Fig. 6: Hypoxia links the UPR with replication stress.

a A549 cells were treated with either control siRNA or PERK siRNA and exposed to 21% O₂ (Norm) or <0.1% O₂ (Hyp) for 16 h. SETX mRNA levels were determined by RT-qPCR. One-way ANOVA Tukey’s multiple comparisons test was used: ****p < 0.0001. b A549 cells were treated with or without PERKi (10 µM) and exposed to DMSO or Thaps (2 µM) for 6 h. SETX mRNA levels were determined by RT-qPCR. One-way ANOVA Tukey’s multiple comparisons test was used: ***p = 0.0003. c A549 cells were either mock-treated or treated with PERK siRNA, transfected with RNase HI^D210NV5 and exposed to hypoxia (<0.1% O₂), CPT (10 µM, 20 min) or DRB (100 µM, 1 h). Staining for V5 was then carried out and the nuclear intensity determined. The two-tailed Student’s t-test was used: ****p < 0.0001. d The percentage of cells displaying 53BP1 foci (>5 per nucleus) as determined by immunofluorescence, in A549 cells exposed to 21% O₂ (Norm) or 6 h of <0.1% O₂ (Hyp). Cells were treated with DMSO, PERKi (10 µM), or DRB (100 µM) and PERKi (10 µM) (PERKi + DRB). The two-tailed Student’s t-test was used: Norm PERKi versus Hyp PERKi (**p = 0.0067), Hyp + PERKi versus Hyp+ PERKi + DRB (**p = 0.0068). e A549 cells were treated with DMSO or PERKi (10 µM) and exposed to normoxia or hypoxia (<0.1% O₂) for 6 h followed by western blotting as indicated. PERK inhibition was confirmed by the absence in the electrophoretic mobility shift of PERK on the western blot. f A549 cells with either mock treatment or PERK siRNA were exposed to hypoxia (<0.1% O₂) for 6 h, cells were fixed and stained for RPA by immunofluorescence assays. Representative images are shown (scale bar: 5 μm). g Quantification of the number of RPA foci per cell from f. Treatment with siRNA PERK is indicated in green. Data from two independent experiments (n = 2) where each dot represents a cell and a minimum of 100 cells were imaged per treatment. Two-tailed Mann–Whitney U test was used: **p = 0.0024. h Replication rates, as determined by IdU incorporation rates, of A549 cells treated with DMSO or PERKi (10 µM) and exposed to 21% O₂ (Norm) or <0.1% O₂ (Hyp) for 6 h. Data from two independent experiments (n = 2) where each dot represents a fibre and a minimum of 100 fibres were analysed per treatment. Two-tailed Mann–Whitney U test was used: ****p < 0.0001. i Hypoxia leads to the activation of the UPR and replication stress. In addition to a shortage of nucleotides, R-loops accumulate in hypoxia and contribute to replication stress. The PERK branch of the UPR signals through ATF4 to increase SETX expression in hypoxia. Hypoxia-induced SETX reduces R-loop accumulation and subsequent replication stress-mediated signalling. a–h Data from three independent experiments (n = 3), mean ± SEM are displayed unless otherwise indicated.