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. 2009 Nov;23(11):1799–1814. doi: 10.1210/me.2009-0165

Fig. 8.

Fig. 8.

RA treatment, through the activation of signaling pathways, influences the regulation of mRNA translation. A, Schematic representation of the reporter genes used for the analysis of mRNA translation regulation. [Adapted from Ref. 26 .]. B, The effect of the RA treatment on the translation of the LCS-EDA luciferase reporters. Neuroblastoma cells were transfected in parallel reactions with either the LCS-EDA or the LCS-EDAmt luciferase reporters, together with a promoterless Renilla luciferase reporter as internal control. After transfection, neuroblastoma cells were treated with 1 μm RA in the presence of LY294002 (10 μm) or U0126 (10 μm) as indicated. The ratio between the activities of the LCS-EDA and the LCS-EDAmt reporters, normalized for the efficiency of the transfection, is represented for control cells and cells treated with RA (1 μm, 24 h) alone (solid bars) or in the presence of the inhibitors LY294002 (10 μm, light gray bars) and U0126 (10 μm, dark gray bars) as indicated. Asterisk on RA-treated sample indicates a statistically significant difference with respect to control (P = 0.004). All the other samples were not statistically different from control. (ANOVA plus Bonferroni post hoc test). C, RA-induced changes in the phosphorylation of eIF4E-BP1. Cells were treated with RA (1 μm, 15 min) alone or in the presence of the inhibitors LY294002 (10 μm) and U0126 (10 μm) as indicated, and cell extracts were prepared. The phosphorylation state of 4E-BP1 was analyzed by Western blot with specific antibodies against the phosphorylated form of the protein (P-4E-BP1). The filter was reprobed finally with antibodies against total 4E-BP1 (4E-BP1). Each lane contains 30 μg of total protein. D, Effect of PI3K and MEK inhibitors in the activation of mTOR and p70S6 kinases by RA. Cells were treated with RA (1 μm, 15 min) alone or in the presence of the inhibitors LY294002 (10 μm) and U0126 (10 μm) as indicated, and whole-cell extracts were prepared. The phosphorylation state of Akt, mTOR, and p70S6 kinases was analyzed by Western blot with specific antibodies against the phosphorylated forms of the kinases (P-Akt, P-mTOR, and P-p70S6). The filter was reprobed finally with antibodies against total Akt (Akt). Each lane contains 30 μg of total protein. LY, LY294002.