The Journal of Neuroscience, May 3, 2006, ():
ARF6 and EFA6A Regulate the Development and Maintenance of Dendritic Spines
J. Neurosci. Choi et al.
26: 4811
Supplemental data
Files in this Data Supplement:
- supplemental material
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Figure S1. The C-terminal (PH+C) region of EFA6A causes an increase in filopodia density in early neurons. A, Cultured hippocampal neurons at DIV 6 were transfected doubly with FLAG-EFA6A PH+C and empty pSUPER.gfp/neo (used as a source of EGFP), or singly with pSUPER.gfp/neo alone, and stained at DIV 9 for EGFP and FLAG. Scale bar = 5 µm. B-D, Quantitation of the effect of EFA6A PH+C on the linear densities of spines, filopodia, and total protrusions. EGFP alone (protrusion density, 34.04 ± 1.02; spine density, 22.81 ± 1.00; filopodia density, 11.23 ± 0.62); EFA6A PH+C (protrusion density, 36.69 ± 1.18; spine density, 21.63 ± 1.08; filopodia density, 15.05 ± 0.80). Data were collected from 800-1200 protrusions on 27-45 dendrites of 31-42 neurons for each group in the two independent experiments. ***p < 0.001 (Student’s t-test).
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Figure S2. Characterization of EFA6A and ARF6 siRNA constructs. A, siRNA-induced reduction in the expression levels of exogenous EFA6A and ARF6 in heterologous cells. HEK293T cells were doubly transfected with combinations of expression constructs (FLAG-EFA6A or ARF6-HA; wild type or siRNA-resistant rescue) + siRNA constructs (pSUPER.gfp/neo EFA6A, ARF6, or alone). Expression levels of EFA6A and ARF6 proteins were characterized by immunoblotting with FLAG and HA antibodies, respectively. EGFP and α-tubulin were blotted for normalization. B, Quantitation of results from (A). Relative expression levels of EFA6A and ARF6 compared to controls (100%; expression constructs only) are indicated. si-EFA6A, 36.07 ± 2.00%; si-EFA6A + Rescue, 100.87 ± 4.83%; si-ARF6, 5.16 ± 1.56%; si-ARF6 + Rescue, 85.41 ± 0.52%. C, EFA6A siRNA reduces EFA6A expression in cultured neurons. Cultured hippocampal neurons were transfected at DIV 13 with pSUPER.gfp/neo alone, pSUPER.gfp/neo EFA6A, or pSUPER.gfp/neo EFA6A + siRNA-resistant rescue construct, and double-labeled at DIV16 for EGFP and EFA6A. Scale bar = 10 µm. D, ARF6 siRNA reduces ARF6 expression in cultured neurons. Cultured neurons were transfected at DIV 13 with pSUPER.gfp/neo ARF6, or pSUPER.gfp/neo alone, and double-labeled at DIV 16 for EGFP and ARF6. Scale bar = 10 µm. E-F, Quantitation of siRNA-induced knockdown of endogenous EFA6A and ARF6 in neurons. AU, arbitrary unit. EFA6A siRNA knockdown (si-vec, 165.03 ± 8.55, n=12 neurons; si-EFA6A, 12.75 ± 3.36, n=12 neurons ; si-EFA6A+Rescue, 232.85 ± 9.88, n=9 neurons); ARF6 siRNA knockdown (si-vec, 121.58 ± 3.38, n=12 neurons; si-ARF6, 22.75 ± 3.40, n=14 neurons). G, siRNA-induced reduction in the expression level of endogenous ARF6 in HEK293T cells. HEK293T cells were transfected with pSUPER.gfp/neo ARF6, or pSUPER.gfp/neo alone, using Lipofectamine 2000. Expression levels of endogenous ARF6 protein were characterized by immunoblotting with ARF6 (1654) antibody. EGFP and α-tubulin were blotted for normalization. H, Quantitation of the results in (G). Relative expression level of endogenous ARF6 compared to control (100%; pSUPER.gfp/neo alone) is indicated. si-ARF6, 24.94 ± 2.44%, n=3, ***p < 0.001 (Student’s t-test).
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Figure S3. Enhanced filopodia proliferation by the C-terminal (PH+C) region of EFA6A in early neurons. A, Cultured hippocampal neurons at DIV 6 were transfected singly with si-EFA6A, or doubly with si-EFA6A + FLAG-EFA6A PH+C (resistant to si-EFA6A), and stained at DIV 9 for EGFP and FLAG. Scale bar = 5 µm. B, Quantitation of the effect of EFA6A PH+C on the linear density of dendritic protrusions. si-vec (34.04 ± 1.02); si-EFA6A (30.58 ± 1.23); si-EFA6A + (PH+C) (36.06 ± 1.17). Data were collected from 600-1200 protrusions on 22-45 dendrites of 20-42 neurons for each group in the two independent experiments. **p < 0.01 (one-way ANOVA).
