Down-regulation of APC in hippocampal neurons. (A and B) DIV14 rat hippocampal neurons treated with control (sh Control) or APC-targeting (APC sh1) shRNAs and stained with phalloidin and APC antibody. (A) Fluorescence intensities were adjusted identically in control (top) and APC shRNA-treated (bottom) images. (B) Enlarged boxed regions from A showing growth cones from control and APC-depleted neurons. The remaining growth cones in APC-depleted neurons are associated with low residual levels of APC. Fluorescence intensity of APC and phalloidin staining in growth cones of APC-depleted neurons (bottom panels) was adjusted to higher levels as compared with control growth cones (top panels) to show localization of the low remaining APC and F-actin signals. (C) Representative images of phalloidin-stained dendritic and axonal growth cones (GC) from DIV6 rat hippocampal neurons treated with control (sh Control) or APC-targeting (APC sh1) shRNAs. (D) Average fluorescence intensity of phalloidin staining in axonal and dendritic growth cones of control and APC-depleted rat hippocampal neurons at DIV6; mean ± SD; n is shown in parenthesis for each sample; ****, P < 0.0001; ***, P < 0.001; ns, not significant. Kruskal–Wallis multiple comparisons test with post hoc Dunn’s test. (E and F) DIV6 mouse hippocampal neurons from wild-type (E) and floxed APC^580S/580S mice (F). APC^580S/580S neurons were transfected with Cre recombinase. Scale bars (apply to all images within indicated panels), 10 µm (A, E, and F) and 5 µm (B and C).