Figure 5. Sprouty4 inhibits neurite outgrowth of dorsal root ganglion neurons (DRG) in response to NGF.

A) Dissociated DRG neurons transfected with GFP in the absence (Control) or in the presence of an excess of Myc-tagged Sprouty4 (Spry4) construct were cultured with NGF (50 ng/ml). After 36 h in culture, neurons were fixed and stained with anti–tubulin antibodies. Scale bar represents 20 m. Arrows indicate neuronal cell bodies and arrowheads denote neurite tips. B) Left panel, histogram showing the inhibition of neurite outgrowth in DRG neurons by exogenous expression of Sprouty4. The results are averages SEM of a representative experiment measured in six wells per experimental group, *, p<0.05 (Student's t test). The experiment was repeated three times with similar results. Right panel, histogram showing the survival of DRG neurons by exogenous expression of Sprouty4. Neuronal survival was evaluated using the nuclear staining DAPI. GFP-positive neurons containing fragmented or condensed nuclear staining were scored as apoptotic cells. The results are averages SEM of a representative experiment performed in triplicate. C) Histogram shows the distribution of neurons carrying neurites in different length categories after transfection with GFP in the absence (Control) or in the presence of Myc-tagged Sprouty4. A total of 43 control- and 40 Sprouty4-transfected neurons from a representative assay were evaluated. Note the noticeable shift to the left of the distribution of neurons that received the Sprouty4 construct.