Mouse α smn

48 h before the experiment, 1.5 × 10⁴ cells were plated onto optically clear bottom 96-well plates (CellCarrier-96, PerkinElmer). Cells were treated with 5 mM EGTA or 2 mM CaCl₂ for 16 h before the experiment. Cells were washed once using PBS, fixed with 4% (v/v) paraformaldehyde for 12 min at room temperature (RT), permeabilized in phosphate-buffered saline (PBS) containing 0.3% (v/v) Triton X-100 and blocked in 5% (w/v) bovine serum albumin (BSA) for 60 min. Cells were incubated with the following primary antibodies overnight in 4°C: mouse α-SMN (BD Transduction Laboratories), 1:200; rabbit α-LC3B-II (Cell Signaling), 1:200. After washing three times with PBS, cells were incubated with Alexa Fluor secondary antibodies (Invitrogen) for 2 h at RT. Alexa Fluor 647 Phalloidin (ThermoFisher) was added to the secondary antibodies for staining of the fibroblasts’ membrane and segmentation purposes in the open-source software image analysis, CellProfiler 4.0.6 (https://cellprofiler.org/) using in-house pipelines. Nuclei were stained with 300 nM 4,6-diamidino-2- phenylindole (DAPI, Molecular Probes). Cells were visualized using a Leica SP8 confocal microscope equipped with a motorised stage for automated acquisition of images, that were acquired at ×20 magnification and ×2.5 digital zoom.

Samples, SMA4 patient (proband II:2) and controls (n = 3), were prepared with 4x Laemmli sample buffer (BioRad) and 2-mercaptoethanol (Sigma) solution 9:1 (v/v). Cell lysates (15 μg) were size fractioned using mini-PROTEAN TGX Precast Gels (BioRad) and then transferred to an Immobilon®-P Polyvinylidene difluoride membrane (Sigma). Membranes were probed with the following primary antibodies: rabbit α-CAPN1 (CST), 1:1,500; mouse α-SMN (BD Transduction Laboratories), 1:2000; rabbit α-p-Akt (CST), 1:1,000; mouse α-Pan-Akt (CST), 1:2000; rabbit α-LC3B-II (CST) 1:1,000; rabbit α-β-actin (CST) 1:2000; rabbit α-caspase-3 (Abcam) 1:4,000 and rabbit α-β-actin (CST) 1:2000. Secondary antibodies used in this study include goat α-rabbit IgG HRP (Sigma), 1:10,000 and goat α-mouse IgG HRP (abcam) 1:5,000. Immobilon™ Western Chemiluminescent HRP Substrate Reagent (Millipore) was added and protein bands were visualized using a ChemiDoc™ MP Imaging System (BioRad).