Fig. 4. PIASx enhanced Osx promoter-driven transcription and Osx transcriptional activity cooperatively with NFAT. (A) Osx upregulated its own promoter and PIASx further enhanced its activity. MC3T3-E1 cells were co-transfected with the expression plasmids pEx3.1Osx, pcDNA3.1-PIASx and pcDNA3.1-PIASxC362S, the reporter plasmid pGL4.18-Osx-Luc and the pRL-TK plasmid at day 1 in DM. 48 hours post-transfection the reporter assay was performed. (B) Endogenous Osx and PIASx are important for Osx promoter activation. Cells were transfected with siRNA at day 1 in DM and 72 hours later transfected with Luc expression and reporter plasmids. 48 hours after Luc transfection, the reporter assay was performed. (C) Effect of PIASx and NFAT on osterix transcriptional activity in the GAL4 transactivation system. MC3T3-E1 cells were transfected with the indicated plasmids at day 1 in DM. (D) Cells were transfected with siRNA at day 1 in the DM and 72 hours later Luc plasmids were transfected. 48 hours post-transfection of Luc plasmids, the reporter assay was performed. (E) Effect of PIASx on the transcriptional activity of Runx2. MC3T3-E1 cells were transfected with the indicated plasmids at day 1 in DM. Cells were transfected with siRNA at day 1 in DM and this was followed 24 hours later by transfection with Luc plasmids (F). 48 hours post-transfection of Luc plasmids the reporter assay was performed. (G) MC3T3-E1 cells were transfected with siRNA at day 1 in DM and this was followed 72 hours later by transfection with Luc plasmids. 48 hours post-transfection of Luc plasmids the reporter assay was performed. Data represent the mean (±s.d.) of three independent experiments, each of which was performed in triplicate.