PACSIN2-wt or S313E/A were transiently transfected into proliferating human podocytes. Cells were fixed with 4% PFA, permeabilized with 0.1% Triton-X100 and stained when mentioned with CellMask Blue (H32720, Thermo Fisher Scientific, Waltham, MA, USA), Hoechst (33342, Merck), anti-β-tubulin III IgG (T2200, rabbit polyclonal, AB_262133, Merck), phalloidin-488 (1:250, A12379, Thermo Fisher Scientific) to stain filamentous actin (F-actin), c-myc (M4439, mouse monoclonal, Merck,) and paxillin (610051, mouse monoclonal; BD Transduction laboratories, Franklin Lakes, NJ, USA) to stain focal adhesions. Alexa Fluor 594 anti-rabbit IgG (A-21207, donkey polyclonal, Invitrogen) and Alexa Fluor 488 anti-mouse IgG (A21202, donkey polyclonal, Thermo Fisher Scientific)) were used as secondary antibodies. Imaging was carried out using the Opera Phenix HCS system (PerkinElmer, Waltham, MA, USA) with a 20× air objective (NA 0.4), followed by processing with CellProfiler 3.1.8 (https://cellprofiler.org/ accessed on 10 January 2023) [16 (link)] to correct for non-uniform illumination, detect the cells or focal adhesions and extract numerical features.
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