Fig. 2: Ether lipids regulate cellular redox-active iron levels in cancer cells.

a Relative lysosomal iron levels (see methods) of PyMT-1099 WT or AGPS KO cells (+/−) pretreatment with TGF-β (n = 5 independent experiments). Fold change calculated relative to the average of untreated PyMT-1099 wild-type (WT) cells. Statistical significance was calculated by one-way ANOVA with Holm-Šídák multiple comparisons test. b Relative lysosomal iron levels of WT, AGPS KO, and AGPS addback pB3 cells (n = 5 independent experiments). Fold change calculated relative to the average of pB3 WT cells. Statistical significance was calculated by one-way ANOVA with Holm-Šídák multiple comparisons test. c Inductively coupled plasma-mass spectrometry (ICP-MS) of cellular iron in PyMT-1099 WT or AGPS KO cells (+/−) pretreatment with TGF-β (n = 6 independent experiments). Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test. d ICP-MS of cellular iron in WT, AGPS KO, and AGPS addback pB3 cells (n = 4 independent experiments). Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test. e Relative lysosomal iron levels in OVCAR8 NT sg, FAR1 KO or AGPS KO cells pretreated with ferric ammonium citrate (FAC, 50 μg/mL) (n = 4 independent experiments). Fold change is calculated relative to the average of NT sg cells. Statistical significance was calculated by one-way ANOVA with Holm-Šídák multiple comparisons test. f ICP-MS of cellular iron in OVCAR8 NT sg, FAR1 KO or AGPS KO cells pretreated with FAC (50 μg/mL) (n = 5 independent experiments). Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test. g Cell viability of OVCAR8 NT sg, FAR1 KO or AGPS KO cells (+/−) FAC pretreatment followed by ML210 treatment for 72 h. Liproxstatin-1 (0.2 μM) was added at the time of ML210. Data shown as mean of n = 3 technical replicates and is representative of three independent biological replicates. h Cell viability in response to ML210 treatment of PyMT-1099 WT or AGPS KO (+/−) pretreatment with TGF-β (10 days) followed by FAC treatment (100 µg/mL) for an additional 24 h. Cells were then treated with ML210 (+/−) liproxstatin-1 (0.2 µM) and cell viability was assessed after 72 h. Data shown as mean of n = 3 technical replicates and is representative of three independent biological replicates. i ICP-MS of cellular iron from primary tumors derived from WT, AGPS KO, and AGPS addback pB3 cells. Mean +/− SEM from 3 independent tumor samples per condition. Group differences were tested by permutation ANOVA followed by one tailed Welch’s t-tests with Holm correction for multiple comparisons. For panels b, d, i: pB3 WT and AGPS KO cells were transduced with the respective vector control plasmids. NT nontargeting, ns not significant.