Fig. 2
From: The small GTPase ARF6 regulates GABAergic synapse development
Effects of ARF1 or ARF6 KD on synaptic structures in cultured hippocampal neurons. a Design of lentiviral shRNA vectors for KD of ARF1 or ARF6. Boxes indicate shRNA target sequences in Arf1 and Arf6. Abbreviations: H1, human H1 promoter; IRES, internal ribosome entry sequence; Ub, ubiquitin promoter. b Arf1 and Arf6 mRNA levels in cultured cortical neurons infected at DIV3 with lentiviruses expressing sh-Arf1 or sh-Arf6 were measured by qRT-PCR. mRNA was prepared at DIV10. Dashed line, 85% KD cutoff level for tests of biological effects. Data are presented as means ± SEMs (n = 3 independent experiments; *p < 0.05 vs. control; Mann-Whitney U test). c Cultured cortical neurons were infected with lentiviruses expressing sh-Arf6 at DIV3 and then immunoblotted with the indicated antibodies at DIV10. d Quantification of ARF6, IQSEC3, gephyrin, and PSD-95 levels from c, normalized to control. Data are presented as means ± SEMs of three experiments (***p < 0.001 vs. control; Mann-Whitney U test). e Representative images of cultured hippocampal neurons transfected at DIV8 with lentiviral constructs expressing EGFP alone (Control), sh-Arf1, sh-Arf6 or cotransfected with sh-Arf1 and sh-Arf6 (sh-Arf1/Arf6). Neurons were analyzed by double-immunofluorescence labeling for EGFP (blue; pseudo-colored) and VGLUT1, PSD-95, GAD67, gephyrin or GABAARγ2 (red) at DIV14. Scale bar, 10 μm (applies to all images). f, g Summary data showing the effects of ARF1 KD, ARF6 KD or ARF1 and ARF6 DKD (double-knockdown) in neurons on synaptic puncta density (f) and synaptic puncta size (g). Data are presented as means ± SEMs (2–3 dendrites per transfected neurons were analyzed and group-averaged; n = 22–30 neurons; *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; non-parametric ANOVA with Kruskal-Wallis test followed by post hoc Dunn’s multiple comparison test)