Fig. 6

NOVA-dependent SEPT8 isoform promotes dendritic arborization and spine morphogenesis. a Detection of palmitoylated SEPT8-X1 by acyl-RAC (as illustrated in Additional file 2:Figure S7A). Top: C-terminal amino acid sequence of SEPT8-X1. Orange box highlights the FIM, with green letters marking palmitoylated cysteines and nearby basic amino acid residues. C469 and C470 were mutated to serine residues in SEPT8-X1-mut. Bottom: COS-1 cells expressing HA-tagged SEPT8 variants were used for the acyl-RAC assay. Immunoblotting of supernatant and thiopropyl-sepharose captured fractions using an antibody against HA is shown. Ten percent of supernatants were loaded compared to the captured fractions. b–d Representative maximum projected confocal images of hippocampal neurons expressing shRNAs and HA-tagged SEPT8 or controls. GFP, which is co-expressed from the shRNA constructs, is used as a neurite tracer. Anti-HA labels exogenously expressed SEPT8 variants. Representative images of dendrites are shown at the bottom of each panel. Scale bar represents 100 μm. e, f Boxplots evaluating dendritic arbor complexity (e) and dendritic spine density (f) in neurons with SEPT8-X1 or NOVA2 KD and SEPT8 rescue. Number of dendritic branching points, total dendritic lengths, and sholl analysis critical values are plotted in e, and number of dendritic spines per 100 μm is plotted in f. Measurements in neurons expressing the control, shX1, and shNova2 shRNAs are highlighted by the gray, pink, and blue boxes, respectively. Quantification was based on 15–17 neurons per group