Fig 2: IRE1a deletion in liver alters the DDR under genotoxic stress.a IRE1a was conditionally deleted the liver using the MxCre and LoxP system (IRE1acKO). Mice were intraperitoneally injected with 50 mg/Kg etoposide (Eto) or 1 mg/Kg tunicamycin (Tm) and sacrificed 6 h and 16 h later. Total mRNA levels of the deleted IRE1, and p21 were measured 6 h later in the liver by real-time-PCR (n = 3–4 mice per group). Xbp1 mRNA splicing (bottom panel) was monitored in the same samples by RT-PCR. b Liver extracts of animals described in a, Ppp2r1a and Bloc1s1 mRNA expression levels were measured 6 h later of Eto treatment by real-time-PCR (n = 3). c Protein liver extracts were obtained from mice treated described in a and the expression levels of indicated proteins were monitored 6 h later of Eto treatment by western blot. Quantification of the levels of p-CHK1 is shown (Right panel). d Mice from a were intraperitoneally injected with 50 mg/Kg Eto and sacrificed 48 h later. Then, livers active-caspase 3 detected by immunohistochemistry (n = 2–3). e Gene expression profile analysis was performed in mRNA from liver extracts of animals described in a. Most significant pathways altered by Eto administration in WT and IRE1a null livers are shown. Three independent biological samples were used. In all panels, data is shown as mean ± s.e.m.; *p < 0.05, **p < 0.01, and ***p < 0.001, based on a–d two-way ANOVA followed Bonferroni’s test. Data is provided as a Source Data file. f Working model: genotoxic stress activates IRE1a in the absence of ER stress markers, selectively engaging RIDD. IRE1a degrades mRNAs involved in the DNA damage response encoding for Ppp2r1a and Ruvbl1, regulating the (de)phosphorylation of the histone H2AX and CHK-1/2. The non-canonical activation of IRE1a involves the participation of the c-Abl kinase that is activated by DNA damage response kinases as ATM. The expression and function of IRE1a is essential to promote survival under DNA damage conditions by controlling cell cycle arrest and DNA repair programs.

Fig 3: c-Abl contributes to the RIDD activation under DNA damage.a c-Abl was knocked down through the stable delivery of an shRNA. Then cells were treated with 10 µM Etoposide (Eto) or 500 ng/mL tunicamicyn (Tm) for 8 h and Xbp1 mRNA splicing was monitored by RT–PCR (n = 3). b MEF ShRNA Scramble and ShRNA c-Abl cells were treated with 10 µM Eto or 500 ng/mL Tm for 8 h, and the decay of Ppp2r1a and Ruvbl1 was measured by real-time-PCR (n = 3). c Trex-IRE1-GFP cells were pre-treated with 10 µM imatinib by 1 h, and then treated with 10 µM Eto, or 500 ng/mL Tm for 8 h and IRE1-GFP foci visualized by confocal microscopy. d Quantification of the percentage of cells positive IRE1-GFP clusters is shown (>200 cells, n = 3). e c-Abl expression in CRISPR control and c-Abl KO cells was monitored by western blot (n = 3). f CRISPR control and c-Abl KO cells were treated with 10 µM Eto or 500 ng/mL Tm for 8 h, and the decay of Ruvbl1 and Ppp2r1a was measured by Real-Time-PCR (n = 3). g HEK-293T cells reconstituted with IRE1a-HA and c-Abl-GFP were exposed to 10 µM Eto for 8 h. Immunoprecipitation (IP) was performed using the HA epitope (IRE1a) and GFP (c-Abl) to assess the possible interaction with c-Abl. h IRE1a KO (Mock) and reconstituted cells with an IRE1a-HA were treated 8 h with 10 µM Eto and stained with a proximity ligation assay (PLA) using an anti-HA or anti-c-Abl antibodies and analyzed by confocal microscopy. Right panel: Number of dots per cell analyzed and percentage of PLA positive cells were quantified (n = 6). i Recombinant IRE1a and c-Abl proteins were incubated at indicated time points and assess its possible interaction by western blot. All data represents the mean ± s.e.m. of three independent experiments, except for co-IP that were performed twice. *p < 0.05, **p < 0.01, and ***p < 0.001, based on (b, f) two-way ANOVA followed Bonferroni’s test, (d, h) One-way ANOVA followed Tukey’s test. Data is provided as a Source Data file.