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. 2004 Jul 5;166(1):17–25. doi: 10.1083/jcb.200405002

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Copyright © 2004, The Rockefeller University Press

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Figure 1. — Inhibition of neurite outgrowth by tomosyn. (A) Effect of overexpression of tomosyn. (Aa) Schematic structure of tomosyn. Gray box (WD), WD40 repeats domain. Black box (VLD), VAMP-like domain. (Ab) Primary cultured rat hippocampal neurons were transfected with HA-tomosyn or a null HA plasmid (Control) and were cultured for 24 h. NG108 cells were transfected with HA-tomosyn or a null HA plasmid (Control), cultured in DME containing 1 mM db-cAMP for 48 h, and allowed to extend neurites. Overexpressing cells were identified by immunostaining of HA (green), and their MT structures were examined by immunostaining of β-tubulin (red). Bars, 20 μm. (Ac) Quantitative analysis of the neurite length of NG108 cells. Percentage of neurites longer than 50 μm. The mean value (± SEM) of percentage of neurites longer than 50 μm from three independent experiments is shown. *, P < 0.01 (t test). (Ad) Quantitative analysis of the number of neurites per cell of NG108 cells. The number of cells, expressed as the percentage of 100 transfected cells bearing 1, 2, 3, or >4 neurites is shown. The mean value (± SEM) of three independent experiments is shown. (B) Effect of tomosyn knock-down by the RNA interference method. (Ba) NG108 cells were transfected with the plasmid expressing both tomosyn siRNA and GFP or the plasmid expressing both scramble siRNA and GFP (Scramble), cultured in DME containing 1 mM db-cAMP for 48 h, and allowed to extend neurites. The cells expressing tomosyn or scramble siRNA were identified by the expression of GFP (green). Their MT structures were examined by immunostaining of β-tubulin (red) and the extent of expression of tomosyn (blue) was examined by immunostaining of tomosyn. Bars, 20 μm. (Bb) Quantification of the effect of tomosyn knock-down. The fluorescence intensity of tomosyn of the NG108 cells expressing GFP were compared with that of the untransfected cells in the same field of view. (Bc) Quantitative analysis of the neurite length of NG108 cells as in Ac. *, P < 0.01. (Bd) Quantitative analysis of the number of neurites per cell of NG108 cells as in Ad.

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