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. Author manuscript; available in PMC: 2014 Sep 1.
Published in final edited form as: Cancer Discov. 2013 Jun 13;3(9):1044–1057. doi: 10.1158/2159-8290.CD-12-0592

Figure 4. TLOC1 increased the cap versus IRES-dependent translation ratio by decreasing IRES-dependent translation.

Figure 4

(A) Illustration of the bi-cistronic translation reporter used to measure translation levels. The renilla luciferase reporter is driven by cap-dependent translation and the firefly luciferase by IRES-dependent translation. (B) TLOC1 over expression significantly increased the ratio of cap-dependent versus IRES-dependent translation by inhibition of IRES-dependent translation (p=0.01, student’s t-test). The ratio of cap/IRES-dependent translation was calculated and is illustrated in the right graph. (C) Overexpression of the transforming TLOC1 truncation mutant ΔC27 significantly (p=0.03, student’s t-test) inhibited IRES-dependent translation as compared to the non-transforming variant ΔN149. (D) Suppression of DDX3X in TLOC1 overexpressing cells significantly reversed the TLOC1 induced change in translation ratio (p=0.03, student’s t-test). (E) TLOC1 and DDX3X binds to 7-methylated GTP beads, and TLOC1. (F) TLOC1 overexpression increased EIF4G protein expression. (G) TLOC1 overexpression in HMLE-MEKDD cells decreased EIF4EBP1-phosphorylation on Threonine 37.45 and Serine 65. Lysates were prepared from TLOC1 or BFP overexpressing HMLE-MEKDD cells, which had been grown with (+GFs) or without (−GFs) growth factors for 24 hrs. Error bars = 1 S.D.