Supplementary Figure 5: ALOX12 is downregulated in human cancers. | Nature Cell Biology

Supplementary Figure 5: ALOX12 is downregulated in human cancers.

From: ALOX12 is required for p53-mediated tumour suppression through a distinct ferroptosis pathway

Supplementary Figure 5

(a) Analysis of ALOX12 mRNA underexpression (blue) in different cancer types derived from Oncomine. (b) Box plots derived from gene expression data in Oncoming comparing the expression of ALOX12 mRNA in different cancer types. For Cervical normal tissues n = 10 independent samples, tumor tissues n = 21 independent samples; Head and Neck normal tissues n = 13 independent samples, tumor tissues n = 41 independent samples; Esophageal normal tissues n = 17 independent samples, tumor tissues n = 17 independent samples; Acute Myeloid Leukemia, normal tissues n = 8 independent samples, tumor tissues n = 285 independent samples. Box plots with centre line at median, box limits at 25th/75th centiles. (c) Kaplan-Meier survival curves were generated for overall survival (months) by stratifying patient samples with Pancreatic Adenocarcinoma (TCGA, Provisional) from cBioPortal (n = 186) based on ALOX12 expression levels. The patients were quartiled for ALOX12 expression [ALOX12 low (n = 45; black) and ALOX12 high (n = 45; red)], Log-rank Mantel-Cox test was used (p = 0.016). (d) Sequence alignment of Alox12 proteins from different species downloaded from HomoloGene and aligned using Clustal Omega Multiple Sequence Alignment. Cancer-associated residues (aa 372, 381, and 562) bolded and shown in red. (e) Representative phase-contrast images of H1299 Tet-on p533KR cells treated with doxycycline (0.5 ug/ml), TBH (60 uM), Ferr-1 (2 uM), and ML-355 (4 uM) as indicated for 8h (Scale bars, 100 µm). The experiments were repeated three times, independently, with similar results. (f) H1299 Tet-on p533KR cells were pre-incubated with doxycycline (0.5 ug/ml) for 12h, then treated with doxycycline (0.5 ug/ml), TBH (60 uM), Ferr-1 (2 uM), and ML-355 (4 uM) as indicated for 8 h. Quantification of cell death is shown. Error bars are mean ± s.d., n = 3 independent experiments. (g) U2OS ALOX12 crispr cells transfected with control, ALOX12, or mutant ALOX12 G381R vectors were pre-incubated with Nutlin (10 uM) for 12 h, then treated with TBH (300uM) and Nutlin (10uM) as indicated for 8h. Quantification of cell death is shown. Error bars are mean ± s.d., n = 3 independent experiments. All P values were calculated using two-tailed unpaired Student’s t-test. Raw data are provided in Supplementary Table 1.

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