Transient TEMPOL treatment of PC-3M cells induces uPAR transcript and protein expression. (A). TEMPOL was added to cells (time 0) and then thoroughly washed away after 24 hours. Proteins were isolated at the time of TEMPOL removal (i.e. 24 h) and at 48, 72 and 96 hours and these proteins were immunoblotted for uPAR expression (upper panel). β-actin loading is shown (lower panel). uPAR transcript expression relative to control (= to 1.0) measured using real-time PCR is shown beneath each band (mean transcript ± s. dev. (B). TEMPOL was added to cell cultures for either a 24 hour period followed by withdrawal for another 24-hour period (labeled as "T × 24 h wd × 24 h"), or for a 48-hour continuous period (labeled as "T × 48 h"). In both treatments, cells were lysed and proteins were extracted after 48 hours. Control cells were not treated. Upper panel shows uPAR protein expression; lower panel shows β-actin expression to verify equivalent loading. (C). NBT staining in untreated cells (left panel) and in cells treated with TEMPOL for 18 hours (right panel). Images photographed at 320× using varel contrast. (D). TEMPOL reduction of superoxide levels is reversed following its removal. Cells were stained using NBT beginning at the time points shown in order to detect intracellular superoxide levels. Bars reflect the degree of NBT staining as a percent of control (untreated) cells. During the 18 hour time point (cross-hatched bar), NBT was present in the cell culture. (E). uPAR cell surface staining (red) in control (left panel) vs. cells 48 hours after TEMPOL treatment (right panel) as above. Nuclei are shown following staining with DAPI (blue). Image taken at 630×.