Fig 4. Involvement of the inhibition of Stat3-mediated transcription in MK591 treatment-induced decrease in c-Myc levels 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 cycloheximide or MK591 as indicated at 37^°C. In (a), time-dependent changes in the levels of c-Myc proteins are shown by Western blot. Beta-actin was used as loading control. Note: MK591 treatment-induced decrease in c-Myc level is slower than cycloheximide treatment. In (b), time-dependent change in c-Myc protein level is shown after treatment of cells with Actinomycin D (a transcription inhibitor) only. In (c), effect of MK591 on c-Myc mRNA expression is shown by real-time PCR in a time-dependent manner. Note: A decrease in mRNA expression (~increased CT value) of c-Myc after MK591 treatment was detected by calculating the change in expression (delta-CT) deducting control values using the same PCR probe set. Normalization of expression was done by using GAPDH as internal control. In (d), effect of MK591 on Stat3-luciferase activity was measured in a time-dependent manner. Stattic (a Stat3 inhibitor) was used as positive control. ** p = <0.005 and *** p = <0.0005. In (e and f), effects of Stattic on protein level and transcriptional activities of c-Myc were detected by Western blot and E-box-luciferase assays respectively. Note: MK591 was also used in parallel experiments for side-by-side comparison with Stattic.