JCS03590 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Laminin is required for axonal growth in DRG neurons both in the presence and in the absence of NTFs. Polyornithine-precoated coverslips were further coated with laminin (0.1-0.3 μg/cm²) (upper panel) or used without laminin coating (lower panel). (A-D and F-I) Neurons were treated with NRTN or GDNF at shown concentrations and cultured for 12 hours. (E,J) Neurons were cultured for 40 hours without NTFs. Bar, 20 μm.

• Supplemental Figure S2 -

  Fig. S2. Roscovitine does not inhibit phosphorylation of Erk1/2 at Thr 202/Tyr 204. (A) NRTN and NGF (100 ng/ml each) were applied for 25 minutes at 20 hours in culture. Neurons were then fixed and stained for Erk1/2 phosphorylated at Thr202 or Tyr204. (B) Roscovitine (Rosc) (50 μM) was added 1 hour before the NTFs, otherwise conditions are the same as in A. (C) Sister cultures that were not stimulated with the NTFs exhibit very low background activation of pErk. Bar, 200 μm.

• Supplemental Figure S3 -

  Fig. S3. Neither SU6656 nor roscovitine significantly affect survival of cultured DRG neurons. The effect of SU6656 (SU) (2 μM) was assessed at 5 days in culture and the effect of roscovitine (Rosc) (50 μM) at 40 hours in culture. The inhibitors were applied when plating neurons. Initial number of neurons was counted at 12 hours in culture. Results expressed as a percentage of the initial value.

• Supplemental Figure S4 -

  Fig. S4. Roscovitine blocks axonal growth in the absence of NTFs at 90 hours. DRG neurons were plated on laminin-precoated substrate in the absence (A,C) or in the presence (B,C) of roscovitine (Rosc) (50 μM). Laminin was precoated at 50 ng/cm². Bar, 20 μm.

• Supplemental Figure S5 -

  Fig. S5. Laminin induces Cdk5 activation in DRG dissociated cultures. A clear activation of Cdk5 by laminin was observed in three independent experiments. Cultures were plated on laminin or in the absence of laminin precoating and maintained for 40 hours.