Fig 3. Effects of MK591 on protein levels of c-Myc and targets in C4-2B cells.
C4-2B cells (~3 x 105 cells per plate) were plated in 60 mm diameter plates and allowed to grow for 48 hours. Then, the old medium was replaced with 2 ml fresh RPMI medium and the cells were treated with MK591 and incubated for 24 hours at 37°C. In (a and b), dose- and time-dependent changes in the levels of c-Myc proteins by treatment with MK591 or ibuprofen are shown by Western blot. Beta-actin was used as loading control. In (c), effect of MK591 on c-Myc-luciferase activity was measured in a time-dependent manner. *** p = <0.0005 and **** p = <0.00005. In (d), effects of MK591 on protein levels of c-Myc-targets are shown by Western blot. Note: Ibuprofen, an inhibitor of cyclooxygenase (30 μM), was used in parallel which showed no appreciable effects on protein levels of either c-Myc or its targets. In (e), time-dependent decrease in mRNA expressions of c-Myc targets was detected by real-time PCR. Change in expression (delta-CT) was calculated by deducting mean values of control for each gene using the same probe set. Normalization of expression was done by using GAPDH as internal control.