Fig. 2.

Functional HIF-1α expression induced by interferon-α depends on active transcription. a Analysis of HIF-1α mRNA level revealed a transcription dependence of IFN-α-induced HIF-1α expression. b-d IFN-α-increased HIF-1α proteins are functional as evidenced by RT-PCR analysis of elevated mRNA levels of HIF-1α-targeting genes (b), luciferase reporter assay with Twist promoter upon treatment with IFN-α (500 and 1000 units/ml) (c) and immunoblotting analysis of higher protein expressed of HIF-1α target genes CA9 and PGK1 under normoxia and hypoxia (d). Cells were treated with 1000 units/ml IFN-α for 24 h (unless indicated otherwise) before lysis and subsequent analyses. **, P < 0.01; ***, P < 0.001 (e) IFN-α treatment activated JAK pathway as shown by phosphorylation of STAT1_Y701 and both JAK (JAKi, 10 μM) and PI3K inhibitors (LY294002, LY, 10 μM) abrogated HIF-1α expression. f IFN-α activated Ras pathway. IFN-α-treated cell lysates (24 h) were subjected to Raf pull-down assay for the level of activated Ras. g Ras farnesyltransferase inhibitor Manumycin A (Man A, 10 μM) inhibited HIF-1α expression