Transactivation of GATA5 promoter activity by USF1 overexpression. A: Co-transfection was performed with plasmids containing GATA5 promoter region from bp −258 to +85 or mutated counterparts, together with expression vector pGS424, psv-USF1, or psv-AUSF in NIH 3T3 cells. Overexpression of USF1 significantly activated the wild-type or Mut −108/−107 GATA5 promoter fragments in which the E-box remains intact, but mutation of the E-box at −115/−114 significantly reduced promoter activity both in the absence (pGS424 or psv-AUSF) or presence (psv-USF1) of USF1 overexpression (P<0.05). B: USF1 activates endogenous GATA5 gene expression. Human bronchial epithelial cells were transfected with expression vector pGS424, psv-USF1, or psv-AUSF. GATA5 mRNA expression level from the transfected cells was quantified by real-time PCR and normalized by μ-actin expression level. C: Reverse transcription (RT)-PCR was performed and the products were visualized on 2% agarose gels.