeLa mCherry-histoneH2B or PLK1–3xMyc expressing mCherry-histoneH2B were transfected with siRNA against Bora or with the different Bora constructs and synchronized by single or double thymidine in the G1-S boundary. Cells were released for 4 h and then transferred for time-lapse analysis to the microscope stage. Live microscopy was performed with Axioimager inverted Z1 (Zeiss) equipped with an environmental chamber (Zeiss) that maintained the cells at 37 °C in a humidified environment of 5% CO₂. Images were taken every 10 min using an Axiocam MRm camera (Zeiss) driven by Axiovision SE64 software (Zeiss). Movies and JPEG files were imported into ImageJ and proceeded using the same software. Nuclear envelope breakdown was judged as such when the nuclear membrane lost a smooth and the linear periphery. The first frame showing a pole ward movement of the chromosomes was defined as anaphase onset.

Thymidine-synchronized HCT116 cells expressing mCherry-histone H2B were released for 5 h, treated either with 100 nM BI6727 or 2 µM of the Eg5 inhibitor STLC. For time-lapse analysis, the treated cells were transferred to the microscope stage and microscopy was performed with Axioimager inverted Z1 (Zeiss) equipped with an environmental chamber (Zeiss) that maintained the cells at 37 °C in a humidified environment of 5% CO₂. Images were taken every 10 min using an Axiocam MRm camera (Zeiss) driven by Axiovision SE64 software (Zeiss). Movies and JPEG files were imported into ImageJ and proceeded using the same software. Nuclear envelope breakdown was judged as such when the nuclear membrane lost a smooth and the linear periphery. The first frame showing a pole ward movement of the chromosomes was defined as anaphase onset.