Supernatants were considered nuclear extracts. Total protein concentration in lysates was this quantified using the Pierce BCA protein assay kit (Pierce, Rockford, IL USA). Equal amounts of protein were loaded onto SDS/PAGE gels and analyzed by Western blot, as described previously [10]. TSP-1 gels were run in non-reducing conditions. Membranes were blocked with 5% non-fat dry milk in TBS-T (20 mM Tris/HCl pH 7.2, 150 mM NaCl and 0.1% Tween 20) and incubated overnight with a monoclonal antibody against human HIF-1�� (dilution 1250; BD Biosciences, San Jose, CA), human TSP-1 (dilution 1300; Thermo Scientific, Runcorn, Cheshire WA7 1PR, UK), human CD36 (1250; Abcam, Cambridge, UK) or Actin (dilution 110000; Sigma-Aldrich, MO, USA).
Protein bands were detected by LAS-3000 (Fujifilm) during incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (dilution 12500; Pierce, Rockford, IL USA) or goat anti-rabbit IgG (15000; Pierce, Rockford, IL USA) following treatment with supersignal west picochemiluminescent substrate (Pierce, Rockford, IL USA). Protein expression was quantified by means of densitometry using Image Gauge Version 4.0 software (Fujifilm Global, Barcelona, Spain). Data were normalized to actin and results are expressed as fold induction vs control group. RNA Extraction and PCR Analysis Total RNA was isolated from U937cells using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). cDNA was synthesised and real-time PCR was performed as described previously [26].
Specific oligonucleotides for HIF-1�� (5��-GAAAGCGCA AGTCCTCAAAG-3�� and 5��-TGGGTAGGAGATGGAGATGC-3��), TSP-1 (5��-AGAGAACAGAGCCCCAC AGA-3�� and 5��-CCCAAAATATCCTGGGAGGT-3��), and CD36 (5��-CAGTTGGAGACCTGCTTATCC-3�� and 5��-GCGTCC TGGGTTACATTTTC-3��) were designed according to reported sequences. Actin (5��-GGAC TTCGAGCAAGAGATGG-3�� and 5��-CTGTACGCCAACACAGTGCT-3`) expression was used as an internal control. The threshold cycle (CT) was determined and relative gene expression was expressed as follows: change in expression (fold)=2?��(��CT) where ��CT=CT (target) – CT (housekeeping), and ��(��CT)=��CT (treated) – ��CT (control). Static Cytometry PBMC and U937-derived macrophages were exposed to hypoxia for 5 h. After treatment, cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton-X100, and then stained with polyclonal antibody against CD36 (1200, Santa Cruz Biotechnology, Santa Cruz, CA).
FITC or TexRed labeled goat anti-rabbit IgG (H + L) (1200, Abcam, Cambridge, UK) were used as the secondary antibody, and 1 ��M Hoechst 33342 (Sigma-Aldrich, Steinheim, Germany) was added to stain nuclei. Fluorescence (18 images per well) was visualized using the fluorescence microscope and the fluorescent signal was quantified using the static cytometer Entinostat software ��Scan�� version 2.03.2. Chromatin Immunoprecipitation U937 cells were differentiated to macrophages.