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. 2015 Mar 13;25(4):496–516. doi: 10.1038/cr.2015.31

Figure 8.

Figure 8

Endophilin A1-p140Cap regulates spine morphogenesis, synapse formation and synaptic function through cortactin. (A) Cultured hippocampal neurons were cotransfected with constructs expressing non-targeting Ctrl-shRNA or EENA1-shRNA and Flag vector, or with constructs expressing EENA1-shRNA and Flag-tagged cortactin at DIV16-17 followed by immunostaining with antibodies against PSD95 (green), Flag (blue) and DsRed at DIV21. Shown are representative confocal images. Scale bar, 5 μm. (B) Quantitative analysis of dendritic spine protrusion density of transfected neurons in A (number of cells analyzed, Ctrl-shRNA: 15, EENA1-shRNA: 14, EENA1-shRNA + cortactin: 18). More than 500 protrusions were measured for each group. All values are shown as mean ± SEM. Statistical test: ***P < 0.001, **P < 0.01; one-way ANOVA followed by Newman-Keuls multiple comparison post hoc tests. (C-E) Quantitative analysis of the fluorescence intensity (C), number (D) and area (E) of PSD95 puncta in dendrites in A (number of cells analyzed, Ctrl-shRNA: 23, EENA1-shRNA: 25, EENA1-shRNA + cortactin: 20; number of puncta analyzed, Ctrl-shRNA: 990, EENA1-shRNA: 578, EENA1-shRNA + cortactin: 923). More than 1 200 μm of dendrite length were analyzed for each group. All values are shown as mean ± SEM. Statistical test: ***P < 0.001; one-way ANOVA followed by Dunnett's multiple-comparison post hoc tests. (F) Cultured hippocampal neurons were cotransfected with constructs expressing non-targeting Ctrl-shRNA or EENA1-shRNA and Flag vector, or with constructs expressing EENA1-shRNA and Flag-tagged cortactin at DIV16-17 followed by immunostaining with the anti-SYP (green), anti-Flag (blue) and anti-DsRed antibodies at DIV21. Shown are representative confocal images. Scale bar, 5 μm. (G, H) Quantitative analysis of the SYP puncta number along dendrites (G) or percentage of dendritic area colocalizing with SYP (H) (number of cells analyzed, Ctrl-shRNA: 16, EENA1-shRNA: 18, + cortactin: 19). More than 650 μm of dendrite length were analyzed for each group. All values are shown as mean ± SEM. Statistical test: ***P < 0.001; one-way ANOVA followed by Dunnett's multiple-comparison post hoc tests. (I) Cultured hippocampal neurons were transfected with Ctrl-shRNA construct or cotransfected with constructs encoding EENA1-shRNA and Flag-tagged p140Cap or Flag-tagged cortactin at DIV9-10 followed by mEPSC recording at DIV14-16. Shown are representative traces of mEPSC recordings. (J) Quantitative analysis of mEPSC frequency and amplitude. n = 3 independent experiments, 27 neurons. Statistical test: *P < 0.05; one-way ANOVA followed by Dunnett's multiple-comparison post hoc tests. ns, not significant. (K) Cumulative distribution of mEPSC frequency. Statistical test: P= 0.023 for EENA1-shRNA versus Ctrl-shRNA, two-sample Kolmogorov-Smirnov test.