Visualizing PCM-1 Dynamics in RPE-1 Cells

Mock‐ or EHD1‐siRNA‐treated RPE‐1 cells on 35 mm glass‐bottom tissue culture dishes (MatTek, Ashland, MA) were transiently transfected with PCM‐1‐GFP for 24 h, and then serum‐starved in DMEM/F12 with 2 mM L‐glutamine, 1× nonessential amino acids and 0.2% FBS for 15 min at 37°C. The cells were imaged in prewarmed Opti‐MEM medium (Gibco) containing 2 mM L‐glutamine with a Zeiss LSM 800 confocal microscope (Carl Zeiss, Jena, Germany). Images were obtained every 5 s for 10 min with the pinhole set to obtain z‐sections of 1 μm. Images and videos were cropped, adjusted for brightness (whole‐image adjustment), and time‐stamped with minimal manipulation for presentation. For quantification, we performed three independent experiments. pEGFP‐N‐PCM1 (Dammermann & Merdes, 2002 (link)) was a kind gift from Dr. Andreas Merdes (Université Toulouse III, Toulouse, France).