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. Author manuscript; available in PMC: 2013 Mar 1.
Published in final edited form as: J Neurochem. 2012 Feb 6;120(6):974–984. doi: 10.1111/j.1471-4159.2012.07651.x

Figure 3.

Figure 3

Effect of silencing GIVA PLA2 expression on Sema3A-induced growth cone collapse. All experiments included a GFP-encoding plasmid to identify transfected cells (dissociated DRG neurons). A, the first panel shows GFP labeling of the growth cone of a transfected neuron (red arrow). The white arrow points at a growth cone of a non-transfected neuron. The second and third panels show phase contrast micrographs of the first frame (0 min exposure to Sema3A) and of the same growth cones after 15 min of Sema3A exposure, respectively. Note enlargement of the transfected growth cone versus area reduction of the non-transfected growth cone. Scale bar 10μm. B, GFP labeling of the same transfected growth cone at three different experimental points: t=0 (first panel); 15 min Sema3A challenge (second panel); and t=30 min, i.e., 15 min Sema3A treatment followed by 15 min 10−8M 12(S)-HETE challenge (third panel). Note that reduction in growth cone size occurs not after Sema3A, but after subsequent 12(S)-HETE challenge. Scale bar 10μm. C, quantitative analysis of growth cone area after transfection and challenge with either Sema3A or 12(S)-HETE. The actual growth cone sizes (in μm2 ± SEM; for n values, see bottom of the graph) at the onset of the challenge are indicated on the top. While the siGIVA growth cones were more variable in size, on average they were not significantly different from those from GFP-only and siScrambled neurons. These same growth cones were subsequently challenged. To assess challenge-induced change, growth cone areas were measured at t=0 min and t=15 min after onset of the challenge. Results are expressed as mean % change in growth cone area ± SEM. Values for n are indicated below the graph. Because the distribution of data was marginally normal, p values from both parametric (ANOVA, FDR) and non-parametric (K-W, Dunn’s procedure) statistical analyses are included in the grey rules whose ends designate the individual measurements to be contrasted (p ANOVA/p K-W). The overall p value was <0.0001 in both analyses.