Figure 5. Effects of silvestrol and BEZ235 on translation of MYC.
A. Incorporation of 35S-labeled methionine in SW480 cells treated with BEZ235 (200nM), Silvestrol (25nM), DOX or solvent control for 24h. Shown are mean+SD (n=3).
B. Polysome fractionation of SW480 cells (Top left), treated with BEZ235 (200nM), DOX, Silvestrol (25nM) or solvent control for 24 h. RNA was isolated from the indicated fractions and relative mRNA content per fraction was measured by RQ-PCR. Top right MYC mRNA distribution, bottom left ACTB mRNA and bottom right TUBB3 mRNA distribution (n=2).
C. Schematic illustration describing the luciferase reporter systems used in (D), (E) and (F). The pmF reporter construct contains the MYC 5’UTR inserted into the control vector pGL3 (Promega) proximal to firefly luciferase coding sequence. The bicistronic pRmF and pRhcvF reporter constructs contain the MYC or the HCV (hepatitis C virus) IRES sequence distal to renilla and proximal to firefly luciferase gene.
D. SW480 cells were transfected with pmF luciferase reporter and treated with BEZ235 (200nM), DOX, silvestrol (25nM), Cymarin (100nM) or solvent control for 24h. Luciferase activity is shown relative to a co-transfected beta-Gal reporter (n=3).
E. SW480 cells were transfected with pRmF luciferase reporter and treated with silvestrol (25nM) or solvent control. Relative firefly luciferase activity was calculated using the ratio of firefly to renilla luciferase (n=3).
F. SW480 cells were transfected with pRhcvF luciferase reporter and analyzed as in panel E (n=3).