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. 2009 Apr 1;29(13):4312–4321. doi: 10.1523/JNEUROSCI.0126-09.2009

Figure 6.

Figure 6.

Ccd1 promotes axon growth. A, Lysates of 293T cells transfected with the U6, U6/ccd1i, pSuper, or pSuper/ccd1i RNAi plasmid together with a plasmid expressing Flag–Ccd1 were immunoblotted with the Flag or ERK1/2 antibody. Expression of shRNAs targeting distinct Ccd1 sequences (U6/ccd1i and pSuper/ccd1i) induced knockdown of Ccd1. B, Granule neurons were transfected with the control U6 (top) or U6/ccd1i RNAi plasmid (bottom) together with a GFP expression plasmid. Five days after transfection, granule neurons were subjected to immunocytochemistry using the GFP antibody. Ccd1 knockdown neurons had shorter axons than control-transfected neurons. Arrowheads, arrows, and asterisks indicate axons, dendrites, and cell bodies, respectively. Scale bar, 100 μm. C, Morphometric analyses of granule neurons transfected and analyzed as in B 3 and 5 d after transfection. Quantification of axon length is shown as mean + SEM. Axon length was significantly reduced in Ccd1 knockdown neurons compared with control-transfected neurons at 5 d in vitro (ANOVA; p < 0.05). Total number of neurons measured = 501. D, Granule neurons were transfected with the control pSuper plasmid (top) or the pSuper/ccd1i RNAi plasmid (bottom) together with the GFP expression plasmid and analyzed as in B. Ccd1 knockdown neurons had shorter axons than control-transfected neurons. E, Morphometric analyses of neurons transfected and analyzed as in D 3 and 5 d after transfection. Axon length was significantly reduced in Ccd1 knockdown neurons compared with control-transfected neurons at 3 and 5 d in vitro (ANOVA; p < 0.05). Total number of neurons measured = 345. F, Granule neurons transfected with the U6, U6/ccd1i, pSuper, or pSuper/ccd1i RNAi plasmid together with the GFP expression plasmid were subjected to immunocytochemistry using the GFP antibody followed by morphometric analysis of total dendrite length. No significant difference in total dendrite length between Ccd1 knockdown or control-transfected neurons was found. Total number of neurons measured = 360.