Figure 5. HIF-1α is directly involved in the hypoxia-modulation of mouse Plgf expression in H5V cells by HRE binding located on second intron. (A) Schematic representation of part of mouse Plgf gene. Areas analyzed by ChIP analysis are indicated by numbered black rectangles. Gray boxes represent exons. An arrow indicates the transcription start site. Black ovals indicate the putative HRE located along the gene. ChIP analysis for H3 (AcH3) and H4 (AcH4) acetylation (B) or for HIF-1α (C) performed starting from chromatin sample of H5V exposed to hypoxia for 12 h. As control, species matched IgG were used. Data obtained by qRT-PCR are expressed as enrichment of chromatin-associated DNA fragments immunoprecipitated by specific antibody compared with input (% Input) and represent the mean ± SEM of two independent experiments performed in triplicate. ^§P < 0.05; *P < 0.01 and ^#P < 0.005 vs normoxic control. Numbers indicate the amplified regions of Plgf gene, as reported in (A). V-PC and V-NC represent Vegf-a positive and negative controls, respectively, containing or not active HRE. (D) qRT-PCR analysis of mouse PlGF mRNA after silencing of HIF-1α in H5V exposed to 1% O₂ for 12 h and determination of PlGF concentration by sandwich ELISA (E) in H5V culture medium harvested after silencing of HIF-1α and 24 h of exposure to 1% O₂. As control, cells transfected with non-targeting (NT) siRNA and mock-transfected cells were used. Data obtained by qRT-PCR are expressed as fold induction and represent the mean ± SEM of two independent experiments performed in triplicate. Data obtained by ELISAs represent the mean ± SEM of two independent experiments performed in triplicate. *P < 0.0001 and ^§P < 0.0005 vs NT and mock in hypoxia. (F) Western blot analysis of mouse HIF-1α performed on H5V protein extracts, after cells exposure to 1% O₂ for 24 h. β-Tubulin detection on the same filter was used for normalization. Densitometry analyses are reported as percentage of arbitrary densitometry units (ADU) of the ratio of HIF-1α and β-Tubulin, assigning the value of 100 to the relative ratio obtained in mock-transfected cells in normoxic condition.