Fig. 3. Inhibiting RhoA signaling increases DCC-dependent SCN axon outgrowth evoked by netrin 1. E13 rat dorsal spinal cord explants were cultured for 14 hours with various amounts of netrin 1 in the presence of 10 µg/ml C3-07 or 10 µM Y27632. (A-I) SCN axons were labeled using the 4D7 monoclonal antibody against Tag1. (J) C3-07 increased total outgrowth per explant by 155% (n=5, P=0.006) at 50 ng/ml netrin 1, 66% (n=4, P=0.041) at 200 ng/ml netrin 1 and 126% (n=4, P=0.003) at 600 ng/ml netrin 1. Y27632 increased outgrowth by 404% (n=5, P<0.001) at 50 ng/ml netrin 1, 137% (n=5, P<0.001) at 200 ng/ml netrin 1 and 236% (n=5, P=0.000) at 600 ng/ml netrin 1 (J). (K) DCC-fb antibody (5 µg/ml) significantly reduced axon outgrowth in the presence of C3-07 or Y27632 at each netrin 1 concentration (n=5, P<0.01). Specifically, total outgrowth with C3-07 was reduced by 74% (n=5, P=0.001) at 50 ng/ml netrin 1, by 70% (n=5, P<0.001) at 200 ng/ml netrin 1 and 79% (n=5, P<0.001) at 600 ng/ml. Total outgrowth with Y27632 was reduced by 57% (n=5, P<0.001) at 50 ng/ml netrin 1, 55% (n=5, P<0.001) at 200 ng/ml netrin 1 and 93% (n=5, P<0.001) at 600 ng/ml netrin 1. ^*P<0.05, ^**P<0.01. Tukey post-hoc tests of means. Error bars indicate s.e.m. Scale bar: 100 µm.