Fig. 2: The unfolded protein response (UPR) is not activated during ferroptosis induced by cystine deficiency.

A MDA-MB-231 (left panel) and Panc-1 (right panel) cells were treated with thapsigargin (Tg), erastin, or cystine starvation with the indicated concentration and duration, then the expression and modification status of key UPR pathway proteins were analyzed with Western blotting. To examine the phosphorylation status of IRE1α and PERK, Phostag PAGE was used to resolve the whole-cell protein lysates. To detect ATF6 cleavage, SDS sample buffer was used to ensure efficient membrane protein extraction. The rest of the analyses used RIPA buffer for protein extraction. HSP90 and β-actin were used as loading controls. Total GCN2 from the same blot in Supplementary Fig. 3A was used as the loading control for the 8% PAGE. B MDA-MB-231 cells were transduced with doxycycline (Dox)-inducible shRNA targeting GFP (ishGFP) or XBP1 (ishXBP1). ishGFP was used as a negative knockdown control and two independent XBP1 shRNAs were used. Top panel: Western blotting analysis of spliced XBP1 (XBP1s) expression upon Tg (300 nM, 6 h) and/or Dox (0.5 mg/ml, 24 h) treatment. β-actin was used as a loading control. Bottom panel: shRNA-transduced and Dox-treated MDA-MB-231 cells were treated with increasing concentrations of erastin. 24 h after erastin treatment, cell viability was measured by CCK8 assay. Viability was set as 100% with 0 μM erastin (DMSO-only). Similarly, the expression of ATF4 (C) or ATF6 (D) was inhibited in MDA-MB-231 cells with two independent non-inducible shRNAs per gene and shGFP as a negative control. The expression level of ATF4 or ATF6 upon Tg treatment was analyzed with Western blotting (top panels) and the effect on erastin-induced ferroptosis was analyzed by CCK8 (bottom panels). Data are presented as mean ± s.d. in the bottom panels of (B), (C) and (D). n = 4 in the bottom panel of (B) and n = 6 in the bottom panels of (C) and (D). n indicates independent repeats. Unpaired, two-tailed Student’s t tests were performed to calculate the P values for all the statistical analyses. N.S. not significant. Source data are provided as a Source Data file.