Neurite Growth and Cell Death Assays

Neuronal Neuro2a (N2a) and HeLa cells were cultures in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with glutamax, 1% penicillin-streptomycin, and 10% fetal calf serum (all Gibco). Cells were co-transfected with Fugene 6 (Promega) for 48 h as per the manufacturer’s protocol. shRNA constructs were from Sigma (#11857 for Gpi1 and as previously described for Oxr1 [14 (link)]). For neurite growth assay, medium was replaced with serum-free medium when transfecting and incubated for 48 h. For cell death assay, cells were treated with 250-μM arsenite (Sigma) for 4 h. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS (Sigma) twice, and blocked with blocking buffer (5% goat serum (VectorLab), 0.5% Triton X-100 (Sigma)) for 1 h at room temperature. Neurites and pyknotic nuclei were visualised with NF200 antibody (Sigma N4142) and DAPI staining (Vectorlabs), respectively.