ATRA induces OLFM4 expression and transactivation of the OLFM4 promoter by RARα/RXRα heterodimer. (A) HL-60 cells were treated with ATRA (1μM), 9-cis-RA (1μM), or vehicle control for 5 days. Medium was replaced with new medium with freshly added RAs or vehicle every 48 hours. OLFM4 mRNA expression relative to β-actin expression was determined by qRT-PCR. (B) HL-60 cells were treated with ATRA (1μM), 5-aza-2′-deoxycytidine (Aza, 5μM), or trichostatin A (TSA, 1μM) alone or in combination for 2 days. OLFM4 mRNA expression relative to β-actin expression was determined by qRT-PCR. *P < .05, **P < .01, when compared with vehicle treatment. (C) Left panel: in vitro–transcribed and –translated RAR-α, RXR-α, or RXR-β protein alone or in combination was incubated with γ-³²P-labeled RARE-DR5 probe of the OLFM4 promoter, then analyzed by electrophoretic mobility-shift assay. Right panel: the in vitro–transcribed and –translated RAR-α and RXR-α protein mixture was incubated with RARE-DR5 probe and subjected to electrophoretic mobility-shift assay. SS indicates supershift band. 100× comp, 100× cold-probe competitions. (D) COS-7 cells were cotransfected for 48 hours with the OLFM4 promoter, luciferase-reporter plasmid (OLFM4-Luc, −959), phRL-TK vector, and expression vectors expressing no cDNA (leftmost set of bars), RAR-α, RXR-α, RXR-β, or combinations of cDNA, as indicated. For the last 24 hours, the cells were treated with 1μM ATRA or vehicle control, then luciferase activities in cell extracts were determined. Values represent the OLFM4 promoter activity relative to TK activity (Renilla luciferase as internal control). Data represent the mean ± SD of 3 independent experiments performed in triplicate. *P < .05, compared with empty vector (no cDNA: leftmost set of bars).