Fig. 5: SETX upregulation is dependent on the PERK branch of the UPR.

a A549 cells were treated with either control siRNA or p53 siRNA and exposed to 21% O₂ (Norm) or <0.1% O₂ (Hyp) for 6 h. SETX mRNA levels were determined by RT-qPCR. One-way ANOVA Tukey’s multiple comparisons test was used: ^nsp = 0.9996, **p = 0.0050. b A549 cells were treated with either control siRNA or ATF4 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 = 0.0023. c A549 cells were exposed to 21% O₂ (Norm) or <0.1% O₂ (Hyp) for 4 h, and ChIP-qPCR experiments were performed to determine fold enrichment of ATF4 at the SETX promoter relative to IgG control. One-way ANOVA Tukey’s multiple comparisons test was used: *p = 0.0416. d SETX mRNA levels in non-treated A549 cells (Norm), Hu (1 mM), <0.1% O₂ (Hyp), tunicamycin (Tuni, 5 µg/mL) or thapsigargin (Thaps, 2 µM) for 16 h. One-way ANOVA Dunnett’s multiple comparisons test was used: ****p < 0.0001. e A549 cells were exposed to Thapsigargin (Thaps) (2 µM) or Tunicamycin (Tuni) (5 µg/mL) for the times indicated, or to Hu (2 mM, 8 h) followed by western blotting. f Percentage of cells displaying RPA foci (>5 per nucleus) in A549 control or Hu (2 mM) treated cells that were also treated with or without Thaps (2 µM) for 24 h. The two-tailed Student’s t-test was used: ****p < 0.0001. g Representative images from f (scale bar: 5 μm). h A549 control or Hu (2 mM) treated cells were also treated with or without Thaps (2 µM) for 24 h followed by western blotting. β-actin was used as the loading control. i A549 control or Hu (2 mM) treated cells were also treated with DMSO, Tuni (5 µg/mL), Thaps (2 µM) or Thaps (2 µM) and PERKi (10 μM) for 24 h followed by western blotting. β-actin was used as the loading control. a–i Data from three independent experiments (n = 3), mean ± SEM are displayed unless otherwise indicated.