Fig. 2: IRE1α deficiency impairs the DDR.

a IRE1α KO (Mock) and IRE1α-HA reconstituted cells were pre-incubated with 1 μM etoposide (Eto) for 16 h and washed three times with PBS and fresh cell culture media was added. The decay of phosphorylated H2AX (P-H2AX) was monitored over time by western blot (middle panel). Quantification of the levels of P-H2AX in cells stimulated with Eto (bottom panel) (n = 3). b IRE1α KO (Mock) and IRE1α-HA reconstituted cells were incubated with 10 μM Eto for 2 h and then washed with PBS and fresh culture media was added. The distribution P-H2AX expression (green) was monitored by indirect immunofluorescence using confocal microscopy. Nuclei were staining with DAPI (Blue). Quantification of P-H2AX per cell is shown (right panel) (n = 3). c WT and IRE1α KO MEFs cells were treated with 10 µM Eto for 3 h to perform the comet assay. Quantification of tail intensity and tail moment (Tail intensity × tail area) is shown (right panel) (n = 3). d WT and IRE1α KO MEFs cells were treated with 5 μM Eto for 3 h to determined cytokinesis-block micronucleus cytome assay (CBMC). Binucleated cells (BN) with micronucleus (MN), nuclear buds (Nbuds) or nucleoplasmid bridges (NPB; see arrows) were visualized and quantified using epifluorescence microscopy (n = 3). e WT and IRE1α KO MEFs cells were treated with 10 μM Eto for 8 h and cell cycle was analyzed by propidium iodide (PI) staining. Quantification of the percentage of cells in G0/G1 and S phases is shown. f WT and IRE1α KO MEFs cells were treated with 10 μM Eto for indicated times. Expression and phosphorylation levels of indicated proteins involved in the DDR were monitored by western blot (left panel). Quantification of the levels of p-CHK1 and p-CHK2 is shown (right panel) (n = 3). In all panels, data is shown as mean ± s.e.m.; *p < 0.05, **p < 0.01, and ***p < 0.001, based on (a, c, d, f) two-way ANOVA followed Bonferroni’s test, b two-way ANOVA followed Tukey’s test. Data is provided as a Source Data file.