Supplementary Figures and Legends.

Transcription

1 Supplementary Figures and Legends.

2 Supplementary Figure 1: Impact of injury on Rb1 and PPARϒ expression. Following ipsilateral axotomy injury, adult DRG expression of Rb1 mrna declined (*p<0.05; n=3; paired two-tailed Student s t-test) (a). There was a nonsignificant trend toward higher PPARϒ mrna expression (NS; n=3; paired two-tailed Student s t-test). Values are means±sem. Western immunoblot showing a decline in DRG protein expression of Rb following injury and a corresponding rise in PPARϒ (N-normal; I-3d injured) (b).

3 Supplementary Figure 2: Full scan images of western blot data in Figures S1 and S4

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5 Supplementary Figure 3: Neurite plasticity following Rb1 sirna knockdown also occurs in adult sensory neurons of mice. Dissociated uninjured (non-preconditioned) adult sensory neurons from adult mice were exposed to scrambled sequence sirna or Rb1 sirna (30 nm) for 36h and stained with an antibody to neurofilament. Examples are illustrated of neurons exposed to control scrambled sequence or Rb1 sirna (a) [Bar=100 μm]. Exposure to Rb1 sirna was associated with rises in neurite outgrowth (b), branching (c), maximum neurite growth (d) and the number of primary neurites (from the soma) (e) [*p<0.05 for b,c; n=4; unpaired two-tailed Student s t-test and *p<0.05 for d,e; n=4,upaired one-tailed Student s t-test]. Values are means±sem.

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7 Supplementary Figure 4: Retinoblastoma knockdown is associated with E2F1 dependent outgrowth, but not Phopho-H2A.X expression. Rb1 sirna is associated with a dose dependent knockdown of Rb1 protein in dissociated adult sensory neurons (a). Phospho-H2A.X levels in control sirna and Rb sirna treated rat primary sensory neuronal cells. Note that there was no difference between neurons exposed to scrambled sequence sirna (control) compared to those exposed to Rb1 sirna. In contrast, increased expression was observed in neurons exposed to cisplatin. The numbers of neurons analyzed is given below the columns (b). Further analysis in Ad- GFP-2A-iCre transfected Rb1 floxed neurons compared to Ad-GFP (control) Rb1 floxed neurons such that only GFP labelled neurons were examined for neurite outgrowth (c) [*p<0.05, n=3; paired one-tailed Student s t-test] and branch number (d) [*p=0.05, n=3; paired one-tailed Student s t-test]. Impact of combined Rb sirna and E2F1 sirna on neurite outgrowth. Addition of E2F1 sirna abrogated the increased neurite outgrowth (e) [*p<0.05, n=4; unpaired one-tailed Student s t-test] and branch number of neurons exposed to Rb1 sirna (f) [*p<0.05, n=4; unpaired two-tailed Student s t-test]. PPARϒ mrna levels in mice with ipsilateral Rb sirna knockdown in mice described in Figure 9 with crush and exposure to either scrambled sequence sirna or Rb1 sirna over 7 days. There was a nonsignificant trend [ns; n=5 scrambled, n=6 Rb sirna; unpaired two-tailed Student s t-test] toward higher DRG PPARϒ expression after exposure to Rb1siRNA (g). Values are means±sem.

8 Supplementary Figure 5a: Full scan images of western blot data in Figures 6a,b

9 Supplementary Figure 5b: Full scan images of western blot data in Figures 6 c,d

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11 Supplementary Figure 6: Neurite plasticity following Rb1 knockdown is blocked by a PPARϒ antagonist. Dissociated uninjured (non-preconditioned) adult sensory neurons exposed to the second of two differing Rb1 sirna constructs providing similar findings (see Figure 5 for data on sequence #1). Bars indicate control neurite outgrowth, Rb1 sirna outgrowth and Rb1 sirna outgrowth with GW9662 at 0.1 or 1.0 μm. Rb1 sirna is associated with rises in total neurite outgrowth (a) [*p<0.05; n=3; ANOVA p<0.001; post hoc Tukey], greater numbers of primary neurites (b) [*p<0.05; n=3; ANOVA p<0.005; post hoc Tukey], longer neurites (c) [*p<0.05; n=3; ANOVA p<0.001; post hoc Tukey] and rises in the number of neurite branches (d) [*p<0.05; n=3; ANOVA p<0.01; post hoc Tukey]. Values are means±sem. The panel below (e) illustrates examples of sensory neurons exposed to an Rb1siRNA constructs without or with two differing doses of GW9662. [Bar=100 μm]