Figure 1: Establishment of a culture-and-replate protocol to investigate mechanisms of regenerative axon growth in vitro.

(a) Schematics of the culture-and-replate protocol. DRG neurons were dissociated from adult mice and cultured for 3 days. Neurons were then replated to initiate axon growth anew. Regenerative axon growth was assessed by measuring axon length from replated neurons at 20 h after replating. (b) In vitro culture recapitulates peripheral axotomy-induced upregulation of several proteins that are encoded by well-known RAGs such as ATF3, c-Jun, GAP43, GADD45a and SPRR1a. Shown are representative immunoblots from DRG neuronal lysates cultured for either 3 h or 3 days. Actin antibodies were used as a loading control. (c) DRG neurons grow long, sparsely branched axons after replating, similar to neurons that were conditioned in vivo by sciatic nerve transection. Note that the mode of axon growth after replating is distinct from that induced by NGF (50 ng ml⁻¹), which stimulates extensive branching but relatively modest lengthening. Scale bar, 200 μm. (d,e) Adult DRG neurons were dissociated, cultured for 3 days and then replated to induce axon growth anew, as depicted in a. Neurons were treated with DBR (20 μM), nocodazole (50 nM) or vehicle control (DMSO) either before (magenta bars in e) or after replating (blue bars in e), as indicated. Schematics (upper left insets in d) depict when the neurons were treated with drugs of interest. Coloured bars indicate the period when neurons were exposed to drugs. White, grey and black arrowheads indicate the starting point of the initial 3-day-culture period, the time of replating and when neurons were fixed for the analysis, respectively. Regenerative axon growth was assessed by measuring axon length after replating. Representative images neurons after replating are shown in d. Quantification of axon length from three independent experiments is shown in e. Scale bar, 200 μm. Error bars represent s.e.m. ***P<0.001, unpaired two-tailed Student’s t-test. Original immunoblot images are shown in Supplementary Fig. S10.