FIGURE 7.

The requirement for Gas7 in the neurite outgrowth of hippocampal neurons. A, knockdown of the endogenous Gas7 in NIH3T3 cells with shGas7 and of N-WASP in Neuro-2a cells with shN-WASP is shown. Cell extracts were prepared from shGas7/GFP-transfected NIH3T3 cells or shN-WASP/GFP-transfected Neuro-2a cells after 48 h of transfection and assayed by Western blot with GFP as a negative control. Both endogenous Gas7 and N-WASP expression were significantly reduced by small hairpin transfection. B, shown is the time-course of Gas7 and N-WASP expression in cultured hippocampal neurons. Lysates were prepared from cultured embryonic hippocampal neurons at the indicated days in vitro (DIV) and assayed by Western blot. N-WASP is expressed constantly from DIV 1–28, peaking after 2 weeks. In contrast, Gas7 expression begins during DIV 3–5 and then continues, with one peak in the second week and a second peak between weeks 3 and 4. C, immunostaining of Gas7-knockdown hippocampal neurons is shown. Cultured hippocampal neurons were transfected with GFP or shGas7/GFP at DIV2 and immunostained at DIV5. Neurite outgrowth was aberrant in Gas7-knockdown neurons as compared with neurons expressing Gas7 normally (GFP panel or the star in shGas7/GFP-transfected panel). Scale bar, 20 μm. D, quantification of morphological changes in neurite outgrowth from C. The ratio of longest neurite length to cell body diameter decreased significantly (shGas7/GFP (n = 42) versus control GFP (n = 45); unpaired Student's t test; ***, p < 0.001). E, quantification of morphological changes in neurite outgrowth is shown. Cultured hippocampal neurons were transfected with GFP, shGas7/GFP, or shN-WASP/GFP at DIV2 and immunostained at DIV4. The ratio of longest neurite length to cell body diameter decreased significantly (shGas7/GFP (n = 28) or shN-WASP/GFP (n = 23) versus control GFP (n = 25); unpaired Student's t test; **, p < 0.01).