mcf10a  (ATCC)

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: CELF interacts with eIF4E at the m 7 G cap, independent of intact eIF4G1. ( a ) Reporter assay quantifying the relative Renilla luciferase expression from the indicated 3′ UTR luciferase reporters in untreated and TGF-β-treated MCF-10A cells. Data were normalized to Firefly luciferase expression and are presented as fold change of this normalized signal relative to CXCR4 reporter in untreated MCF-10A cells. ( b ) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of indicated Renilla luciferase reporters (pRL-TK) in untreated and TGF-β-treated MCF-10A cells. Data were normalized to endogenous ACTB expression. ( c ) As in (a), with the indicated 3′ UTR luciferase reporters driven from an EMCV internal ribosomal entry site (IRES) in untreated and TGF-β-treated MCF-10A cells. ( d ) As in (b), for reporter assays in panel (c). ( e ) Right six lanes—immunoblots of indicated immunoprecipitates from whole-cell lysates derived from MCF-10A cells treated with TGF-β for 72 h. One half of each total immunoprecipitate was digested with RNase A prior to immunoblotting with the indicated antibodies. Right six lanes—as in the left six lanes, but lysates were digested with coxsackievirus 2A protease to cleave eIF4G1 before immunoprecipitation. CT = C-terminal; FL = full length; NT = N-terminal. ( f ) m 7 GTP cap analog binding assays utilizing cytosolic extracts derived from MCF-10A cells treated with TGF-β for 72 h. As above, one half of each extract was digested with coxsackievirus 2A protease to cleave eIF4G1 before the assay. ( g ) Proximity ligation assays using the indicated pairs of antibodies on MCF-10A cells treated with TGF-β for 72 h. In all panels, results are representative of at least three independent experiments and error bars depict mean ± standard deviation (SD) of aggregate replicates performed in triplicate. NS: not significant; * P -value < 0.05 (Student’s t-test).

Article Snippet: The MCF-10A cell line was obtained from the ATCC (Manassas, VA) and cultured as described previously [ ].

Techniques: Reporter Assay, Luciferase, Expressing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Derivative Assay, Immunoprecipitation, Binding Assay, Ligation, Standard Deviation

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: CELF1 stimulates translation of GRE-containing EMT effector mRNAs in the context of reduced eIF4G1 function. ( a ) RNA crosslinking-immunoprecipitation/qRT-PCR of GRE-containing mRNAs ( EGR3, FOSB, JUNB, SNAI1 ) from TGF-β-treated MCF-10A cells using anti-CELF1, anti-eIF4E, and anti-eIF4G1 antibodies or mouse and rabbit IgG. ACTB and GAPDH are non-GRE-containing negative control mRNAs. ( b ) RNA crosslinking-immunoprecipitation/qRT-PCR of GRE-containing mRNAs ( EGR3, FOSB, JUNB, SNAI1 ) from TGF-β-treated MCF-10A cells using tandem anti-eIF4E/anti-CELF1 immunoprecipitation, tandem anti-eIF4E/anti-eIF4G1 immunoprecipitation, or tandem immunoprecipitation with mouse and rabbit IgGs. ACTB and GAPDH are non-GRE-containing negative controls. ( c, d ) Efficiency of in vitro translation of indicated capped and polyadenylated Renilla luciferase reporter mRNAs in mock or 2A protease-digested cell-free extract. ( e, f ) Efficiency of in vitro translation of reporter mRNAs as described in panels (c) and (d), but with mock-depleted ( Beads ) cell-free extract, eIF4G1-immunodepleted ( ID ) cell-free extract, or eIF4G1-immunodepleted cell-free extract reconstituted by addition of 20 nM enriched eIF4G1 and/or an equivalent concentration of recombinant CELF1. In panels (c–f), all extracts were derived from TGF-β-treated MCF-10A cells transiently transfected with shRNAs targeting either GLB1 (c, e) or CELF1 (d, f). CXCR4 = control, WT = wild-type 3′ UTR, ΔGRE =3′ UTR with deletion of GRE. In all panels, results are representative of at least three independent experiments. Error bars depict mean ± standard deviation (SD) of aggregate replicates performed in triplicate. NS: not significant; * P -value < 0.05 ( a, b, e, f : ANOVA with Dunnet’s post-hoc test; c, d : Student’s t-test).

Article Snippet: The MCF-10A cell line was obtained from the ATCC (Manassas, VA) and cultured as described previously [ ].

Techniques: Cross-linking Immunoprecipitation, Quantitative RT-PCR, Negative Control, Immunoprecipitation, In Vitro, Luciferase, Concentration Assay, Recombinant, Derivative Assay, Transfection, Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: Phosphorylation of eIF4E is required for CELF1-driven EMT in MCF-10A cells. ( a ) Immunoblots of lysates derived from MCF-10A cells stably expressing either HA-tagged WT or S209A mutant murine EIF4e and shRNA targeting human EIF4E or control shRNA, and either mock transfected or transiently transfected with a CELF1 overexpression construct for 72 h. GAPDH = loading control. ( b ) Immunoblot of indicated immunoprecipitates from lysates derived from TGF-β-treated MCF-10A cells, stably expressing either HA-tagged WT or S209A mutant murine Eif4e and shRNA targeting human EIF4E or control shRNA. IgG: negative immunoprecipitation control. ( c ) Polysomal profiles from MCF-10A cells in which endogenous EIF4E expression had been knocked down via shRNA and then rescued via stable transduction of either WT or S209A mutant Eif4e . ( d ) qRT-PCR validation of polyribosomal enrichment and depletion of indicated mRNAs via total and polysomal mRNA from MCF-10A cells stably expressing WT or S209A mutant Eif4e , treated with TGF-beta for 72 h. ( e ) MCF-10A cells in which endogenous EIF4E expression had been knocked down via shRNA and then rescued via stable transduction of either WT or an S209A mutant Eif4e were transiently transfected with CELF1 expression construct. After 72 h, extracts were assessed via immunoblot for relative protein expression of CELF1-regulated EMT effectors. In all panels, results are representative of at least three independent experiments. Error bars in panel (d) depict mean ± standard deviation (SD). NS: not significant; * P -value < 0.05 (Student’s t-test).

Article Snippet: The MCF-10A cell line was obtained from the ATCC (Manassas, VA) and cultured as described previously [ ].

Techniques: Phospho-proteomics, Western Blot, Derivative Assay, Stable Transfection, Expressing, Mutagenesis, shRNA, Control, Transfection, Over Expression, Construct, Immunoprecipitation, Transduction, Quantitative RT-PCR, Biomarker Discovery, Standard Deviation

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: CELF1 directly binds eIF4E via interactions via the canonical dorsal cleft region and the lateral hydrophobic patch. ( a ) Schematic of CELF1 domain structure and candidate eIF4E binding motifs. RRM, RNA-recognition motif. ( b ) Immunoblots of immunoprecipitations from lysates of MCF-10A cells transfected with WT or indicated mutant GFP-CELF1 plasmids for 72 h. ( c ) Immunoblots of binding assays using affinity-purified phosphomimic eIF4E ( GST-EIF4E S209D ), affinity-purified WT CELF1 ( 6xHis-CELF1 ), or affinity-purified mutant CELF1 ( 6x-His-CELF1 Δ365–71 ). ( d ) Immunoblots of immunoprecipitations derived from lysates of MCF-10A cells stably expressing an shRNA targeting the 3′ UTR of EIF4E and co-expressing either WT or EIF4E W73A mutant, treated with TGF-β or transiently transfected with a GFP-CELF1 plasmid for 72 h. IgG: negative control. ( e ) Immunoblots of binding assays in which affinity-purified 6xHis-CELF1 was mixed with affinity-purified phosphomimic ( GST-EIF4E S209D ) or mutant ( GST-EIF4E S209D/W73A ) eIF4E. ( f ) HSQC-NMR spectra of 15 N-labeled eIF4E S209D , alone or mixed with a seven-fold excess of CELF1 YAAAALP-containing peptide (sequence shown). Representative shifts are magnified. ( g ) Dorsal surface view of the crystal structure of eIF4E complexed with m 7 GTP (PDB 1IPC - ), depicting chemical shifts observed in HSQC-NMR. The canonical eIF4E binding cleft is colored in red, and chemical shifts induced by the CELF1 peptide are indicated in blue. Shifts overlapping the canonical binding cleft are indicated in purple. ( h ) As in (g), rotating the eIF4E structure ninety degrees along a roughly fifteen-degree bearing for depiction of the lateral surface mediating non-canonical binding. Coloring and annotations are as in (g). ( i ) Purified, untagged CELF1 was mixed with purified eIF4E, eIF4E S209D , eIF4E S209D/W73A (disrupts canonical dorsal binding), or eIF4E S209D/I63A/I79A (disrupts non-canonical lateral binding) and then immunoprecipitated with IgG (negative control) or anti-CELF1 antibody and immunoblotted with the indicated antibodies. Results in (a–e, i) are representative of at least three individual experiments.

Article Snippet: The MCF-10A cell line was obtained from the ATCC (Manassas, VA) and cultured as described previously [ ].

Techniques: Binding Assay, Western Blot, Transfection, Mutagenesis, Affinity Purification, Derivative Assay, Stable Transfection, Expressing, shRNA, Plasmid Preparation, Negative Control, Labeling, Sequencing, Purification, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: Interaction of CELF1 and eIF4E is required for CELF1-driven EMT and experimental metastasis. ( a ) Immunoblot analysis of indicated EMT markers in lysates derived from MCF-10A cells transfected with WT or indicated mutant GFP-CELF1 plasmids for 72 h. ( b ) Immunoblot analysis of indicated EMT markers in lysates derived from MCF-10A cells expressing either WT or W73A mutant human EIF4E and shRNA targeting the 3′ UTR of human EIF4E and induced to undergo EMT via stable expression of GFP-CELF1 or TGF-β treatment for 72 h. ( c ) Immunoblot analysis of indicated EMT markers and GFP-CELF1 in lysates derived from parental MCF-10AT1 cells ( left column ) and MDA-MB-468 ( right column ), or each cell line stably transduced with either WT or Δ365–71 mutant GFP-CELF1 . GAPDH = loading control in panels (a), (b), and (c); black line in panels (a) and (b) denotes lysates derived from the same experiment, but gels processed in parallel. All results (a–c) are representative of at least three independent experiments. Quantification of relative in vitro cellular migration ( d, f ) and invasion ( e, g ) in transwell assays in parental MCF-10AT1 and MDA-MB-468 cells, respectively, or stably transduced with either WT or Δ365–371 mutant GFP-CELF1 . Data represents mean ± SD of at least three independent experiments, each performed in triplicate. * P -value < 0.05 (ANOVA with Dunnet’s post-hoc test). ( h, i ) Parental MCF-10AT1 cells, or cells stably overexpressing either WT or Δ365–71 mutant GFP-CELF1 , were injected into the tail vein of athymic nude mice. The incidence and progression of metastasis were measured by luciferin injection and bioluminescence imaging of Firefly luciferase (h), and ex vivo excised lungs on day 15 (i). ( j ) Representative hematoxylin and eosin (H&E) ( top ) and immunohistochemical (IHC) ( bottom ) staining, respectively, of the lungs from mice shown in panel (h). Scale bar, 200 µm ( top ); 50 µm ( bottom ). Black arrows ( bottom ) indicate micrometastases. Dotted lines indicate area shown in corresponding H&E staining of serial sections shown in panel (j). For (h–j), representative images are from n = 4 for parental, n = 4 for WT GFP-CELF1 , and n = 6 for mutant GFP-CELF1 Δ365–71 experimental groups.

Article Snippet: The MCF-10A cell line was obtained from the ATCC (Manassas, VA) and cultured as described previously [ ].

Techniques: Western Blot, Derivative Assay, Transfection, Mutagenesis, Expressing, shRNA, Stable Transfection, Transduction, Control, In Vitro, Migration, Injection, Imaging, Luciferase, Ex Vivo, Immunohistochemical staining, Staining