PtK1 cells were electroporated with EmeraldGFP-tagged LifeAct using the Neon transfection system and plated on acid-treated glass coverslips. Cells were cultured for 48-hours at 37 ° C, 5% CO2 and imaged using a Nikon Ti motorized inverted microscope with Perfect Focus System, Yokagawa CSU-X1 spinning disk confocal and Spectral Applied Research Borealis modification, 491 solid-state laser, 100× 1.45 N.A. objective, Hamamatsu ORCA-AG cooled CCD camera, and Metamorph software. Cells were in L-15 medium containing 10% FBS, 20 mM HEPES, and 0.03 U/ml Oxyrase. Experiments using U0126 were carried out using a Nikon TE2000 inverted microscope with Perfect Focus System, Yokogawa CSU-10 spinning disk confocal, 488 solid-state laser, 60× 1.4 N.A. objective, Andor Clara cooled CCD camera, and NIS Elements software.
Cells were imaged for 10 minutes before perturbation and 20 minutes following perturbation (30 min total). PtK1 cell boundaries were detected from the fluorescence microscopy data using a threshold method. Once identified, the edge was then subdivided into 1 micron long segments and their respective velocities were calculated as described previously (25 (link)). For inhibitor-treated experiments, results were obtained from 3–4 independent experiments. For BrafV600E overexpression experiments, results were obtained from 2 independent experiments.
Cells were imaged for 10 minutes before perturbation and 20 minutes following perturbation (30 min total). PtK1 cell boundaries were detected from the fluorescence microscopy data using a threshold method. Once identified, the edge was then subdivided into 1 micron long segments and their respective velocities were calculated as described previously (25 (link)). For inhibitor-treated experiments, results were obtained from 3–4 independent experiments. For BrafV600E overexpression experiments, results were obtained from 2 independent experiments.