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. 2021 Jan 20;20(2):e13297. doi: 10.1111/acel.13297

FIGURE 4.

FIGURE 4

Continuous or intermittent Oltipraz exposure during adult life preserves Nrf2 target gene signal responsiveness and chromatin structure. (a)Experimental timeline. Control flies were kept on food without Oltipraz (#1). Long‐term oltipraz treatment includes continuous (#2) and intermittent (#3) strategies. Another two groups of flies were exposed to 1‐week Oltipraz treatment during week 1 (#4) or week 5 (#5). Note that by the end of week 5 all flies were transferred to normal food for 3 days before being subjected to the later experiments. (b)CncC reporter activity in Oltipraz the five groups of flies. Flies carrying the CncC responsive ARE: GFP reporter (Chatterjee & Bohmann, 2012) were subjected to different Oltipraz treatment regimens described in panel a. ARE: GFP reporter activity were assessed under basal and stressed (5 μM oral DEM) conditions. (c) RT‐qPCR quantifying CncC‐regulated mRNAs gstD1, gclC, and gclM in the five groups of flies described in panel a. ***, p < 0.001, **, p < 0.01; *, p < 0.05 by 2‐way ANOVA followed by a multiple comparison test. ns, not significant. (d) Survival of the five experimental groups after exposure to 5 μM DEM was recorded, and the data were analyzed by Mantel‐Cox log‐rank test. ****, p < 0.0001. (e) Chromatin organization at promoter regions of two CncC target genes gclC, gclM and a housekeeping gene rp49 was assessed by DNase I ‐qPCR assay. * = p < 0.05; **, p < 0.01, ***, p < 0.001 by 1‐way ANOVA followed by a multiple comparison test. ns, not significant. (f) Flies carrying the cncMI12862 − GFSTF . 1 MIMIC element which express epitope tagged CncC from the endogenous promoter, as described in Figure 3, where exposed to control food or treated continuously, intermittently or only during week 5 with oltipraz (see panel a). Subsequently, DEM treatment and ChIP‐PCR was conducted as described for the experiment in Figure 3. Note that DEM inducible CncC binding is observed in 5‐week old flies that had continuously and intermittently received Oltipraz, but not in the controls. ***, p < 0.001, **, p < 0.01; *, p < 0.05 by 2‐way ANOVA followed by a multiple comparison test. ns, not significant.