Fig. 5: IRE1α expression confers protection against genotoxic stress in fly models. | Nature Communications

Fig. 5: IRE1α expression confers protection against genotoxic stress in fly models.

From: Genotoxic stress triggers the activation of IRE1α-dependent RNA decay to modulate the DNA damage response

Fig. 5

a D. melanogaster larvae were fed with 100 μM etoposide (Eto) or 50 μg/ml tunicamycin (Tm) for 4 h and then dXbp1s mRNA evaluated by real-time PCR and normalized to the expression levels of dRpl32 gene (n = 3). b dIre1 mRNA was knocked down by expressing a specific RNAi constructs under the control of tubulin-Gal4 driver. D. melanogaster larvae were fed with 100 μM Eto or 50 μg/ml Tm for 4 h and the decay of RIDD targets dSparc, dPontin, and dMys mRNA was evaluated by RT-qPCR and normalized to the expression levels of dRpl32 mRNA (n = 3). c Control and dIre1 knockdown larvae were fed with 500 μM Eto and allowed to reach adulthood for survival analysis. The number of hatched flies was quantified (n = 20 per group) (n = 3). d A dIre1-RNAi expressing fly line was generated to specifically target dIre1 in the imaginal disc of D. melanogaster. The wing SMART assays test was used to monitor genomic alterations after targeting dIre1 in flies. Larvae in first instar were grown in food supplemented with the DNA damaging agent 0.125 mg/ml doxorubicin (Dox) or 0.5% DMSO as control. Adult flies from control and dIre1 RNAi larvae were fixed and the left wing analyzed for the number of mwh clones (right panel) (n = 3). e Using the same experimental setting described in d, imaginal discs were collected, fixed and caspase-3 positive cells detected by immunofluorescence. Nucleus was stained with TO-PRO3 to visualize total number of cells. Quantification of active caspase-3 cells per imaginal disc is presented (right panel) (n = 3). f Mutant knockout dIre1 cells (dIre1 clone) in the eye-antenna imaginal disc were marked with GFP (see methods). Quantification of the ratio clone size/disc size is presented (right panel) (n = 10 clones). In all panels, data is shown as mean ± s.e.m.; *p < 0.05, **p < 0.01, and ***p < 0.001, based on a one-way ANOVA followed Tukey’s test, b–f two-way ANOVA followed Bonferroni’s test. Data is provided as a Source Data file.

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