Hypoxia inducible factor 1alpha hif 1α

The enzymatic activity of superoxide dismutase (SOD) was measured by a SOD assay kit (Sigma, Switzerland) and glutathione content was measured by GSH-Glo Glutathione assay kit (Promega, Madison, WI) according to protocols provided by the companies. A 20/20 luminometer (Turner BioSystems, Sunnyvale, CA) was used to detect total glutathione levels while a Spectra Max Plus (Sunnyvale, CA) was used to detect the total SOD activity. Myocardial protein content of cleaved caspase-3 (Santa Cruz Biotechnology), hypoxia-inducible factor 1alpha (Hif-1α) (Novus Biologicals, Littleton, CO) from LV homogenates were determined by Western blot analysis as described previously (Zhang et al. 2007 (link)). Briefly, total protein from LV tissue was extracted by T-PER tissue protein extraction reagent (Thermo Scientific, Rockford, IL). Protein samples (25–30 μg) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes. The membranes were probed with corresponding primary antibodies. Appropriate HRP-conjugated secondary antibodies were used and the antibody-antigen complexes in all membranes were detected by the ECL PLUS Detection Kit (Thermo Scientific). The expression of these proteins was quantified with Scion Image (NIH, Bethesda, MD) and adjusted to β-actin.

Tumors and lungs were zinc fixed and paraffin-embedded. Tumor sections were stained using the Opal staining system and analyzed with InForm software using the phenotyping tool according to the manufacturer's instructions (PerkinElmer, Rodgau, Germany). Tumor sections were stained with the following antibodies: cleaved caspase (Cell Signaling, Cambridge, U.K.); Ki67 (abcam, Cambridge, U.K.); hypoxia-inducible factor 1-alpha (HIF1α) (Novus); panCytokeratin (abcam); CD31 (BD); alpha smooth muscle actin (αSMA) (Sigma-Aldrich); spectral DAPI (PerkinElmer); neural/glial antigen 2 (NG2) (R&D systems, Minneapolis, USA). For metastases at least nine independent sections of each lung were stained with Mayer's hemalum (Merck, Darmstadt, Germany) and analyzed. Secondary antibody controls for each antibody species were routinely included (Supplementary Figure 1).

The following antibodies and reagents were used: TER119, CD3 (17A2), CD4 (GK1.5), CD8a (53–6.7), CD19 (6D5), CD31 (MEC13.3), CD45 (30-F11), Ly6C (HK1.4), Ly6G (RB6-8C5), CD11b (M1/70) (Biolegend), CD31, PCNA (Santa Cruz Technologies), Cytokeratin 14 (Covance), CD11b, CD31, Ki67 (Abcam), α-SMA, β-Tubulin, β-Actin (Sigma), Vimentin (Lifespan Biosciences), N-Cadherin, E-cadherin, p21, p53 (Cell Signaling), hypoxia inducible factor 1 alpha (HIF1α) (Novus Biologicals) and PIMO (Hypoxiprobes).