Pitx2 induces expression of SMC differentiation marker genes. (A) Luciferase assays were performed in undifferentiated A404 cells cotransfected with expression plasmids for Pitx2 isoforms and the SM α-actin promoter-enhancer-luciferase construct. RLA, relative luciferase activity. (B) Undifferentiated A404 cells were infected with adenovirus expressing Pitx2a or empty adenovirus, and expression of SMC differentiation marker genes was determined by real-time RT-PCR. (C) Efficiency and specificity of Pitx2 siRNA were examined in COS cells cotransfected with Flag-Pitx2a expression plasmid and Pitx2 siRNA expression plasmid or control plasmids. pMighty-Empty contained no target sequence and pMighty-αScr targeted scrambled sequence. (D) A404 cells were induced to differentiate into SMCs by RA treatment for 1 d (early) or by RA treatment for 3 d followed by puromycin selection for 2 d (late). Cells were transfected with the SM α-actin promoter-enhancer-luciferase construct and a siRNA expression plasmid for Pitx2 (pMighty-αPitx2), Prx1 (pMighty-αPrx1), or pMighty-αScr. Luciferase assays were performed. (E and F) A404 cells were induced to differentiate into SMCs by RA treatment and were transfected with siRNA duplexes for Pitx2 or EGFP. Expression of SMC differentiation marker genes was determined by real-time RT-PCR (E) or Western blotting (F). Values represent the mean ± SEM of three independent experiments.