Cell profiler

Neurons were fixed for 10 min at room temperature in 4% paraformaldehyde in PBS. Permeabilization and blocking of nonspecific epitopes were performed simultaneously using 0.1% Triton X-100, 1% BSA, and 10% FBS in PBS for 45 min. Subsequently, the primary antibodies mouse anti-tubulin beta 3 (TUBB3) (1:1000, Cat# BLD-801202, Biolegend, San Diego, CA, USA), rabbit cleaved caspase 3 (CC3) (1:400, Cat# 9661S, Cell Signaling), rabbit LC3 (1:1000, Cat# NB600-1384, Novus Biological), and mouse FUS (1:500, Cat# AMAB90549, Sigma) were applied overnight at 4 °C in 0.1% BSA in PBS. The next day, the cells were washed with 0.1% BSA in PBS and incubated with the secondary antibody for 1 h at room temperature. Finally, cells were washed three times with 0.1% BSA in PBS containing 0.005% Tween-20, including Hoechst counter-staining for nuclei in the second washing step. Neurons were imaged with either a Zeiss ApoTome or a Zeiss LSM780/FCS laser scanning confocal microscope as indicated, and image analysis was performed with a Cell Profiler.

Normal and HGPS fibroblasts were transfected with GFP-SCP2 plasmid (a kind gift from Dr. Ralf Erdmann, RUB, Germany) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA). At 48 hours post transfection, cells were fixed and immunostained with PMP70 antibody. Zeiss RGB fluorescence images were separated into respective channels in CellProfiler and named accordingly. The peroxisomes were identified and segmented by a primary object identification module, whereas cell borders were manually defined with the manual free drawing object module. Peroxisomes intensity was constrained using Otsu threshold and segmented by intensity automatic settings. A relate object module was implemented to quantify the percentage of PMP70 puncta not colocalized with GFP-SCP2.