Fig. 6.

Silencing of HIF-1α in DC regulated MMP2, VEGF, and expression of CD83. a Monocytes were transfected with siRNA for HIF-1α for 48 h (siHIF-1). HIF-1α-silenced monocytes were cultured for 5 days with IL-4 and GM-CSF and induced for further maturation by treatment with LPS for 48 h under hypoxia. b The expression of HIF-1α was evaluated by real-time RT-PCR and Western blot. Transfection of siRNA targeting HIF-1α reduced HIF-1α expression by 80% or greater. c FACS analysis showed that silencing of HIF-1α did not alter the forward and side scatter properties of DCs. d H-DCs were co-cultured with allogeneic T cells at a ratio of 1:5, 1:10, and 1:20 for 5 days under hypoxia. HIF-1α silencing reduced the ability of H/H-mDCs to stimulate allo-T cell. Results are expressed as the means ± SD of three independent experiments. *P < 0.05 is shown. e H-DCs were cultured with necrotic cells labeled with PKH26 for 2 h at 37 or 4°C under hypoxia. FACS analysis showed that silencing of HIF-1α did not affect phagocytosis by H-DCs. f–h Only the invasion of H/H-imDCs was slightly increased by silencing of HIF-1α. The average of three independent experiments is shown. Bars show the means plus SD. *P < 0.05 is shown. i mRNA expression was measured by real-time RT-PCR. mRNA expression of MMP2 in HIF-1α silencing DCs was enhanced (left panel). Expression of MMP9 mRNA was not affected (right panel). j IL-12 and VEGF secretion by H-DCs was measured by ELISA