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. 2011 Nov 15;51(10):1882–1892. doi: 10.1016/j.freeradbiomed.2011.08.023

Fig. 6.

Fig. 6

c-Myc and Mxi-1 are involved in different regulation of IL-8 expression by HIF isoforms. Real-time PCR was performed to examine (A) Mxi-1 and (B) c-Myc mRNA after 24 h incubation of cells under severe (0.5% O2) hypoxia. Note the tendency toward increase in Mxi-1 and decrease in c-Myc expression under hypoxia. (C, D) ChIP assay was performed to check c-Myc binding to the IL-8 promoter ((C) region − 802 to − 611 and (D) region − 417 to − 226) under hypoxic conditions (6 h). Antibodies against IgG and c-Myc (anti-c-Myc) were used. Binding sites of c-Myc within the IL-8 promoter were amplified using real-time PCR as described under Materials and methods. Note a strong decrease in c-Myc binding under hypoxic conditions. (E) Real-time PCR was performed to examine c-Myc mRNA after 48 h transduction with AdHIF-1α. AdHIF-1α diminished c-Myc mRNA. (F) Cells were transduced for 48 h with AdHIF-1α, AdHIF-2α, or AdGFP as a control. As shown by Western blotting, in contrast to HIF-1α, overexpression of HIF-2α resulted in increased expression of c-Myc protein; the representative immunoblot is shown. (G) ELISA was performed to assess the IL-8 protein level after 24 h transfection with siRNA against Mxi-1 mRNA or scrambled siRNA as a control and incubation under normoxic or hypoxic conditions for the next 24 h. (H) ELISA was done to assess the IL-8 protein level after 24 h transfection with siRNA against Mxi-1 mRNA or scrambled siRNA as a control and 48 h transduction with AdHIF-1α or AdGFP. Silencing of Mxi-1 by siRNA reversed both the AdHIF-1α- and the hypoxia-dependent diminishment of IL-8. (I) ELISA was done to assess IL-8 protein level after 24 h transfection with siRNA against c-Myc mRNA or scrambled siRNA as a control and 48 h transduction with AdHIF-2α or AdGFP. c-Myc siRNA partially reversed HIF-2α-induced IL-8 expression. Each bar represents the mean ± SD of two to four independent experiments performed in duplicate or (I) a representative experiment is shown. *p < 0.05, comparing normoxia vs hypoxia or AdGFP vs AdHIF-1α or AdHIF-2α; #p < 0.05, comparing scrambled siRNA vs siRNA against Mxi-1.