Phenol red free and co2 independent medium

Hela Kyoto cells were transiently transfected with pIRESneo3-mCherry-NLS and pIRESpuro3-AcGFP-NES using FuGENE6. After 6 h, non-targeting control or PPP1R12A-siRNA was transfected using DharmaFECT 1. Time-lapse movies were performed 40 h after siRNA transfection. Prior to the start of recording, the culture medium was changed to phenol-red free and CO₂ independent medium (Invitrogen). Images were acquired as 3 z-planes 2 μm apart every 2 min at 37 °C using a Nikon TE2000 microscope equipped with a Plan Fluor × 60/1.4 DIC H Lens (Nikon), a PerkinElmer ERS Spinning disk system, a Digital CCD C4742-80-12AG camera (Hamamatsu), and controlled by Volocity 6.0.1 software. In each experiment, the number of cells and rupture events were quantified. Nuclear envelope rupture events were defined as the sudden efflux of mCherry-NLS into the cytoplasm and the sudden influx of AcGFP-NES into the nucleus without mitotic entry. Duration of each event was calculated as the time from marker influx/efflux until the mCherry-NLS began to accumulate in the nucleus again. To quantify mCherry-NLS signals over time, mean mCherry-NLS pixel intensity was measured by averaging four small circular regions manually placed in the nucleus or in the cytoplasm of a cell using ImageJ (https://imagej.nih.gov/ij/).

HeLa Kyoto cells stably expressing AcGFP-LAP2β and H2B-mCherry were transfected with siRNA and cultured for 50 h before recording. Prior to start of recording, the culture medium was changed to phenol-red free and CO₂ independent medium (Invitrogen). Images were acquired as 4 z-planes 3 μm apart every 3 min at 37 °C using a Nikon TE2000 microscope equipped with a Plan Fluor × 60/1.4 DIC H Lens (Nikon), a PerkinElmer ERS Spinning disk system, a Digital CCD C4742-80-12AG camera (Hamamatsu), and controlled by Volocity 6.0.1 software. Cell images were projected with z-stacks and analysed by ImageJ. For analysis of cell migration, MDA-MB-231 cells were transfected with either control or PPP1R12A-siRNA using Lipofectamine 2000 (Invitrogen). The following day the cells were trypsinized and seeded in CO₂ independent media (Invitrogen) in 24 well MatTek dishes (MatTek Corporation). Six to eight hours after seeding the cells were imaged for 20 h using phase contrast microscopy using an adapted Zeiss LSM510 microscope. Cell tracking was performed using the Manual Tracking plugin in Fiji software.