To obtain direct evidence that YB-1 binds to the MACC1 promoter, a ChIP assay was performed in A549 cells. PCR amplifications were performed using two primer sets designed to flank the potential YB-1 binding sites on the MACC1 promoter, referred to as YB-1 binding site 1 and YB-1 binding site 2 (Figure (Figure4A).4A). YB-1:MACC1 promoter binding was validated using the YB-1 binding site 1 primers, but not the YB-1 binding site 2 primers (Figure (Figure4F).4F). These results indicated that YB-1 directly bound to specific regions of the MACC1 promoter (-1860 to -1856), thereby up-regulating the expression of MACC1.