FIGURE 2.

LPS-IFNγ induces HIF-1 expression and transactivation capacity. A, electrophoretic mobility shift assay analysis of the HIF-1 DNA binding activity of nuclear extracts from RAW 264.7 cells untreated (C) or treated with LPS-IFNγ for different times or DFO for 20 h; s.c. and u.c. are the specific and unspecific competition, respectively, of the LPS-IFNγ/4-h sample. The arrow indicates the inducible HIF-1 complex, whereas the arrowhead indicates the constitutive (const) complex. The binding activity of the constitutively expressed transcription factor oct-1 was used to assess equal loading. B, immunoblot analysis of the nuclear extracts from RAW 264.7 cells untreated (C) or treated with LPS-IFNγ for different times using anti-HIF-1α antibody. The blots were reprobed using the antibody against TFIID as a loading control. C, relative luciferase activity (RLA) in RAW 264.7 cells untreated (C) or exposed to LPS/IFNγ. The cells were transiently transfected with the empty pGL3 vector (ev) or a construct in which luciferase was controlled by an HRE multimer; when appropriate, the cells were also co-transfected with an expression vector coding for a dominant negative mutant of the constitutive HIF-1 β subunit (ΔARNT). The cells were co-transfected using a control vector containing the Renilla luciferase gene. Luciferase activity was determined after 24 h, corrected for transfection efficiency on the basis of Renilla luciferase activity, and normalized to the activity recorded in untreated cells (arbitrarily defined as 1). *, p < 0.001 versus untreated controls; **, p < 0.001 versus cells treated with LPS/IFNγ. All of the results are representative of at least three independent experiments. The values indicate the -fold difference ±S.D. in relation to the untreated controls.