Effect of E12 and PIASy on α-SMA gene expression.

<p>(A) Effect of E12 on α-SMA gene expression. Mouse MCs (0.25×10⁵) were plated in 12-well plates, and six hours later, transfected with the E12 expression plasmid (100, 200, 500 ng plasmid DNA/well). After 48 hours, cell lysates were prepared, and α-SMA was visualized by Western blot analysis. Results are means ± SD (n = 5). (B) Mouse MCs (0.15×10⁵) were plated in 24-well plates, and six hours later, cotransfected with 25, 50, and 150 ng of the E12 expression plasmid, and 150 ng of the reporter construct. Luciferase activities in lysates prepared 36 hours post-transfection were measured. The activity of the reporter plasmid alone was arbitrary given a value of 1, and the activities of the other transfections were adjusted relative to this assay. Means of three independent experiments in triplicate are shown. (C) Mouse MCs were cotransfected with 150 ng of the PIASy expression plasmid, 25 ng of the E12 expression plasmid, and 150 ng of the reporter construct. Luciferase assay was performed as described in the legend for B. (D) Extracts from mouse MCs transfected as in C were prepared for Western blotting with anti-myc, anti-flag, and anti-GAPDH antibodies (Ab). (E) Mouse MCs were cotransfected with 25 ng of the E12 expression plasmid, and 150 ng of the reporter construct, together with or without increasing amounts of PIASy or PIASy^CA as indicated. Luciferase assay was performed as described in the legend for B. *P<0.05, v.s. control.