Pico high content imager

Images were acquired at 10× in a Pico high-content imager (Molecular Devices, San Jose, CA, USA) and stitched as described for neurite tracing. Stitched images were then analyzed with a custom script written in the MetaXpress image-analysis software (Molecular Devices, San Jose, CA, USA, version 6.6.2.46) as follows. GFP-positive cells were identified using the “auto-threshold” tool in the GFP channel. The threshold was set to ≥ 3 times the background intensity. The resulting GFP-positive area mask was then overlaid on the SOD1 channel (i.e., to measure SOD1 intensity only in cells with positive GFP signal, and therefore transduced with the lentiviral constructs). The SOD1 intensity corresponding to the GFP-positive mask was calculated as a total intensity value for each well. The total intensity for each well was then divided by the total area of the GFP+ mask to control for different numbers and different sizes of GFP-positive cells. Cells expressing Nb61 or Nb61-PEST were compared to cells expressing GFP alone (n = 3 technical replicates) using GraphPad Prism (v9.3). The analyst was blinded to the conditions.

Images were acquired at 10× in a Pico high-content imager (Molecular Devices, San Jose, CA, USA) and stitched to create one image containing three fields of view across the well of a 384-well plate. Stitched images were then analyzed with a custom neurite-tracing script written in the MetaXpress image-analysis software (Molecular Devices, San Jose, CA, USA, version 6.6.2.46). In brief, neurite detection was set to be ≥ 3 times the intensity of the background, to be in the width range of 0–5 μm, and to be at least 2 μm long to be counted. All calculated lengths were summed across the stitched fields, covering approximately 50% of the area of a well of a 384-well plate. The analyst was blinded to the conditions.