Fig 1. Constitutive expression of HIF-1α in BMDCs leads to increased iNOS expression without affecting surface levels of co-stimulatory or co-inhibitory molecules.

(a) Hif1a-dPA loxP-stop-loxP mice express a transgene containing a floxed transcriptional stop cassette upstream of a mutated HIF-1α with two proline to alanine substitutions (P402A and P564A; HIF-1α-dPA). (b) Average cell concentration following 10 days of BM culture in the presence of GM-CSF (data shown is representative of 4 independent experiments). (c) BMDC frequencies, based on FSC/SSC and CD11c⁺CD11b⁺ profiles, are similar between WT and HIF-1α-Tg cultures. (d) Constitutive HIF-1α expression was confirmed in HIF-1α-Tg BMDCs by Western blot before or after stimulation with CpG for 16-20h. (e) Expression of iNOS (a direct HIF target) by Western blot in CpG-stimulated, WT and HIF-1α-Tg BMDCs. (f) BMDCs were left unstimulated or stimulated with CpG, and stained for various DC activation markers and (g) other surface molecules including CD103, OX40L, PD-L1, PD-L2, and CCR7. For all plots, the solid black line indicates WT BMDCs and the solid red line indicates HIF-1α-Tg BMDCs. FMO controls are indicated by the shaded grey histogram. For experiments with CpG stimulation of BMDC, stimulation was performed by adding CpG to BMDCs at a concentration of 10μM and incubating for 16-20h. Western blot data are representative of two independent experiments, and flow cytometry data are representative of at least five independent experiments. For (b), n.s.: not significant using Student’s t-test (two-tailed).