Introduction 11 26 29 37 38 14 26 29 38 11 26 38 11 14 26 38 11 14 26 38 25 49 11 14 26 29 38 39 6 13 32 33 44 2 6 32 44 21 4 31 3 2 2 Materials and methods Human adipocyte culture Human subcutaneous preadipocytes, derived from adipose tissue pooled from seven female subjects, were obtained (together with culture media) from Zen-Bio (USA). The patients had a mean body mass index of 25 (range 22.5–28.2) and average age of 41 years (range 27–51 years). 2 2 v v 2 v v 2 2 2 2 2 2 2 2 2 2 2 RNA extraction and cDNA synthesis Total RNA was isolated from cells using Trizol, and 1 μg of RNA was treated with DNase I (Invitrogen) according to the manufacturer’s instructions. RNA concentration was quantified from the absorbance at 260 nm; all samples had a 260/280 nm absorbance ratio of 1.7–1.9. ST Real-time PCR Quantitative real-time polymerase chain reactions (PCRs) were carried out in a final volume of 12.5 μl consisting of 12.5–50 ng of reverse-transcribed cDNA mixed with optimal concentrations of primers and probe and qPCR™ core kit (Eurogentec, UK) in 96-well plates on a Mx3005P detector (Stratagene, USA). 41 42 GLUT1 (93 bp): 5′-ATACTCATGACCATCGCGCTAG-3′ (forward), 5′-AAAGAAGGCCACAAAGCCAAAG-3′ (reverse) and 5′-FAM-TGGAGCAGCTACCCTGGATGTCCTATCTGA-TAMRA-3′ (probe); FIAF (117 bp): 5′-GATGGCTCAGTGGACTTCAACC-3′ (forward), 5′-CCCGTGATGCTATGCACCTTC-3′ (reverse) and 5′-FAM-CCAGACCCAGCCAGAACTCGCCGT-TAMRA-3′ (probe); HIF-1α (75 bp): 5′-TCCAGTTACGTTCCTTCGATCA-3′ (forward), 5′-TTTGAGGACTTGCGCTTTCA-3′ (reverse) and 5′-FAM-CACCATTAGAAAGCAGTTCCGCAAGCC-TAMRA-3′ (probe); MIF (74 bp): 5′-AGCCCGGACAGGGTCTACA-3′ (forward), 5′-GCGAAGGTGGAGTTGTTCCA-3′ (reverse) and 5′-FAM-CTATTACGACATGAACGCGGCCAATGT-TAMRA-3′ (probe); POLR2A (81 bp): 5′-ATGGAGATCCCCACCAATATCC-3′ (forward), 5′-CATGGGACTGGGTGCTGAAC-3′ (reverse) and 5′-FAM-TGCTGGACCCACCGGCATGTTC TAMRA-3′ (probe). Typically, the amplification started with 2 min at 50°C, 10 min at 95°C and then 40 cycles of the following: 15 s at 95°C and 1 min at 60°C. 2 2 \documentclass[12pt]{minimal}
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\begin{document}$$ 2^{{ - \Delta \Delta \operatorname{Ct} }}  $$\end{document} Measurement of HIF-1α and adipokines by ELISA w v 2 Adiponectin, IL-6, leptin, MIF and VEGF were measured in cell culture media using commercial ELISA kits (R&D Systems). The assays were conducted in 96-well microplates according to the manufacturer’s instructions. Measurement of HIF-1α by Western blotting Samples were prepared as described above for the HIF-1α ELISA assay. Fifteen micrograms of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. The membranes were blocked and probed with polyclonal goat anti-human HIF-1α (R&D Systems) or mouse monoclonal anti-α-tubulin (Sigma, UK) as the primary antibody, then subjected to HRP-conjugated anti-goat IgG (R&D systems) or anti-mouse IgG (Santa Cruz Biotechnology) as the secondary antibody. Specific proteins were visualised with the enhanced chemiluminescence reagent (Amersham, UK). Statistical analysis t Results HIF-1α expression during differentiation of human adipocytes 1 1 Fig. 1 a b c n  1 2 2 2 2 2 2 Fig. 2 2 a b 2 1 n a P P b P 2  2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 Fig. 3 2 a b c 2 n P P  3 2 2 2 4 4 2 4 4 Fig. 4 2 a b 2 n P P P  4 4 4 2 2 2 2 2 2 5 2 2 5 Fig. 5 2 a b c 2 2 n P P P  2 5 2 5 2 2 2 6 2 6 2 Fig. 6 2 a b 5 2 n P P P  2 6 2 2 6 Adipokine secretion in hypoxia 2 7 2 7 Fig. 7 2 a b–e 6 2 n P P P  7 2 7 Discussion 7 9 38 11 14 26 38 15 45 38 13 32 44 16 4 21 3 32 2 2 2 2 2 2 40 2 2 2 20 24 27 2 2 17 43 1 5 48 12 2 34 46 47 2 2 2 4 50 21 51 35 8 10 23 21 18 2 22 18 36 28 2 4 19 30 2 38