Monocytes were incubated with 25 nM hM CSF for 7 days to differentiate into monocyte derived macro technical support phages. Culture of cell lines THP 1, HL 60, and U937 cells were cultured in Roswell Park Memorial Insti tute media 1640 supplemented with 10% heat inactivated FCS, 100 units ml penicillin G, 100 ug ml streptomycin, and 50 uM 2 ME. Human microvascular endothelial cells were purchased from the Cen ter of Disease Control and were cultivated in endothelial basal medium supplemented with 5% heat inacti vated FCS, 100 units ml penicillin G, 100 ug ml streptomycin, 1% 200 mM L glutamine, 0,01% endothelial growth factor, 0,2% hydrocortisone and grown in 0. 2% gelatine coated 75 cm2 culture flasks or 24 well plates, respectively. Induction of hypoxia and stimulation Cells were incubated in a Inhibitors,Modulators,Libraries humidified hypoxic chamber at 5% CO2 level and less than 1% O2 balanced with N2.

Normoxic controls were incubated at 5% CO2 in a humidified atmosphere with 18% O2. Stimulation was done with PMA 10 ng ml. For kinetic analyzes under hypoxia, monocytes were incubated in a water jacket chamber sealed with a Clark type oxygen electrode as described previously. RNA isolation and quantitative real time PCR of selected genes Inhibitors,Modulators,Libraries After cell lysis, total RNA was extracted and the quality was assessed on a Bioanalyzer. The cDNA was synthesized by reverse transcription using TaqMan Reverse Transcription Reagents. qPCR was carried out using the LightCycler Fast Start DNA Master SYBR Green I Kit. Data were normal ized to the expression Inhibitors,Modulators,Libraries of b actin.

All primers used were obtained from TIB Molbiol, b actin, Immunoblotting Cell lysis, for whole cell extracts of monocytes, 106 Inhibitors,Modulators,Libraries cells were lysed in 20 ul Laemmli buffer. For the preparation of nuclear cytoplasmic fraction, 4 106 cells were lysed using the Nuclear Extract Kit from Active Motif, according to the manufacturers instructions. Immunodetection of proteins, 20 ul of whole cell extract or nuclear cytoplasmic fraction was separated by SDS PAGE and blotted onto polyvinylidene difluoride membranes. Blotted proteins were analyzed as indicated and visualized by enzymatic chemiluminescence. Statistical analyzes Data shown are reported as the mean SD of at least six experiments. Differences between normally distribu ted groups were compared using the Students t test and in non normally Inhibitors,Modulators,Libraries distributed groups with the Mann Whitney U test for independent groups and with the Wilcoxon t test for dependent samples.

Results HIF 1a is stabilised under hypoxia in human monocytes but remains in the cytoplasm In order to investigate first the stabilisation of HIF 1a as a function of pO2 values and duration of incubation, MACS isolated CD14 monocytes were incubated in a Clark type electrode for 5 h with a subse quent reoxygenation time of 12 minutes. Immunoblot analyzes revealed that monocyte stabilisation blog post of HIF 1a begins when hypoxia commences.