Figure 5: Nrf2 inhibits redox-sensitive neuronal developmental signalling pathways. | Nature Communications

Figure 5: Nrf2 inhibits redox-sensitive neuronal developmental signalling pathways.

From: Neuronal development is promoted by weakened intrinsic antioxidant defences due to epigenetic repression of Nrf2

Figure 5

(a) Sholl analysis of neurons transfected DIV4–7 with DN-βCat: βcat ARM-Myc; Rac-N17: Rac N17-Myc. *P<0.05 compared with control (Con): two-way analysis of variance (ANOVA) plus Dunnett’s post-hoc, n=3, 24 cells per condition. (b,c) Total dendritic length (b) and branch tip number (c) calculated for the cells analysed in a. *P<0.05, one-way ANOVA plus Dunnett’s post-hoc. (d) YFP-PSD-95 puncta density measured. *P<0.05, n=4 (32–40 cells quantified per condition). Example pictures to the right (scale bar, 10 μm). (e) Sholl analysis of neurons treated daily±D-JNKI1 (2 μM) DIV4–6. *P<0.05, two-way ANOVA plus Sidak’s post-hoc, n=4 (40 cells per treatment). (f,g) Dendritic length (f) and branch tip number (g) measured in the cells treated as in e. *P<0.05 Student’s t-test, n=4 (20 cells per treatment). (h) YFP-PSD-95 puncta density measured. *P<0.05 (n=3, 24 cells per condition). Example pictures to the right (scale bar, 10 μm). (i) Neurons were transfected with a TCF/LEF-luciferase reporter of Wnt signalling, a pTK-renilla Con, Dvl1 plasmid, plus Con or Nrx-directed shRNA vector (ratio: 2:1:2:2). 48 h post-transfection, neurons were treated with H2O2 for 16 h, and reporter activity measured (normalized to Renilla). *P<0.05, n=3: two-way ANOVA with Bonferroni’s post-hoc test here and for j. #Difference between H2O2-stimulated values and the corresponding Con. (j) Neurons were transfected with the Wnt reporter, Renilla, Dvl1 plasmid, plus Con or Nrf2 vectors (ratio: 2:1:2:2). H2O2-induced Wnt activity measured as for i. *P<0.05, n=4. (k) Western analysis of H2O2 (25 μM)-induced c-Jun (Ser-73) phosphorylation±D-JNKI-1 (DJI) pretreatment. *P<0.05 (n=3). (l) Western analysis of H2O2-induced c-Jun (Ser-73) phosphorylation in neurons nucleofected with Con or Nrf2-encoding plasmids. *P<0.05, two-tailed t-test (n=3). (m,n) Nrf2 activity buffers neuronal redox potential. Neurons expressing Grx1-roGFP plus either Con (globin) or Nrf2-encoding plasmids were imaged during H2O2 treatment. Ratios (ex=387±5 and 494±10; em=530±10 in both cases) for each cell were calculated and normalized to the maximal ratio obtained by treating cells with high H2O2 (100 μM). *P<0.05, two-way ANOVA, plus Bonferronni’s post-hoc, n=6 (30 (Con) and 35 (Nrf2) cells analysed). (n) Example traces of single experiments relating to m.

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