prl tk cxcr4 6x reporter plasmid  (Addgene inc)

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: WT or GRE deletion mutant CRLF1 and SNAI1 3′ UTRs were fused downstream of the Renilla luciferase coding sequence in the broadly used pRL-TK CXCR4 6x reporter plasmid ([ ], Plasmid #11 308, Addgene, Cambridge, MA) as described before [ ].

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: WT or GRE deletion mutant CRLF1 and SNAI1 3′ UTRs were fused downstream of the Renilla luciferase coding sequence in the broadly used pRL-TK CXCR4 6x reporter plasmid ([ ], Plasmid #11 308, Addgene, Cambridge, MA) as described before [ ].

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

Journal: International Journal of Biological Sciences

Article Title: The Interaction of CircESR1 and HNRNPAB Regulates Cell Cycle Transition of Breast Cancer Cell

doi: 10.7150/ijbs.126014

Figure Lengend Snippet: Estrogen promotes HNRNPAB expression via SP1. (A) Immunoblot assessment of HNRNPAB in 2 human normal breast epithelial cell lines and 11 human BC cell lines. (B) CPTAC database analyzed the relative HNRNPAB and ERα protein level in 105 BC patients. P value was determined by Pearson correlation analysis. (C) The relative expression of TFF1 and HNRNPAB mRNAs after stimulated with 10 nM E 2 in estrogen-deprived MCF-7 cells for 48 h analyzed by qRT-PCR. (D) Immunoblot assessment of HNRNPAB expression in MCF-7 with or without 10 nM E 2 in estrogen-deprived MCF-7 cells for 72 h, or in short-term oestrogen deprivation (STED) MCF-7 cells for 7 days and parental cells. (E) The schematic of HNRNPAB promoter region sequence selected from the transcription start site of -2 kb~+100 bp. (F) MCF-7 cells were transfected with pGL3 basic reporter vector containing HNRNPAB promoter, and pRL-TK reporter control vector containing luciferase activity, as well as control siRNA or different siRNA sequences of JUN , FOS , FOXA1 and SP1 . The relative fluorescence activity was measured. (G) CPTAC database analyzed the relative HNRNPAB and SP1 protein level in 68 ER+ BC patients. P value was determined by Pearson correlation analysis. (H) 293T cells were transfected with control or SP1 vector, and pRL-TK reporter control vector containing luciferase activity, as well as pGL3 basic reporter vector containing different regions of HNRNPAB promoter. The relative fluorescence activity was measured. (I) The schematic of three predicted conserved SP1 binding sites on the upstream -425 bp~+100 bp regions of the human HNRNPAB gene. Red boxes represent the predicted SP1 binding sites. (J) The DNA regions enriched by SP1 in ChIP assay were verified by PCR. (K) 293T cells were transfected with control or SP1 vector, and pRL-TK reporter control vector containing luciferase activity, as well as pGL3 basic reporter vector or containing “b” or “b” mutant regions. The relative fluorescence activity was measured. (L) The relative expression of SP1 and HNRNPAB mRNAs in MCF-7 cells bearing control or SP1 shRNAs analyzed by qRT-PCR. (M) Immunoblot assessment of SP1 and HNRNPAB expression in MCF-7 cells with or without SP1 shRNAs in the presence or absence of 10 nM E 2 for 48 h. Data was shown as mean ± S.D. from three independent experiments. Unpaired two-tailed Student's t test (C, H, K) and one-way ANOVA followed by Tukey's multiple comparisons test (F, L). ns, P >0.05; ***, P <0.001; ****, P <0.0001.

Article Snippet: For transcription factor mediated HNRNPAB expression, 0.2 μg pGL3 Basic luciferase reporter (RRID: Addgene_48743), 50 nmol JUN or FOS or FOXA1 or SP1 siRNAs and 0.02 μg pRL-TK plasmid (RRID: Addgene_11313) were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Sequencing, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay, Fluorescence, Binding Assay, Mutagenesis, Two Tailed Test