Figure 2. GREs confer context-dependent translational regulation to mRNA transcripts.

(a) MEME algorithm output of an enriched GRE within the 3′-UTRs of the putative positive regulator set and schematic representation of GRE location in 3′-UTR of indicated transcripts. (b) Immunoblot analysis of proteins encoded by GRE-containing mRNAs in untreated and TGF-β-treated MCF7 and MCF10A cells. HSP90 serves as a representative loading control. The relative changes in total mRNA expression (MCF10A±TGF-β) of each GRE-containing gene in total mRNA as quantified by qRT-PCR are indicated on the right. (c) Reporter assay quantifying the relative tRFP expression from 70 3′-UTR reporters in TGF-β-treated MCF10A cells relative to untreated controls. Wild-type (WT) and mutant (MUT) ZEB1 UTRs (black) serve as positive and negative controls, respectively. Data were normalized to relative tGFP expression. (d) Reporter assay quantifying the relative tRFP expression from indicated WT 3′-UTRs (black) and those in which the GRE (or miR-200 binding sites for ZEB1) (red) have been deleted in TGF-β-treated MCF10A cells relative to untreated controls. Data were normalized to relative tGFP expression. (e) qRT-PCR of relative mRNA expression of indicated WT and GRE-mutant tRFP reporters in untreated and TGF-β-treated MCF10A cells. (f) Reporter assay quantifying the relative Renilla luciferase expression from the indicated 3′-UTR luciferase reporters in TGF-β-treated MCF10A cells relative to untreated controls. Renilla activity was normalized to Firefly luciferase activity and this ratio is expressed relative to the parental control reporter (pRL-TK CXCR4 6x). (g) qRT-PCR of relative mRNA expression of indicated Renilla Luciferase reporters (pRL-TK) in untreated and TGF-β-treated MCF10A cells. (h) Reporter assay quantifying the relative Renilla luciferase expression from the indicated 3′-UTR luciferase reporters driven from an internal ribosomal entry site in TGF-β-treated MCF10A cells relative to untreated controls. Renilla activity was normalized to Firefly luciferase activity. Data are normalized to a control reporter containing the pRL-TK CXCR4 6x 3′-UTR. (i) qRT-PCR of relative mRNA expression of indicated Renilla luciferase reporters driven by EMCV IRES (pFR-EMCV) in untreated and TGF-β-treated MCF10A cells. All panels (excluding a) are representative of a minimum of three experimental replicates. For immunoblots depicted, samples were derived from the same experiment and gels were processed in parallel. Error bars in d depict s.d. of the mean, error bars in e,f,h depict s.e.m. NS indicates non-significant, *P≤0.05, (Student's t-test). Full scans of blots are shown in Supplementary Fig. 8.