96 well cell carrier plates

A customized siRNA library targeting enzymes of the ubiquitination system was purchased from Dharmacon. HeLa cells stably expressing EGFP–KIND2-S181D were transfected with the siRNA using Lipofectamine RNAiMAX at a final concentration of 40 nM in 96-well cell carrier plates (Perkin Elmer) according to the manufacturer’s protocol. After 48 h of transfection, cells were fixed with 3% PFA, stained with DAPI and imaged in an Opera Phenix High-Content Screening system (Perkin Elmer) with a ×20 objective. At least five random fields of view were collected per well, and the GFP fluorescence intensities of 800–1,200 cells were quantified per well using Harmony (v.4.9).

Cells were plated on coverslips and fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). Cells were permeabilized using 0.5% Triton-X in PBS and blocked using 10% bovine calf serum in PBS, followed by immunostaining. Immunofluorescence images were obtained using a Nikon Eclipse 80i fluorescence microscope using NIS elements software to deconvolve and focus stacked images or the Operetta™ high content imager (PerkinElmer, Waltham, MA) as indicated.
For Operetta™ imaging, cells were plated in 96-well cell-carrier plates (PerkinElmer) and imaged in a single focal plane at ×20 magnification. Using Columbus™ (PerkinElmer) software, the protein intensity in the cytoplasm and nucleus were quantified. The nucleus and cytoplasm were defined by DAPI staining and CellMask™ Blue (Thermo Fisher Scientific) staining, respectively. The percentage of protein in the nucleus was calculated as the ratio of signal intensity present in the nucleus divided by the signal intensity present in the entire cell. The percentage of protein present in the nucleus was calculated for every cell and median values reported per well. To compare treatment conditions, the average of these median values was calculated for multiple wells within each independent experiment.