Figure 4: CELF1 is a major regulator of GRE-containing mRNAs encoding drivers of EMT.

(a) Immunoblot analysis of CELF and MBNL proteins in untreated and TGF-β-treated MCF7 and MCF10A cells. HSP90 serves as a loading control. (b) Immunoblot analysis of epithelial and mesenchymal markers in untreated and TGF-β-treated MCF10A cells transiently transfected with the indicated siRNAs. HSP90 serves as a loading control. (c) Quantification of relative cellular migration and invasion in transwell assays in untreated and TGF-β-treated MCF10A cells transiently transfected with the indicated siRNAs or overexpression constructs. (d) Immunoblot analysis of epithelial and mesenchymal markers in untreated and TGF-β-treated MCF10A cells transiently transfected with vehicle or CELF1 overexpression construct. HSP90 serves as a loading control. (e) Quantification of relative cellular migration and invasion in transwell assays in untreated and TGF-β-treated MCF10A cells transiently transfected with vehicle or CELF1 overexpression construct. (f) RNA crosslinking-immunoprecipitation/qRT-PCR of GRE-containing mRNAs from untreated and TGF-β-treated MCF10A cells using two different anti-CELF1 antibodies or mouse IgG. ACTB is a non-GRE-containing negative control. (g) RNA crosslinking-immunoprecipitation/qRT-PCR of tRFP reporters containing either the wild-type 3′-UTRs for the indicated genes or corresponding mutant 3′-UTRs in which the GRE has been deleted by site-directed mutagenesis. Reporters were immunoprecipitated from untreated and TGF-β-treated MCF10A cells using anti-CELF1 antibody or mouse IgG. (h) Immunoblot analysis of indicated epithelial and mesenchymal cell markers and CELF1 in untreated MCF10A cells 72 h post-transient transfection with CELF1 driven by the CMV Immediate Early promoter in combination with siRNAs targeting Firefly Luciferase or each of the ten GRE-containing mRNAs. HSP90 serves as a loading control. (i) Quantification of relative cellular migration and invasion in transwell assays in untreated MCF10A cells 72 h post-transient transfection with CELF1 driven by the CMV Immediate Early promoter in combination with siRNAs targeting Firefly Luciferase or each of the ten GRE-containing mRNAs. (j) Immunoblot analysis of indicated epithelial and mesenchymal cell markers and CELF1 in untreated MCF10A cells 72 h post-transient transfection with pL6.3 expression vectors engineered to overexpress Renilla Luciferase or each of the indicated individual GRE-containing coding sequences in combination with siRNA-targeting CELF1. HSP90 serves as a loading control. (k) Quantification of relative cellular migration and invasion in transwell assays (bottom) in untreated MCF10A cells 72 h post-transient transfection with pL6.3 expression vectors engineered to overexpress Renilla Luciferase or each of the indicated individual GRE-containing coding sequences in combination with siRNA-targeting CELF1. All panels 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. In c,e,i,k, error bars depict s.d. of the mean. In f,g error bars depict s.e.m. *P≤0.05 (Student’s t-test). See also Supplementary Fig. 3. Full scans of blots are shown in Supplementary Fig. 10.