Dox-dependent TS expression in TFTS66 cells.

<p><b>A.</b> TS antigens in cell lysates of TFTS66 transformant were detected by immunoblotting using anti-human TS mouse monoclonal antibody. The standard cell lysates (0 ng/ml Dox) were titrated, and a standard curve for detection was obtained from the signal intensity on the digitized image (upper panel). Using the highly linear detection characteristics (p = 0.997), TS expression levels were quantified: rectangle, TFTS66; circle, the control transformant, TFC7. <b>B.</b> TS expression in TFTS66 cells exposed to various concentrations of Dox was assessed by immunoblotting and similarly quantified. The symbols are shaded according to the Dox concentrations. Some of the data points are also shown in Fig 2A. <b>C.</b> The quantity of TS protein (TS_total, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123076#sec002" target="_blank">Materials and methods</a>) and its catalytic activity were enzymatically assayed in lysates prepared from TFTS66 cells exposed to 0, 0.5 and 1.0 ng/ml Dox. The results are plotted as a function of the TS expression level determined by immunoblotting: open rectangle, Dox0; shaded rectangle, Dox0.5; closed rectangle, Dox1.0. <b>D.</b> TS expression in TFTS66 cells was observed using fluorescent immunocytochemistry. Cells grown on chamber slides were fixed and reacted with TS-specific antibody. Cellular distribution of TS antigens was visualized by red fluorescent signals. Cells were also counterstained with Hoechst 33342. Results obtained in TFTS66 (0, 0.1 and 1.0 ng/ml Dox) and its parental line, DLD-1, are shown (magnification X100).