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Additional file 1: of Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1α to drive lung adenocarcinoma metastasis

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posted on 29.01.2019, 05:00 by Yi-Hua Jan, Tsung-Ching Lai, Chih-Jen Yang, Yuan-Feng Lin, Ming-Shyan Huang, Michael Hsiao
Table S1. Correlation of clinicopathological features of NSCLC patients with AK4 and HIF-1α expression. Figure S1. (related to Fig. 1) Ingenuity upstream analysis of consensus AK4 metabolic gene signature between GSE31210 and TCGA LUAD. A, Venn diagram analysis of AK4 metabolic gene signature in GSE31210 and TCGA LUAD datasets. Activation z score more than 2 or less than − 2 is predicted to be significant activation or inhibition respectively. B, Left panel, Ingenuity upstream analysis of consensus AK4 metabolic signature. Right panel, heatmap illustrates HIF-1 α -regulated genes that are positively or negatively correlated with AK4 expression in consensus AK4 metabolic signature. Figure S2. (related to Fig. 3) AK4-induced EMT is HIF-1α-dependent. A, WB analysis of AK4, HIF-1α, GnT-V, E-cadherin, Vimentin, Snail from CL1-0 vector- or AK4-expressing cells transduced with shNS or shHIF-1α in Hx.B, Invasion assay of CL1-0 vector- or AK4-expressing cell transduced with shNS or shHIF-1α in Hx. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. *P ≤ 0.05; **P ≤ 0.01. Figure S3. (related to Fig. 4) Differentially expressed genes in glycolysis/gluconeogenesis and glutathione metabolism in CL1-0 upon AK4 overexpression. A, Relative expression level of genes in KEGG glycolysis and gluconeogenesis pathway from CL1-0 AK4 versus CL1-0 Vec microarray data. B, Relative expression level of genes in KEGG glutathione metabolism pathway from CL1-0 AK4 versus CL1-0 Vec microarray data. Figure S4. (related to Fig. 6) MTT assay cell viability assay of digitoxigenin, lanatoside C, digoxin, proscillaridin, and withaferin-A in CL1-0, CL1-5, CL1-0 Vec, and CL1-0 AK4. Figure S5. (related to Fig. 6) Withaferin-A treatment suppresses metastasis in A549 orthotopic lung cancer mouse model. A, 5 A549-GL cells were orthotopically injected into the left lung of NSG mice that were treated over an interval of one day with DMSO vehicle control or withaferin-A: 1.0 mg/kg; 4.0 mg/kg. Luminescence was measured using a noninvasive, bioluminescence imaging system (IVIS spectrum) at days 1 (top) and 28 (bottom). B, Luminescence, fluorescence, gross view (formalin-fixed) and H&E staining images in the lungs of mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 28 after orthotopic injection of A549-GL cells (top). Quantification of tumor weight in the lung of mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 28 after orthotopic injection of A549-GL cells (bottom). C, Luminescence, fluorescence, gross view (formalin-fixed) and H&E staining images in the livers of mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 28 after orthotopic injection of A549-GL cells (top). Quantification of liver nodule number in the mice treated with DMSO vehicle control or withaferin-A (1.0 mg/kg or 4.0 mg/kg) at day 28 after orthotopic injection of A549-GL cells (bottom). The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. *P ≤ 0.05; **P ≤ 0.01. (PDF 3400 kb)

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