Fig. 3

CBE treatment reduces the neuronal NFE2L2 response induced by microglia. A The NFE2L2 activity was measured in SK-ARE-luc2 cells treated with vehicle (water) or 200 μM CBE for 48 h, then treated with vehicle or 5 μM and 15 μM tBHQ for 24 h. Data are expressed as fold changes (FC) of RLU/μg protein extracts, versus vehicle and presented as mean ± SD of n=4 independent samples measured in triplicate. Statistical significance was determined by one-way ANOVA followed by Dunnet’s multiple comparison test versus vehicle and unpaired t-test between CBE or vehicle in equal concentration of tBHQ. ns, not significant, *p<0.05, **p<0.01. B The NFE2L2 activity was measured in SK-ARE-luc2 cocultured for 48 h with different cell lines (MCF-7; C6; BV-2; RAW 249.7) and primary cultures (murine peritoneal macrophages, mMP); human macrophages, hMP). Data are expressed as fold changes (FC) of RLU mean values versus SK-ARE-luc2 monoculture and presented as mean ± SD for n=9 independent samples measured in triplicate. Statistical significance was determined by two-way ANOVA followed by Sidak’s multiple comparisons test. ***p<0.001. C Scheme of the experiments reported in D and E aimed at testing the effect of CBE on neuronal NFE2L2 activation in SK-ARE-luc2/BV-2 cocultures. D Data are expressed as fold changes (FC) of RLU versus vehicle and presented as mean ±SD of n=9 independent samples measured in triplicate. *p<0.05, ***p<0.001 calculated by unpaired t-test. E Data report the semiquantitative analysis of the average fluorescence of SK-ARE-luc2 nuclei, following Nfe2l2 immunostaining, and presented as violin-plot with median (dash line) and quartiles (dot lines) of the analysis of N=6000 nuclei measured in 20 fields/group (see Supplementary Fig. 4). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. ***p<0.001. F Scheme of the experiments reported in G–I, aimed at testing the effects of CBE in primary cocultures of neurons and microglia (μglia). G Luciferase activity from ARE-luc2 primary neurons treated with 200 μM CBE for 48 h in monoculture, or cocultured with wild type primary microglia. ARE-luc2 primary neurons cocultured with wild type primary microglia treated with vehicle (H) or with 200 μM CBE for 48 h (I). Data are expressed as fold changes (FC) of RLU versus vehicle treated samples (G), versus neuron monoculture (H) and versus vehicle treated cocultures (I), and presented as mean ±SD of n=5 independent samples measured in triplicate. *p<0.05, ***p<0.001 calculated with the unpaired t-test