Figure 4: Regenerative axon growth from adult sensory neurons requires mTOR-independent protein synthesis.

(a,b) Adult DRG neurons were dissociated, cultured for 3 days and then replated to initiate axon growth anew, as depicted in Fig. 1a. Neurons were treated with rapamycin (20 nM), cycloheximide (5 μg ml⁻¹) or DMSO (control) either before (magenta bars in b) or after replating (blue bars in b), as indicated, and axon length was measured at 20 h after replating. The schematics and symbols (upper left insets in a) are identical to Fig. 1d. Representative images of replated neurons are shown in a and quantification of axon length from three independent experiments is shown in b. Scale bar, 200 μm. Error bars represent s.e.m. n.s., statistically insignificant difference, unpaired two-tailed Student’s t-test. (c) Representative images of control (upper panels) and rapamycin-treated (lower panels) adult DRG neurons immunostained with TuJ1 and phospho-S6 antibodies. Note that rapamycin treatment completely prevented S6 phosphorylation but had no effect on axon growth. Scale bar, 200 μm. (d) Representative immunoblots from adult DRG neurons cultured for 3 days in the presence or absence of rapamycin. Actin antibodies were used as a loading control. Note that rapamycin treatment completely prevented S6 phosphorylation. Original immunoblot image is shown in Supplementary Fig. S11.