Fig. 6.

siRNA knock-down of endogenous HIF-3α leads to downregulation of many HIF target genes. Hep3B cells were transfected with a negative control siRNA (luc, pGL2 luciferase control duplex siRNA) or with HIF-3α siRNAs (three independent siRNAs a, b, c alone or in combination) twice at an interval of 24 h and cultured in 1% oxygen for 24 h after the second transfection. The expression of HIF-3α (all variants), HIF-1α, HIF-2α (a) and Epo (c) at the mRNA level was analyzed by qPCR and the expression of HIF-1α and HIF-2α (b) and Epo (c) at the protein level by Western blotting and ELISA, respectively. The effect of the HIF-3α siRNA on selected HIF target genes was analyzed by qPCR (d). The data represent means ± SD from at least four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. In the case of the Epo ELISA analysis, the data represent the results of a typical experiment