Journal: The Journal of Experimental Medicine

Article Title: Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas

doi: 10.1084/jem.20110283

Figure Lengend Snippet: STAT3 somatic mutations identified in inflammatory hepatocellular tumors

Article Snippet: A full-length STAT3 open reading frame cloned in pCMV6-XL4 vector was purchased from OriGene (SC124165; available from GenBank/EMBL/DDBJ under accession no. NM_139276 ).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas

doi: 10.1084/jem.20110283

Figure Lengend Snippet: Gain-of-function mutations in STAT3 in IHCA. (A) Distribution of STAT3 mutations identified in IHCA is represented according to the different protein domains. Asterisk indicates the two mutations that are found on the same allele in tumor #379. Stat3-C location is indicated in gray. (B) STAT3 mutants L78R (L 78 ), E166Q (E 166 ), Y640F (Y 640 ), D502Y K658Y (D 502 /K 658 ), G656_Y657insF (G 656 ), Y657_M660dup (Y 657 ), and Stat3-C (S C ) or control STAT3 WT (WT) and empty plasmid (EP) were transfected in Hep3B, HepG2, and Huh7 cells expressing a STAT3-driven luciferase (Luc) reporter construct. STAT3 activation was measured after 6 h of serum starvation and, when indicated, was treated for 3 h with 100 ng/ml IL-6. Shown is the Luc activity (mean) determined from triplicate co-transfections (± SD) relative to pSIEM-luc alone (EP) without IL-6. (C) Graphs are qRT–PCR results showing the induction of endogenous CRP , STAT3 , and SOCS3 mRNA after overexpression of mutant STAT3 relative to unstimulated EP-transfected Hep3B cells (EP; mean ± SD). (D) Endogenous CRP mRNA expression in Hep3B cells transfected with increasing amounts of expression plasmids encoding WT or E 166 STAT3 mutant (dotted line: mock-transfected cells). (E) Activity of the STAT3 mutants, including D502Y (D 502 ), K658Y (K 658 ), and the double mutant D502Y K658Y (D 502 /K 658 ) were evaluated using STAT3-driven Luc in Hep3B cell line. Data are from triplicate transfections (± SD) relative to pSIEM-luc alone (EP). *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student’s t test.

Article Snippet: A full-length STAT3 open reading frame cloned in pCMV6-XL4 vector was purchased from OriGene (SC124165; available from GenBank/EMBL/DDBJ under accession no. NM_139276 ).

Techniques: Plasmid Preparation, Transfection, Expressing, Luciferase, Construct, Activation Assay, Activity Assay, Quantitative RT-PCR, Over Expression, Mutagenesis, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas

doi: 10.1084/jem.20110283

Figure Lengend Snippet: Subcellular localization and role of tyrosine 705 phosphorylation in STAT3 IHCA mutants. (A) Immunochemistry of phospho-STAT3-Y705 in STAT3-mutated IHCA and adjacent nontumor liver (NTL). (B–D) Vectors driving the expression of STAT3 mutants, including L78R (L 78 ), E166Q (E 166 ), Y640F (Y 640 ), G656_Y657insF (G 656 ), or WT (WT) STAT3, were transfected into Hep3B cells. (B) Expression of total and phosphorylated ( p Y705) STAT3 proteins were assessed by Western blot and bands were quantified with ChemiDoc using the Quantity One software; bars show the expression of p Y705 STAT3 relative to total STAT3. (C and D) Subcellular localization of total and phosphorylated ( p Y705) STAT3 proteins were analyzed by Western blot (C) and by immunofluorescence (D). Results are representative of three independent experiments. Bars, 20 µm. (E) CRP mRNA expression after transfection of WT, Y705F (Y 705 ), Y640F (Y 640 ), Y640F/Y705F (Y 640 /Y 705 ), G656_Y657insF (G 656 ), or G656_Y657insF/Y705F (G 656 /Y 705 ) STAT3 in unstimulated Hep3B cells. Data are from triplicate transfections (± SD) relative to EP. *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student’s t test.

Article Snippet: A full-length STAT3 open reading frame cloned in pCMV6-XL4 vector was purchased from OriGene (SC124165; available from GenBank/EMBL/DDBJ under accession no. NM_139276 ).

Techniques: Expressing, Transfection, Western Blot, Software, Immunofluorescence, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas

doi: 10.1084/jem.20110283

Figure Lengend Snippet: Dimerization of STAT3 IHCA mutants. (A) Flag- and Myc-tagged constructs encoding either WT or mutant Y640 STAT3 were co-transfected (1:1) into Hep3B cells treated, or not treated, with 100 ng/mlIL-6. STAT3 dimer formation was detected after immunoprecipitation using the anti-Flag antibody, followed by immunoblot (IB) analysis with Myc, Flag, and p Y705-STAT3–specific antibodies. Results are representative of three independent experiments. (B) Effects of increasing WT STAT3 expression (wedge) on the constitutive activity of Y 640 mutant STAT3 without IL-6. The graph plots the mean level of expression of SOCS3 and CRP relative to cells transfected with EP (± SD). Units are plasmid micrograms. (C) Several STAT3 IHCA mutations cluster near the TAD of STAT3 (crystal structure; NCBI Protein database accession no. 1BG1; ). Residues 640 and 656–660 (shown in green), which are mutated in IHCA tumors, the phosphorylated tyrosine (Tyr-705, black), and TAD residues 711–716 (blue), are shown in stick representation. One polypeptide chain and both TADs are shown as a cartoon. The twofold related molecule, except the TAD, is shown as a surface representation. For residues of the TAD, Tyr-710 of the twofold related molecule is labeled, whereas residues 711–716 are not, for clarity. (D) DNA-binding activity of 10 µg of nuclear extracts of Hep3B cells engineered to express WT, Y 640 , or G 656 STAT3 mutants. Bars indicate the DNA-binding activity to consensus STAT3 oligonucleotides relative to activity measured in cells transfected with EP. *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student’s t test. Data are from triplicate transfections (±SD).

Article Snippet: A full-length STAT3 open reading frame cloned in pCMV6-XL4 vector was purchased from OriGene (SC124165; available from GenBank/EMBL/DDBJ under accession no. NM_139276 ).

Techniques: Construct, Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Expressing, Activity Assay, Plasmid Preparation, Labeling, Binding Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas

doi: 10.1084/jem.20110283

Figure Lengend Snippet: Role of IL-6–JAK pathway and Src kinases on STAT3 IHCA mutant activation. STAT3 mutants Y 640 and E 166 were overexpressed in Hep3B cells. Activation of STAT3 is shown by Luc activity (mean) determined from triplicate transfections (± SD) relative to pSIEM-luc alone (EP) with serum starvation. (A) Hep3B cells were exposed to increasing concentrations of IL-6. (B) STAT3 mutants (E 166 and Y 640 ) or empty vector (EP) were co-transfected with JAK1 siRNA (+) or control siRNA (−) into Hep3B cells exposed to 100 ng/ml IL-6. (C) STAT3 mutants (E 166 and Y 640 ) or empty vector (EP) were co-transfected with gp130 siRNA (+) or control siRNA (−) into Hep3B cells. (D) STAT3 mutants (E 166 and Y 640 ) or empty vector (EP) were co-transfected with JAK2 siRNA (+) or control siRNA (−) into Hep3B cells. (E) STAT3 mutants (E 166 and Y 640 ) or empty vector (EP) were co-transfected with JAK1 siRNA (+) or control siRNA (v) into Hep3B cells. (F) STAT3 mutants (E 166 and Y 640 ) or empty vector (EP) were transfected into Hep3B cells exposed to Src inhibitor 1 (SrcI-1; 10 µM; 9 h). *, P < 0.05; **, P < 0.01; ***, P < 0.001, two-tailed Student’s t test. Data are from triplicate transfections (± SD). (G) Expression profiles of STAT3-mutated IHCA and gp130-mutated IHCA. Comparison was performed for the 28 genes validated by qRT-PCR that were significantly differentially expressed between IHCA and nontumorous livers. 5 STAT3-mutated IHCAs (black) were compared with 11 gp130-mutated IHCAs (in white). Results are expressed as mean ± SD; differences between groups are not significant (two-tailed Mann-Whitney test).

Article Snippet: A full-length STAT3 open reading frame cloned in pCMV6-XL4 vector was purchased from OriGene (SC124165; available from GenBank/EMBL/DDBJ under accession no. NM_139276 ).

Techniques: Mutagenesis, Activation Assay, Activity Assay, Transfection, Plasmid Preparation, Two Tailed Test, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Scientific Reports

Article Title: EGF-mediated reduced miR-92a-1-5p controls HTR-8/SVneo cell invasion through activation of MAPK8 and FAS which in turn increase MMP-2/-9 expression

doi: 10.1038/s41598-020-68966-4

Figure Lengend Snippet: Effect of STAT1 and STAT3 silencing and inhibition by Fludarabine and Stattic in HTR-8/SVneo cells on the expression of miR-92a-1-5p in presence or absence of EGF. ( a ) Expression of miR-92a-1-5p by qRT-PCR in HTR-8/SVneo cells transfected with STAT1 siRNA treated with or without EGF for 24 h as compared to untreated scrambled control siRNA transfected cells. ( b ) Expression of miR-92a-1-5p by qRT-PCR in HTR-8/SVneo cells transfected with STAT3 siRNA treated with or without EGF for 24 h as compared to untreated scrambled control siRNA transfected cells. ( c ) Expression of miR-92a-1-5p by qRT-PCR in HTR-8/SVneo cells pre-treated with or without Fludarabine (4 h) and subsequently treated with or without EGF for 24 h. ( d ) Expression of miR-92a-1-5p by qRT-PCR in HTR-8/SVneo cells pre-treated with or without Stattic (1 h) and subsequently treated with or without EGF for 24 h. Each bar represents relative expression after normalization with the miR-191 used as an internal miRNA normalizer. Data in ( a ), ( b ), ( c ) and ( d ) is mean ± SEM of three independent experiments performed in triplicates. Synthetically synthesized STAT3 binding sites present in the promoter region of miR-92a-1-5p (BS1 corresponding to 492–510 nt and BS2 corresponding to 777–795 nt position from miRNA precursor encoding region, table and ) were cloned upstream of the PKG promoter of the firefly luciferase gene in pmirGLO Dual-Luciferase vector. ( e ) shows the relative luciferase activity determined from HEK-293T cells co-transfected with clones possessing the synthetically synthesized STAT3 binding sites in pmirGLO Dual-Luciferase vector with STAT3 mammalian expression vector, as indicated. The data are represented as the mean of three experiments ± S.E.M. performed in duplicates.

Article Snippet: Alternately, HEK-293T cells were co-transfected with 250 ng of luciferase reporter plasmid harboring the STAT3 binding site along with 310 ng of STAT3 mammalian expression vector (OriGene Technologies, MD, USA) using Lipofectamine RNAiMAX reagent in OptiMEM.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Transfection, Synthesized, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay

Journal: Scientific Reports

Article Title: EGF-mediated reduced miR-92a-1-5p controls HTR-8/SVneo cell invasion through activation of MAPK8 and FAS which in turn increase MMP-2/-9 expression

doi: 10.1038/s41598-020-68966-4

Figure Lengend Snippet: Graphical representation of the effect of reduced miR-92a-1-5p expression during EGF-mediated increased HTR-8/SVneo cell invasion. EGF leads to reduced expression of miR-92a-1-5p in HTR-8/SVneo trophoblastic cells; reduced miR-92a-1-5p leads to increased invasion of HTR-8/SVneo cells by targeting MAPK8 and FAS, which increases expression of MMP-2 & MMP-9, and decreases expression of TIMP1. EGF activated STAT1 and STAT3 may lead to reduced expression of miR-92a-1-5p by binding to the miRNA precursor encoding region as inhibition of STAT1 and STAT3 abrogates reduction of miR-92a-1-5p expression in EGF treated HTR-8/SVneo cells.

Article Snippet: Alternately, HEK-293T cells were co-transfected with 250 ng of luciferase reporter plasmid harboring the STAT3 binding site along with 310 ng of STAT3 mammalian expression vector (OriGene Technologies, MD, USA) using Lipofectamine RNAiMAX reagent in OptiMEM.

Techniques: Expressing, Binding Assay, Inhibition