A, effect of echinomycin on RON promoter activity: cells were
transfected with pGL3 control vector or –400-bp wild-type RON promoter.
24 h following transfection, cells were treated with varying concentrations of
echinomycin for 16 h, and luciferase activity was measured following
normalization to protein levels. B, ChIP assay on the chromatin
fragments from breast cancer cells using control IgG or HIF-1α
antibodies was performed as described under “Experimental
Procedures.” C, illustration: RON promoter-luciferase reporter
construct depicting the HIF-1α binding site (HRE) and Sp1 binding sites.
D, effect of HRE binding site mutation on RON promoter activity:
cells were transfected with either pGL3 control vector or –400-bp
wild-type RON promoter or HRE binding site mutant RON promoter-luciferase
reporter constructs and luciferase activity was measured after 48 h following
normalization to protein levels. To determine the effect of HIF-1α
expression knockdown on RON promoter activity control or –400-bp
wild-type RON promoter activity was analyzed in control shRNA or shRNA
HIF-1α-expressing clones.