Fig. 3: Oxygen levels regulate GPX4 protein levels and sensitivity to GPX4 inhibition.
From: Lymph node environment drives FSP1 targetability in metastasizing melanoma
a, Immunoblot of HIF-1α and GPX4 in B16-F0 under 1% O2 for 16, 24 and 48 h. b, Quantification of GPX4 from a. c, Immunoblot of HIF-1α and GPX4 after reoxygenation. B16-F0 cells were cultured under 1% O2 for 24 h, then re-exposed to 21% O2 for 2, 4 or 8 h. d, Quantification from c. e, Confocal microscopy analysis of GPX4 (green) and mitochondria (MitoView; magenta) under 21% and 1% O2 for 24 h. Scale bars, 50 μm. f, Subcellular fractionation of GPX4 under 21% and 1% O2 for 24 h. g,i, GPX4 protein levels in B16-F0 and LN7-1134BL cells treated with proteasome inhibitors (BTZ (10 nM) (g) or MG-132 (0.5 μM) (i)) under 21% or 1% O2 for 24 h. h,j, Quantification of the experiments in g and i, respectively. k, Gpx4 mRNA levels with or without BTZ (10 nM) under 21% or 1% O2 for 24 h. l, GPX4 immunoprecipitation and ubiquitination in the LN7-1134BL line under 21% or 1% O2 for 16 h. LE, long exposure; SE, short exposure. m, The cell viability of B16-F0 and LN metastatic lines treated with ML-210 under 21% or 1% O2 for 48 h. n, The total GSH levels in parental and LN metastatic lines under 21% or 1% O2 for 24 h. n = 3 (a–e, g–l and n) and n = 2 (f) independent experiments. For m, n = 3 technical replicates, representative of 1 of 3 independent experiments. For b, d, h, j, k and n, data are mean ± s.d. Statistical analysis was performed using Kruskal–Wallis tests followed by Dunn’s multiple-comparison test (b, d, h and j) and one-way ANOVA followed by Tukey’s (k) or Šidák’s (m and n) post hoc test.
