41966) supplemented with 10% fetal bovine serum (FBS), 5% horse serum, 2 mm l-glutamine and 1% penicillin–streptomycin–fungizone (all supplements from Invitrogen). Cells at 80-90% confluency were transfected with the EGFP, KCC2-FL, KCC2-ΔNTD and KCC2-C568A expression vectors using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. At 24 or 48 h after transfection, cells were fixed with 4% paraformaldehyde and then permeabilized and blocked in 7% non-fat dry milk and 0.1% Triton

X-100 in PBS. Incubation with primary antibodies was done at 4°C overnight. See Table 1 for antibody details. The following day, the cells were rinsed and secondary antibodies were incubated for 1.5 h. Endogenous actin was visualized with FITC- or TRITC-phalloidin (Sigma-Aldrich) diluted to 50 μg/mL in the same solution as the secondary antibody. Thereafter the cells were rinsed Ferroptosis mutation in PBS and mounted in Vectashield Hard Set mounting medium (Vector Laboratories), before analysis by fluorescent (Zeiss AxioExaminer D1; 40 × objective) or confocal (Leica TCS-SP;

40 × objective) microscopy. Transfected C17.2 cells were extracted in ice-cold lysis buffer [50 mm Tris, pH 7.4, 150 mm sodium chloride, 1% NP-40, 1 mm EDTA and 1 ×  protease inhibitor cocktail (Roche)] and the extracts were incubated with R428 3 μg of a rabbit (Upstate) or monoclonal (NeuroMab) KCC2 antibody. Immunoprecipitates were collected on Protein G Sepharose Fast flow beads (GE Healthcare Biosciences, Uppsala, Sweden) by overnight rotation, washed with lysis buffer, resuspended in 2 × Laemmli sample buffer, and subjected to SDS-PAGE followed by Western

blot analysis using anti-4.1N and anti-KCC2 antibodies at a 1 : 2000 dilution. This method has been described previously (Lindqvist et al., 2010; see also Liang et al., 2007). Briefly, subconfluent C17.2 cells were transfected and then allowed to reach 100% confluency. The cells were then treated with 10 μm Mitomycin C (Sigma-Aldrich) for 3 h to arrest the cell cycle. A scratch was introduced through the cell layer using a pipette tip. The medium was changed to serum-reduced (1% FBS) to keep the cells Idoxuridine from dividing, and a line was drawn underneath the culture dish perpendicular to the scratch. Pictures were taken just above or below the line under a light phase-contrast microscope (Nikon Eclipse TE200; 10 × objective), immediately (T = 0) and after 18 h (T = 18 h). For quantification of β-tubulin III/TuJ1, phospho-histone-3, doublecortin, PSA-NCAM and Caspase-3 (Fig. 4 and Supporting information, Fig. S3), the length and width of the neural tube was measured based on micrographs using the measuring tool in Adobe Photoshop CS (Adobe Systems Inc., San Jose, CA, USA). Positive cells were counted manually and a mark was made on each cell to avoid double counting. The number of cells was divided by the total area of the neural tube. The area unit for the neural tube measurements is mm2.