anti-klf4 Search Results


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  • 97
    Abcam anti klf4
    Overexpression of Sox9 or <t>KLF4</t> concentration dependently inhibits TCF-luciferase activity. HEK293, SW480, A549, and T47D cells were co-transfected with either Sox9/TCF-luciferase or KLF4/lTCF-luciferase constructs at concentrations ranging from 0 to 200
    Anti Klf4, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology anti klf4
    Proteolytic regulation of <t>KLF4</t> by estrogen signaling contributes to its overexpression in breast cancer. A , KLF4 mRNA level is down-regulated in breast cancer. Oncomine was used to analyze the previously published microarray data. B , KLF4 protein level
    Anti Klf4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 333 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    klf4  (Abcam)
    84
    Abcam klf4
    Expression of Pluripotency-Associated Genes in Putative iPS Clones (A) (B) HCF- and BJ-derived iPS clones were analyzed for expression of pluripotency markers by immunostaining. HCF#1 and BJ#SA cells were positive for pluripotency markers SSEA4, TRA-1-60, TRA-1-81, OCT4, SOX2, <t>KLF4</t> and NANOG, while no notable staining was observed for SSEA1. Cells were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI). Control (m) and Control (r); control cells treated with FITC-conjugated secondary antibodies against mouse IgG and rabbit IgG. Scale bars indicate 20 µm. (C) HCF- and BJ-derived iPS-like clones were analyzed for pluripotency-associated gene expression by RT-PCR. Total cellular RNA from parental BJ and HCF fibroblasts and no template (water) samples were included as controls. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcript was amplified as an internal RNA control.
    Klf4, supplied by Abcam, used in various techniques. Bioz Stars score: 84/100, based on 1425 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GeneTex klf4
    Model of mechanism by which Mule may control T-cell proliferation through ubiquitination and degradation of <t>KLF4.</t> ( a ) WT T cells at steady-state. In the absence of antigenic stimulation, Mule expression in T cells is low due to self-ubiquitination and degradation and its E3 ligase activity is insufficient to remove KLF4. KLF4 transactivates E2F2, which acts as a transcriptional repressor together with CDKI p21 and p27 to block entry into the cell cycle. ( b ) Antigen-stimulated WT T cells. In response to TCR engagement by antigen, Mule expression is rapidly increased and sustained due to inhibition of its self-ubiquitination and degradation. Mule ubiquitinates KLF4 and promotes its degradation such that insufficient KLF4 remains to successfully transactivate E2F2, p21 and p27. T cells can thus transcribe genes promoting cell cycle entry. ( c ) In antigen-stimulated Mule -deficient T cells, KLF4 cannot be degraded. The accumulating KLF4 protein transactivates E2F2, p21 and p27, leading to repressed expression of cell cycle genes. These T cells then fail to proliferate efficiently.
    Klf4, supplied by GeneTex, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam anti klf4 antibody epr19590
    Model of mechanism by which Mule may control T-cell proliferation through ubiquitination and degradation of <t>KLF4.</t> ( a ) WT T cells at steady-state. In the absence of antigenic stimulation, Mule expression in T cells is low due to self-ubiquitination and degradation and its E3 ligase activity is insufficient to remove KLF4. KLF4 transactivates E2F2, which acts as a transcriptional repressor together with CDKI p21 and p27 to block entry into the cell cycle. ( b ) Antigen-stimulated WT T cells. In response to TCR engagement by antigen, Mule expression is rapidly increased and sustained due to inhibition of its self-ubiquitination and degradation. Mule ubiquitinates KLF4 and promotes its degradation such that insufficient KLF4 remains to successfully transactivate E2F2, p21 and p27. T cells can thus transcribe genes promoting cell cycle entry. ( c ) In antigen-stimulated Mule -deficient T cells, KLF4 cannot be degraded. The accumulating KLF4 protein transactivates E2F2, p21 and p27, leading to repressed expression of cell cycle genes. These T cells then fail to proliferate efficiently.
    Anti Klf4 Antibody Epr19590, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    klf4  (ATGen)
    90
    ATGen klf4
    Reprogramming HFFs using synthetic modified mRNA. (A) Synthetic modified mRNAs are translated into proteins. Cells transfected with mRNA cocktail expressing reprogramming factors <t>Klf4,</t> cMyc, Oct4, LIN28, and Sox2. The 15-h time point was chosen for analysis
    Klf4, supplied by ATGen, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Cell Signaling Technology Inc klf4
    <t>KLF4</t> suppressed HCC migration via regulation of MGLL. A. MGLL was knocked down in SNU449 and BEL7402 cells with or without KLF4 overexpression. Cell lysates were then subjected to western blotting analysis using the indicated antibodies. B-E. Effects of MGLL on migration were examined by transwell and wound healing assays. The results are representative of three independent experiments. *P
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    91
    Bioss anti klf4
    Immunofluorescence identification of the generated iPS. The newly derived mRNA iPS colonies were stained for the specialized markers of pluripotency: Oct4, Sox2, Nanog, SSEA-1, <t>Klf4</t> and c-Myc. Nuclei were counter-stained with DAPI; (magnification 200×).
    Anti Klf4, supplied by Bioss, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GeneTex anti klf4
    DDX3 suppresses stemness gene signature. ( a ) Lower expression of DDX3 along with overexpression of stemness markers was observed in the poorly differentiated cell line. Cell lysates (50 μg) of HepG2, Hep3B, HuH-7 and SK-Hep-1 cells were analyzed by western blotting with antibodies against DDX3, Nanog, Oct4, c-Myc, Sox2, <t>KLF4,</t> Bmi1, CK19 and β-actin. ( b ) Knockdown of DDX3 led to up-regulation of stemness markers. Cell lysates (50 μg) of stable shLuc, shDDX3 #2 and shDDX3 #3 HepG2 cells were analyzed by western blotting with antibodies described in ( a ). ( c ) DDX3 overexpression suppressed stemness markers. Plasmid pcDNA3-SRα/FLAG or pcDNA3-SRα/FLAG-DDX3 was transfected into SK-Hep-1 cells. At 48 h post transfection, cell lysates were prepared and subjected to immunoblotting with antibodies described in ( a ) and anti-FLAG antibody. β-actin was used as internal control in ( a – c ). ( d ) DDX3 knockdown was correlated with up-regulation of hepatic CSC surface markers. mRNA expressions of DDX3, CD133, CD13, EpCAM, CD90 and GAPDH in shLuc, shDDX3 #2 and shDDX3 #3 cells were detected by qRT-PCR. GAPDH was used as internal control. Fold change of each mRNA transcript in shDDX3 #2 and shDDX3 #3 cells was relative to that of shLuc cells. ( e ) DDX3 overexpression resulted in suppression of hepatic CSC surface markers. SK-Hep-1 cells were transfected with plasmid pcDNA3-SRα/FLAG or pcDNA3-SRα/FLAG-DDX3 as described in ( c ). At 48 h post transfection, total RNA was extracted and subjected to qRT-PCR analysis. GAPDH was used as internal control. Fold change of each mRNA transcript in FLAG-DDX3-expressing cells was relative to that of vector control cells. All experiments were performed at least three times, and the error bar indicates ± 1 s.d. of the mean. Statistical analyses were carried out using t test (* p
    Anti Klf4, supplied by GeneTex, used in various techniques. Bioz Stars score: 91/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Proteintech anti klf4
    <t>KLF4</t> is a direct target of miR-543 and KLF4 expression is inversely correlated with miR-543 expression in CRC tissues (A) Western blot analysis of KLF4 protein expression after transfection in HCT116 and SW-480 cells. (B) Luciferase activities of wild-type and the mutant pmirGLO- KLF4 -3′-UTR reporter in HCT116 and SW-480 cells. (C) The predicted miR-543 binding site on the KLF4 mRNA 3′-UTR and the corresponding mutations in 3′-UTR of KLF4 . (D, E) KLF4 expression on protein level (D) and mRNA level (E) was determined in CRC tissues (T) and adjacent nontumorous tissues (ANT). (F) Spearman's correlation analysis was performed to detect the association between the expression level of miR-543 and KLF4 in GC tissues. (G, H) KLF4 expression on protein level (G) and mRNA level (H) was determined in three CRC cell lines (HCT116, SW-480 and HT29) and nontumorous mucosa. Error bars represent mean ± SD from three independent experiments. * p
    Anti Klf4, supplied by Proteintech, used in various techniques. Bioz Stars score: 91/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GE Healthcare anti klf4
    Active p38/MAPK pathway is necessary for FBXO32-mediated <t>KLF4</t> degradation. ( a ) p38 inhibitor leads to KLF4 accumulation. MCF7 cells plated in six-well plate were treated with DMSO or indicated inhibitors for 6 h. The protein level of KLF4 was detected by immunoblot. ( b ) KLF4 protein half-life is elongated under p38 inhibition. MCF7 cells plated in six-well plate were treated with DMSO or p38 inhibitors (SB203580 and SB239063) for 6 h. Then CHX was added 0, 2, 4 or 6 h before harvest. KLF4 protein level was tested by immunoblotting. ( c ) p38 inhibitor abolishes FBXO32-mediated KLF4 degradation. HEK293T cells were transfected with His-tagged KLF4 and Myc-tagged FBXO32. Twenty-four hours after transfection, the cells were treated with DMSO or p38 inhibitor for 6 h followed by treatment with CHX for indicated time. Western blot was performed to examine the protein level of KLF4. ( d , f ) p38 inhibitor disrupts KLF4 and FBXO32 interaction. HEK293T cells were transfected with Flag-tagged KLF4 and Myc-tagged FBXO32 or empty vector. Twenty-four hours after transfection, the cells were treated with DMSO or SB203580 for 6 h followed by 4-h treatment with MG132. The cell lysates were precipitated with anti-Flag M2 affinity gel and the precipitates were analyzed by western blot ( d ). MCF7 cells cultured in 10 cm dishes were treated with either DMSO or SB203580 for 6 h. The cell lysates were subjected to co-IP analysis by KLF4 antibody. The immunoprecipitates were blotted by FBXO32 antibody ( f ). ( e, g ) p38 inhibitor decreased FBXO32-mediated KLF4 ubiquitination. Indicated plasmids were co-transfected into HEK293T cells. Twenty-four hours after transfection, the cells were treated with DMSO or SB203580 for 6 h followed by 6- h treatment with MG132. The cells were subjected to ubiquitination assay and the ubiquitinated KLF4 were detected by immunoblotting with anti-HA antibody ( e ). FBXO32 overexpressed MCF7 cells cultured in 10 cm dishes were treated with either DMSO or SB203580 for 6 h. The cell lysates were subjected to ubiquitination analysis. Ubiquitinated KLF4 proteins were revealed by immunoblotting with ubiquitin antibody ( g ).
    Anti Klf4, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    R&D Systems klf4
    Erk5 Signaling Stabilizes Naive Pluripotency and Suppresses Transition to Primed (A) Erk5 −/− mESCs were transfected with empty vector or Erk5 cDNA and Nanog, Oct4, Klf2, Rex1, and Esrrb mRNA levels determined by qRT-PCR following 3-day LIF withdrawal. Data represent average ± SD (n = 3). (B) Erk5 −/− mESCs were transfected with empty vector or Erk5 cDNA. Fgf5 and Brachyury mRNA levels were determined by qRT-PCR following 3 days in the presence or absence of LIF. Data represent average ± SD (n = 3). (C) Erk5 −/− mESCs were transfected with empty vector or Erk5 constructs and Klf2 protein expression determined by immunoblotting and normalized. Data are presented as average ± SD (n = 3). (D) Erk5 −/− mESCs were transfected with empty vector or Erk5 constructs and stimulated with H 2 O 2 , and Erk5 kinase activity was determined. Erk5 and Erk1/2 expression levels were determined by immunoblotting. Data represent average ± SD (n = 3). Intervening lanes were removed, indicated by a dotted line. (E) Erk5 +/+ mESCs were transfected with empty vector or Mek5DD cDNA. Nanog, Oct4, Klf2, Rex1, and Esrrb mRNA levels were then determined by qRT-PCR following 3 days of LIF withdrawal. Data represent average ± SD (n = 3). (F) Erk5 +/+ mESCs were transfected with empty vector or Mek5DD, and Fgf5 and Brachyury mRNA levels were determined by qRT-PCR after 4 or 5 days in the presence or absence of LIF, respectively. Data represent average ± SD (n = 3). (G) Stat3 pTyr705, total Stat3, Erk1/2 pThr202/Tyr204, and total Erk1/2 levels in Erk5 +/+ or Erk5 −/− mESC clones were determined by immunoblotting (n = 3). (H) Erk5 +/+ and Erk5 −/− mESCs were treated with 1 μM AZD4547 or ruxolitinib. Nanog, Dnmt3b, Klf2, <t>Klf4,</t> and Erk1/2 levels were then determined by immunoblotting. Intervening lanes were removed, as indicated by a dotted line (n = 3). See also Figure S2 .
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    92
    Epitomics anti klf4
    TXL increases the expression of tight junction proteins and <t>KLF4</t> in hypoxic aorta. A, Endothelial cells were treated with the indicated stimuli for 24 hours. Western blot analysis of VE-cadherin, beta-catenin, occludin, claudin-1, ZO-1, and KLF4 expression is shown (left panel). Densitometric scanning (right panel). Values are the mean ± SD from 3 independent experiments. * P
    Anti Klf4, supplied by Epitomics, used in various techniques. Bioz Stars score: 92/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Stemgent anti klf4
    Expression and Localization of pluripotent associated factors in control and CDX2-KD blastocysts. ( A , A′ and B , B′ ) Immunofluorescent image of OCT4 at cell nucleus was fully positive in control and CDX2-KD blastocysts. ( C , C′ and D , D′ ) Immunofluorescent image of SOX2 at cell nucleus was positive in ICM and negative in TE in control and CDX2-KD blastocysts. ICM staining was shown in inserted picture. ( E , E′ and F , F′ ) Immunofluorescent image of <t>KLF4</t> at cell nucleus was fully positive in control and CDX2-KD blastocysts. ( G , G′ and H , H′ ) Immunofluorescent image of CDX2 at cell nucleus was fully positive in control blastocyst, and was full negative in CDX2-KD blastocyst. Bar = 100 μm.
    Anti Klf4, supplied by Stemgent, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    MBL International klf4
    <t>KLF4</t> and A2bAR are correlated significantly in human adipose tissue. Subcutaneous ( A , n = 73) and visceral ( B , n = 19) adipose tissues were obtained from obese individuals. As described under “Experimental Procedures,” mRNA was extracted
    Klf4, supplied by MBL International, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore klf4
    <t>Klf4</t> directly regulates the expression of crucial EMT genes. ( A ) Venn-diagrams depicting the commonly regulated genes between the data obtained by Klf4 chromatin immunoprecipitation-deep sequencing (ChIP-Seq), gene expression profiling of TGFβ-induced EMT (EMT Up and Dn, respectively), and gene expression profiling of siRNA-mediated Klf4 depletion in the presence of TGFβ for 2 days (Klf4 Up and Dn, respectively), all performed in NMuMG cells. The numbers of genes directly up-regulated (left diagram; 30 genes) or down-regulated (right diagram; 56) during EMT are shown (see Table S1 for the gene names). ( B ) Chromatin immunoprecipitation with antibody against Klf4 followed by quantitative PCR (ChIP-qPCR) to demonstrate the occupancy of Klf4 at the promoters of the N-cadherin ( Cdh2 ), vimentin ( Vim ) and β-catenin ( Ctnnb1 ) genes. The qPCR data were normalized to ChIP-qPCR of an intergenic region. ( C ) Wiggle-tracks to show the binding of Klf4 at the promoters of the N-cadherin ( Cdh2 ), vimentin ( Vim ), β-catenin ( Ctnnb1 ), Jnk1 ( Mapk8 ), vascular endothelial growth factor A ( Vegfa ) and endothelin1 ( Edn1 ) genes by using UCSC genome browser. These files were generated from Klf4 ChIP-Seq data of NMuMG cells. Statistical values were calculated using an unpaired/paired, two-tailed t-test and experiments were performed at least three times. * = p≤0.05; ** = p≤0.01.
    Klf4, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 262 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Overexpression of Sox9 or KLF4 concentration dependently inhibits TCF-luciferase activity. HEK293, SW480, A549, and T47D cells were co-transfected with either Sox9/TCF-luciferase or KLF4/lTCF-luciferase constructs at concentrations ranging from 0 to 200

    Journal: Biochimica et biophysica acta

    Article Title: KLF4 and SOX9 Transcription Factors Antagonize ?-Catenin and Inhibit TCF-Activity In Cancer Cells

    doi: 10.1016/j.bbamcr.2012.06.027

    Figure Lengend Snippet: Overexpression of Sox9 or KLF4 concentration dependently inhibits TCF-luciferase activity. HEK293, SW480, A549, and T47D cells were co-transfected with either Sox9/TCF-luciferase or KLF4/lTCF-luciferase constructs at concentrations ranging from 0 to 200

    Article Snippet: The blots were probed with anti-Sox9 (Santa-Cruz Biotechnologies, CA), anti-KLF4 (Abcam, Cambridge, MA), anti-β-catenin (Millipore, Bedford, MA), anti-β-actin (Santa-Cruz) antibodies.

    Techniques: Over Expression, Concentration Assay, Luciferase, Activity Assay, Transfection, Construct

    TCF does not abolish Sox9 and KLF4 binding. Nuclear protein extracts were prepared from SW480 colon cancer cells and EMSA were performed as described in Methods. Oligonucleotides at increasing concentration (10-, 25-, 50-, or 100-fold excess) were incubated

    Journal: Biochimica et biophysica acta

    Article Title: KLF4 and SOX9 Transcription Factors Antagonize ?-Catenin and Inhibit TCF-Activity In Cancer Cells

    doi: 10.1016/j.bbamcr.2012.06.027

    Figure Lengend Snippet: TCF does not abolish Sox9 and KLF4 binding. Nuclear protein extracts were prepared from SW480 colon cancer cells and EMSA were performed as described in Methods. Oligonucleotides at increasing concentration (10-, 25-, 50-, or 100-fold excess) were incubated

    Article Snippet: The blots were probed with anti-Sox9 (Santa-Cruz Biotechnologies, CA), anti-KLF4 (Abcam, Cambridge, MA), anti-β-catenin (Millipore, Bedford, MA), anti-β-actin (Santa-Cruz) antibodies.

    Techniques: Binding Assay, Concentration Assay, Incubation

    Sox9 and KLF4 oligonucleotides abolish TCF-binding activity in cancer cells. Nuclear protein extracts (10 μg) were prepared from the indicated cancer cells and gel shift assays were performed as described in Methods. Excess of TCF (lane 2), Sox9

    Journal: Biochimica et biophysica acta

    Article Title: KLF4 and SOX9 Transcription Factors Antagonize ?-Catenin and Inhibit TCF-Activity In Cancer Cells

    doi: 10.1016/j.bbamcr.2012.06.027

    Figure Lengend Snippet: Sox9 and KLF4 oligonucleotides abolish TCF-binding activity in cancer cells. Nuclear protein extracts (10 μg) were prepared from the indicated cancer cells and gel shift assays were performed as described in Methods. Excess of TCF (lane 2), Sox9

    Article Snippet: The blots were probed with anti-Sox9 (Santa-Cruz Biotechnologies, CA), anti-KLF4 (Abcam, Cambridge, MA), anti-β-catenin (Millipore, Bedford, MA), anti-β-actin (Santa-Cruz) antibodies.

    Techniques: Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay

    Overexpression of Sox9 or KLF4 inhibits both TCF-binding and activity. Panel A, SW480, A549, and T47D cells were co-transfected with either Sox9/TCF-luciferase or KLF4/TCF-luciferase expression vectors. Forty-eight hours later, cells were lysed and luciferase

    Journal: Biochimica et biophysica acta

    Article Title: KLF4 and SOX9 Transcription Factors Antagonize ?-Catenin and Inhibit TCF-Activity In Cancer Cells

    doi: 10.1016/j.bbamcr.2012.06.027

    Figure Lengend Snippet: Overexpression of Sox9 or KLF4 inhibits both TCF-binding and activity. Panel A, SW480, A549, and T47D cells were co-transfected with either Sox9/TCF-luciferase or KLF4/TCF-luciferase expression vectors. Forty-eight hours later, cells were lysed and luciferase

    Article Snippet: The blots were probed with anti-Sox9 (Santa-Cruz Biotechnologies, CA), anti-KLF4 (Abcam, Cambridge, MA), anti-β-catenin (Millipore, Bedford, MA), anti-β-actin (Santa-Cruz) antibodies.

    Techniques: Over Expression, Binding Assay, Activity Assay, Transfection, Luciferase, Expressing

    Sox9 and KLF4 oligonucleotides decreased TCF-binding activity in HEK293 cells overexpressing β-catenin. Nuclear extract proteins were prepared from non-transfected HEK293 (Panel A) or HEK293 cells transfected with 1 μg of β-catenin

    Journal: Biochimica et biophysica acta

    Article Title: KLF4 and SOX9 Transcription Factors Antagonize ?-Catenin and Inhibit TCF-Activity In Cancer Cells

    doi: 10.1016/j.bbamcr.2012.06.027

    Figure Lengend Snippet: Sox9 and KLF4 oligonucleotides decreased TCF-binding activity in HEK293 cells overexpressing β-catenin. Nuclear extract proteins were prepared from non-transfected HEK293 (Panel A) or HEK293 cells transfected with 1 μg of β-catenin

    Article Snippet: The blots were probed with anti-Sox9 (Santa-Cruz Biotechnologies, CA), anti-KLF4 (Abcam, Cambridge, MA), anti-β-catenin (Millipore, Bedford, MA), anti-β-actin (Santa-Cruz) antibodies.

    Techniques: Binding Assay, Activity Assay, Transfection

    BPNet predicts ChIP-nexus signal at base resolution. A) ChIP-nexus experiments were performed on Oct4, Sox2, Nanog, and Klf4 in mouse embryonic stem cells. After digestion of the 5’ DNA ends with lambda exonuclease, strand-specific stop sites were mapped to the genome at base resolution. Bound sites exhibit a distinct footprint of aligned reads, where the positive (+) strand peak occurs many bases before the negative (-) strand peak. B) Profile heatmaps of Oct4 and Sox2 ChIP-nexus data (red and blue for positive and negative strand respectively, color depth represents normalized signal intensity) at the top 500 Oct4-Sox2 motif sites. C) The average ChIP-nexus data (positive and negative strand at top and bottom) and ChIP-seq data (grey) at the top 500 Oct4-Sox2 and Sox2 motif sites for Oct4 (red) and Sox2 (blue). The ChIP-nexus data have higher resolution and show less unspecific binding of Oct4 to the Sox2 motif. D) Architecture of the convolutional neural network (BPNet) that was trained to simultaneously predict the number of ChIP-nexus reads at each strand for all TFs from 1 kb DNA sequences. E) Observed and predicted ChIP-nexus read coverage of the positive strand (top) and the negative strand (bottom) for the Lefty enhancer located on the held-out test chromosome 8. F) BPNet predicts the positions of local high signal in the profiles at replicate-level accuracy as measured by the area under precision-recall curve (auPRC) at resolutions from 1 bp to 10 bp in held-out test chromosomes 1, 8 and 9. G) More convolutional layers (x-axis) increase the number of input bases considered for profile prediction at each position (receptive field) and this yields increasingly more accurate profile shape predictions on the tuning chromosomes 2-4 (measured in auPRC as above).

    Journal: bioRxiv

    Article Title: Deep learning at base-resolution reveals cis-regulatory motif syntax

    doi: 10.1101/737981

    Figure Lengend Snippet: BPNet predicts ChIP-nexus signal at base resolution. A) ChIP-nexus experiments were performed on Oct4, Sox2, Nanog, and Klf4 in mouse embryonic stem cells. After digestion of the 5’ DNA ends with lambda exonuclease, strand-specific stop sites were mapped to the genome at base resolution. Bound sites exhibit a distinct footprint of aligned reads, where the positive (+) strand peak occurs many bases before the negative (-) strand peak. B) Profile heatmaps of Oct4 and Sox2 ChIP-nexus data (red and blue for positive and negative strand respectively, color depth represents normalized signal intensity) at the top 500 Oct4-Sox2 motif sites. C) The average ChIP-nexus data (positive and negative strand at top and bottom) and ChIP-seq data (grey) at the top 500 Oct4-Sox2 and Sox2 motif sites for Oct4 (red) and Sox2 (blue). The ChIP-nexus data have higher resolution and show less unspecific binding of Oct4 to the Sox2 motif. D) Architecture of the convolutional neural network (BPNet) that was trained to simultaneously predict the number of ChIP-nexus reads at each strand for all TFs from 1 kb DNA sequences. E) Observed and predicted ChIP-nexus read coverage of the positive strand (top) and the negative strand (bottom) for the Lefty enhancer located on the held-out test chromosome 8. F) BPNet predicts the positions of local high signal in the profiles at replicate-level accuracy as measured by the area under precision-recall curve (auPRC) at resolutions from 1 bp to 10 bp in held-out test chromosomes 1, 8 and 9. G) More convolutional layers (x-axis) increase the number of input bases considered for profile prediction at each position (receptive field) and this yields increasingly more accurate profile shape predictions on the tuning chromosomes 2-4 (measured in auPRC as above).

    Article Snippet: The following antibodies were used: α-Oct3/4 (Santa Cruz, sc-8628), α-Sox2 (Santa Cruz, sc-17320), α-Sox2 (Active Motif, 39843), α-Nanog (Santa Cruz, sc-30328), α-Klf4 (R & D Systems, AF3158), α-Klf4 (Abcam, ab106629), α-Esrrb (Abcam, ab19331), α-Pbx 1/2/3 (Santa Cruz, sc-888), and α-Zic3 (Abcam, ab222124).

    Techniques: Chromatin Immunoprecipitation, Binding Assay

    Strict spacings between motifs are likely due to retrotransposons Most over-represented instances of strict spacings between motifs are due to ERVs . A) To show that TF binding occurs with strict spacings in retrotransposons and that this is likely ancestral, the RLTR9E N6 motif is shown as an example. Sequences of the individual instances in the genome were sorted by the Kimura distance from the consensus motif, with the most similar sequences on top (which are likely more ancestral). Nanog, Sox2 and Klf4 ChIP-nexus binding footprints are shown in the same order on the right (+ strand reads in red, - strand reads in blue), revealing that the binding site spacing is largely constant across all sequences. B) Analysis of the most frequent distances between motif pairs (with > 500 co-occurrences, distance measured at the trimmed motifs’ centers). The top 1% most frequent distances mapped in 83% to ERVs and were often larger than 20 bp. C) Histograms depicting the frequency of center-to-center motif pair spacings across the 11 representative motifs. Colors represent ERV classes which overlap with the corresponding motif pairs.

    Journal: bioRxiv

    Article Title: Deep learning at base-resolution reveals cis-regulatory motif syntax

    doi: 10.1101/737981

    Figure Lengend Snippet: Strict spacings between motifs are likely due to retrotransposons Most over-represented instances of strict spacings between motifs are due to ERVs . A) To show that TF binding occurs with strict spacings in retrotransposons and that this is likely ancestral, the RLTR9E N6 motif is shown as an example. Sequences of the individual instances in the genome were sorted by the Kimura distance from the consensus motif, with the most similar sequences on top (which are likely more ancestral). Nanog, Sox2 and Klf4 ChIP-nexus binding footprints are shown in the same order on the right (+ strand reads in red, - strand reads in blue), revealing that the binding site spacing is largely constant across all sequences. B) Analysis of the most frequent distances between motif pairs (with > 500 co-occurrences, distance measured at the trimmed motifs’ centers). The top 1% most frequent distances mapped in 83% to ERVs and were often larger than 20 bp. C) Histograms depicting the frequency of center-to-center motif pair spacings across the 11 representative motifs. Colors represent ERV classes which overlap with the corresponding motif pairs.

    Article Snippet: The following antibodies were used: α-Oct3/4 (Santa Cruz, sc-8628), α-Sox2 (Santa Cruz, sc-17320), α-Sox2 (Active Motif, 39843), α-Nanog (Santa Cruz, sc-30328), α-Klf4 (R & D Systems, AF3158), α-Klf4 (Abcam, ab106629), α-Esrrb (Abcam, ab19331), α-Pbx 1/2/3 (Santa Cruz, sc-888), and α-Zic3 (Abcam, ab222124).

    Techniques: Binding Assay, Chromatin Immunoprecipitation

    PWM scanning and ChExMix BPNet and TF-MoDISco discover more motifs than ChExMix, MEME or HOMER as well as map motif instances with greater accuracy than PWM scanning. A) Motifs discovered by ChExMix, HOMER, and MEME for Oct4, Sox2, Nanog and Klf4 ChIP-nexus peaks that are closest to the 11 primary representative BPNet motifs (top row). Green checkmark denotes whether the discovered motif is similar to the BPNet motif. B) Number of motif instances located up to 500 bp (top) or 100 bp (bottom) away from the ChIP-nexus peak summits showing a strong ChIP-nexus footprint. Only motif instances in peaks from held-out test chromosomes (1, 8 and 9) were used for the evaluation. (x-axis) top N motif instances from each of the methods were sorted in descending order of scores (PWM log odds score or CWM contrib score). For BPNet-augm, the center of the genomic region for which the contribution scores were computed was randomly jittered up to 200 bp away from the peak summit. This augmentation prevents BPNet from using the positional information of the peak summit. In the final column (Nanog replicate), the Nanog ChIP-nexus footprint was measured by a separate biological replicate using a different antibody (α-Nanog from Abcam, ab214549), which was not used during training or evaluation. C) The pairwise spacing of Nanog motif instances located up to 100 bp away from the ChIP-nexus peak summits in all possible strand orientations (rows) for different methods and/or thresholds (columns). Results for all chromosomes are shown.

    Journal: bioRxiv

    Article Title: Deep learning at base-resolution reveals cis-regulatory motif syntax

    doi: 10.1101/737981

    Figure Lengend Snippet: PWM scanning and ChExMix BPNet and TF-MoDISco discover more motifs than ChExMix, MEME or HOMER as well as map motif instances with greater accuracy than PWM scanning. A) Motifs discovered by ChExMix, HOMER, and MEME for Oct4, Sox2, Nanog and Klf4 ChIP-nexus peaks that are closest to the 11 primary representative BPNet motifs (top row). Green checkmark denotes whether the discovered motif is similar to the BPNet motif. B) Number of motif instances located up to 500 bp (top) or 100 bp (bottom) away from the ChIP-nexus peak summits showing a strong ChIP-nexus footprint. Only motif instances in peaks from held-out test chromosomes (1, 8 and 9) were used for the evaluation. (x-axis) top N motif instances from each of the methods were sorted in descending order of scores (PWM log odds score or CWM contrib score). For BPNet-augm, the center of the genomic region for which the contribution scores were computed was randomly jittered up to 200 bp away from the peak summit. This augmentation prevents BPNet from using the positional information of the peak summit. In the final column (Nanog replicate), the Nanog ChIP-nexus footprint was measured by a separate biological replicate using a different antibody (α-Nanog from Abcam, ab214549), which was not used during training or evaluation. C) The pairwise spacing of Nanog motif instances located up to 100 bp away from the ChIP-nexus peak summits in all possible strand orientations (rows) for different methods and/or thresholds (columns). Results for all chromosomes are shown.

    Article Snippet: The following antibodies were used: α-Oct3/4 (Santa Cruz, sc-8628), α-Sox2 (Santa Cruz, sc-17320), α-Sox2 (Active Motif, 39843), α-Nanog (Santa Cruz, sc-30328), α-Klf4 (R & D Systems, AF3158), α-Klf4 (Abcam, ab106629), α-Esrrb (Abcam, ab19331), α-Pbx 1/2/3 (Santa Cruz, sc-888), and α-Zic3 (Abcam, ab222124).

    Techniques: Chromatin Immunoprecipitation

    In silico analysis of motif interactions reveals TF cooperativity and motif syntax. A) In the in silico analysis on synthetic sequences, Motif A is inserted into 128 different randomized background sequences. Next, BPNet is used to predict the average TF binding profile of TF A averaged across all sequences (averaging out randomly created binding effects in the background sequences). The profile summit positions and their magnitude hA are determined as a reference point (dotted lines). Motif B is then inserted at a specific distance from Motif A into a new set of random sequences and the average predicted profile height at the reference summit (dotted lines) is measured ( hAB ). The fold-change of TF binding ( hAB / hA ) is used to quantify the interaction between motifs as a function of distance (d). B) In the genomic in silico motif interaction analysis, naturally occurring instances of Motif A and Motif B as determined by CWM scanning are used. First, the average predicted profile height and position of TF A is determined in the presence of Motif B ( hAB ). Then the sequence at motif B is replaced with random bases, and the predicted profile height of TF A is measured in the absence of motif B at the reference point ( hA at dotted lines). The same binding fold-change hAB / hA as a function of d is used to quantify the interaction. C) Examples from the synthetic in silico analysis showing protein-range interactions involving Nanog and Sox2 (left) or nucleosome-range interactions exerted by the Oct4-Sox2 motif (bound by Oct4) on the binding of Sox2, Nanog or Klf4 on their respective motifs (right). Results are shown for the +/+ orientation of the two motifs. The fold change of predicted binding signal, as illustrated in A, was corrected for interference from nearby motifs (Figure S16A). D) Similar results are obtained in the genomic in silico mutagenesis analysis using the average of all motif orientations. E) Quantification of the results shown in D as heat map. The distances

    Journal: bioRxiv

    Article Title: Deep learning at base-resolution reveals cis-regulatory motif syntax

    doi: 10.1101/737981

    Figure Lengend Snippet: In silico analysis of motif interactions reveals TF cooperativity and motif syntax. A) In the in silico analysis on synthetic sequences, Motif A is inserted into 128 different randomized background sequences. Next, BPNet is used to predict the average TF binding profile of TF A averaged across all sequences (averaging out randomly created binding effects in the background sequences). The profile summit positions and their magnitude hA are determined as a reference point (dotted lines). Motif B is then inserted at a specific distance from Motif A into a new set of random sequences and the average predicted profile height at the reference summit (dotted lines) is measured ( hAB ). The fold-change of TF binding ( hAB / hA ) is used to quantify the interaction between motifs as a function of distance (d). B) In the genomic in silico motif interaction analysis, naturally occurring instances of Motif A and Motif B as determined by CWM scanning are used. First, the average predicted profile height and position of TF A is determined in the presence of Motif B ( hAB ). Then the sequence at motif B is replaced with random bases, and the predicted profile height of TF A is measured in the absence of motif B at the reference point ( hA at dotted lines). The same binding fold-change hAB / hA as a function of d is used to quantify the interaction. C) Examples from the synthetic in silico analysis showing protein-range interactions involving Nanog and Sox2 (left) or nucleosome-range interactions exerted by the Oct4-Sox2 motif (bound by Oct4) on the binding of Sox2, Nanog or Klf4 on their respective motifs (right). Results are shown for the +/+ orientation of the two motifs. The fold change of predicted binding signal, as illustrated in A, was corrected for interference from nearby motifs (Figure S16A). D) Similar results are obtained in the genomic in silico mutagenesis analysis using the average of all motif orientations. E) Quantification of the results shown in D as heat map. The distances

    Article Snippet: The following antibodies were used: α-Oct3/4 (Santa Cruz, sc-8628), α-Sox2 (Santa Cruz, sc-17320), α-Sox2 (Active Motif, 39843), α-Nanog (Santa Cruz, sc-30328), α-Klf4 (R & D Systems, AF3158), α-Klf4 (Abcam, ab106629), α-Esrrb (Abcam, ab19331), α-Pbx 1/2/3 (Santa Cruz, sc-888), and α-Zic3 (Abcam, ab222124).

    Techniques: In Silico, Binding Assay, Sequencing, Mutagenesis

    Long motifs discovered by TF-MoDISco come from retrotransposons TF-MoDISco discovered long motifs that originated from retrotransposons. A) Among all motifs discovered by TF-MoDISco, 18 motifs display unusually high information content (IC) of > 30 bits (green). The expected short motifs are shown in grey. B) Histogram of the overlap of short motifs (grey) and long motifs (green) with repeat elements shows that long motifs overlap > 80% with annotated retrotransposons. C) Long motifs with their PFM, ID, fraction of motif instances overlapping with a repeat, and the most frequent (top class) RepeatMasker annotation. Highlighted within the repeat elements are potential motif instances of Oct4-Sox2, Sox2, Nanog and Klf4 as indicated by the CWMs.

    Journal: bioRxiv

    Article Title: Deep learning at base-resolution reveals cis-regulatory motif syntax

    doi: 10.1101/737981

    Figure Lengend Snippet: Long motifs discovered by TF-MoDISco come from retrotransposons TF-MoDISco discovered long motifs that originated from retrotransposons. A) Among all motifs discovered by TF-MoDISco, 18 motifs display unusually high information content (IC) of > 30 bits (green). The expected short motifs are shown in grey. B) Histogram of the overlap of short motifs (grey) and long motifs (green) with repeat elements shows that long motifs overlap > 80% with annotated retrotransposons. C) Long motifs with their PFM, ID, fraction of motif instances overlapping with a repeat, and the most frequent (top class) RepeatMasker annotation. Highlighted within the repeat elements are potential motif instances of Oct4-Sox2, Sox2, Nanog and Klf4 as indicated by the CWMs.

    Article Snippet: The following antibodies were used: α-Oct3/4 (Santa Cruz, sc-8628), α-Sox2 (Santa Cruz, sc-17320), α-Sox2 (Active Motif, 39843), α-Nanog (Santa Cruz, sc-30328), α-Klf4 (R & D Systems, AF3158), α-Klf4 (Abcam, ab106629), α-Esrrb (Abcam, ab19331), α-Pbx 1/2/3 (Santa Cruz, sc-888), and α-Zic3 (Abcam, ab222124).

    Techniques:

    BPNet predictions and sequence contribution scores at known enhancers Additional BPNet predictions across known enhancer regions. A) Observed and predicted ChIP-nexus read counts for the Oct4 distal enhancer. B , C , D) Previously validated binding motifs for Oct4-Sox2 were re-discovered by BPNet. ChIP-nexus read counts and BPNet contribution scores for three enhancers are shown. B) The Oct4-Sox2 motif site in the Klf4 E2 enhancer was validated by deleting the site using CRISPR/Cas9 ( 72 ). C , D) The Oct4-Sox2 binding motifs in the Nanog and Fbx15 enhancers were confirmed previously using reporter assays of constructs with various motif mutations ( 73 , 74 ).

    Journal: bioRxiv

    Article Title: Deep learning at base-resolution reveals cis-regulatory motif syntax

    doi: 10.1101/737981

    Figure Lengend Snippet: BPNet predictions and sequence contribution scores at known enhancers Additional BPNet predictions across known enhancer regions. A) Observed and predicted ChIP-nexus read counts for the Oct4 distal enhancer. B , C , D) Previously validated binding motifs for Oct4-Sox2 were re-discovered by BPNet. ChIP-nexus read counts and BPNet contribution scores for three enhancers are shown. B) The Oct4-Sox2 motif site in the Klf4 E2 enhancer was validated by deleting the site using CRISPR/Cas9 ( 72 ). C , D) The Oct4-Sox2 binding motifs in the Nanog and Fbx15 enhancers were confirmed previously using reporter assays of constructs with various motif mutations ( 73 , 74 ).

    Article Snippet: The following antibodies were used: α-Oct3/4 (Santa Cruz, sc-8628), α-Sox2 (Santa Cruz, sc-17320), α-Sox2 (Active Motif, 39843), α-Nanog (Santa Cruz, sc-30328), α-Klf4 (R & D Systems, AF3158), α-Klf4 (Abcam, ab106629), α-Esrrb (Abcam, ab19331), α-Pbx 1/2/3 (Santa Cruz, sc-888), and α-Zic3 (Abcam, ab222124).

    Techniques: Sequencing, Chromatin Immunoprecipitation, Binding Assay, CRISPR, Construct

    No evidence that Nanog binds with a partner Lack of evidence for a Nanog binding partner. A) Median ChIP-nexus signal, predicted BPNet signal, and DeepLIFT contribution of Oct4, Sox2, Nanog, and Klf4 show no signal across the genomic instances matching the putative Nanog-Sox heterodimer motif (RMWMAATWNCATTSW) ( 69 ). The signal for Oct4-Sox2, Sox2, Nanog , and Klf4 motif instances are shown as control. B) Since the Nanog motif resembles the known Pbx binding motif, we performed Pbx ChIP-nexus experiments to test whether Pbx might be a binding partner for Nanog. However, the average Nanog, Pbx and Sox2 ChIP-nexus binding profiles show no detectable footprints for Pbx or Sox2 on the three Nanog motifs, arguing against Pbx or Sox2 being stable interaction partners. However, an unknown interaction partner cannot be ruled out. C) The average Pbx ChIP-nexus footprint on the known Pbx motif from JASPAR ( 139 ) (top 1000 based on PWM-match score) confirms that the Pbx ChIP-nexus experiment worked (left). Likewise, Sox2 shows specific binding to its identified Sox2 motif (right). Note that the y-axis is set to the same RPM scale in A and B to allow comparisons of signal strength.

    Journal: bioRxiv

    Article Title: Deep learning at base-resolution reveals cis-regulatory motif syntax

    doi: 10.1101/737981

    Figure Lengend Snippet: No evidence that Nanog binds with a partner Lack of evidence for a Nanog binding partner. A) Median ChIP-nexus signal, predicted BPNet signal, and DeepLIFT contribution of Oct4, Sox2, Nanog, and Klf4 show no signal across the genomic instances matching the putative Nanog-Sox heterodimer motif (RMWMAATWNCATTSW) ( 69 ). The signal for Oct4-Sox2, Sox2, Nanog , and Klf4 motif instances are shown as control. B) Since the Nanog motif resembles the known Pbx binding motif, we performed Pbx ChIP-nexus experiments to test whether Pbx might be a binding partner for Nanog. However, the average Nanog, Pbx and Sox2 ChIP-nexus binding profiles show no detectable footprints for Pbx or Sox2 on the three Nanog motifs, arguing against Pbx or Sox2 being stable interaction partners. However, an unknown interaction partner cannot be ruled out. C) The average Pbx ChIP-nexus footprint on the known Pbx motif from JASPAR ( 139 ) (top 1000 based on PWM-match score) confirms that the Pbx ChIP-nexus experiment worked (left). Likewise, Sox2 shows specific binding to its identified Sox2 motif (right). Note that the y-axis is set to the same RPM scale in A and B to allow comparisons of signal strength.

    Article Snippet: The following antibodies were used: α-Oct3/4 (Santa Cruz, sc-8628), α-Sox2 (Santa Cruz, sc-17320), α-Sox2 (Active Motif, 39843), α-Nanog (Santa Cruz, sc-30328), α-Klf4 (R & D Systems, AF3158), α-Klf4 (Abcam, ab106629), α-Esrrb (Abcam, ab19331), α-Pbx 1/2/3 (Santa Cruz, sc-888), and α-Zic3 (Abcam, ab222124).

    Techniques: Binding Assay, Chromatin Immunoprecipitation

    KLF4 acts as a transcriptional suppressor of SRA. ( A ) 293T cells were transfected with pGL4.10 vector containing SRA promoter-linked Firefly gene (pSRA) together with pGL4.70 vector containing Renilla gene, and then treated with formononetin for 12 h. The transcriptional activity of SRA was measured by a dual luciferase assay system and normalized to that of Renilla, n=5. ( B ) 293T cells were infected with Ad-KLF4, and then transfected with pSRA together with pGL4.70 vector, followed by treatment with formononetin for 12 h. The transcriptional activity of SRA was measured by a dual luciferase assay system and normalized to that of Renilla, n=5. ( C ) 293T cells were transfected with pGL4.10 vector containing mutant SRA promoter-linked Firefly gene (pSRA-KLFREmut) together with pGL4.70 vector, and then treated with formononetin for 12 h. The transcriptional activity of SRA was measured as described in (B), n=5. ( D ) 293T cells were firstly infected with Ad-KLF4, and then transfected with pSRA-KLFREmut together with pGL4.70 vector, followed by treatment with formononetin for 12 h. The transcriptional activity of SRA was measured as described in (B), n=5. ( E ) After treatment of formononetin, the ability of KLF4 binding to SRA promoter was assayed by the chromosome-immunoprecipitation experiment, n=5. Data are presented as mean ± SEM, * P

    Journal: Theranostics

    Article Title: Formononetin attenuates atherosclerosis via regulating interaction between KLF4 and SRA in apoE-/- mice

    doi: 10.7150/thno.38115

    Figure Lengend Snippet: KLF4 acts as a transcriptional suppressor of SRA. ( A ) 293T cells were transfected with pGL4.10 vector containing SRA promoter-linked Firefly gene (pSRA) together with pGL4.70 vector containing Renilla gene, and then treated with formononetin for 12 h. The transcriptional activity of SRA was measured by a dual luciferase assay system and normalized to that of Renilla, n=5. ( B ) 293T cells were infected with Ad-KLF4, and then transfected with pSRA together with pGL4.70 vector, followed by treatment with formononetin for 12 h. The transcriptional activity of SRA was measured by a dual luciferase assay system and normalized to that of Renilla, n=5. ( C ) 293T cells were transfected with pGL4.10 vector containing mutant SRA promoter-linked Firefly gene (pSRA-KLFREmut) together with pGL4.70 vector, and then treated with formononetin for 12 h. The transcriptional activity of SRA was measured as described in (B), n=5. ( D ) 293T cells were firstly infected with Ad-KLF4, and then transfected with pSRA-KLFREmut together with pGL4.70 vector, followed by treatment with formononetin for 12 h. The transcriptional activity of SRA was measured as described in (B), n=5. ( E ) After treatment of formononetin, the ability of KLF4 binding to SRA promoter was assayed by the chromosome-immunoprecipitation experiment, n=5. Data are presented as mean ± SEM, * P

    Article Snippet: Rabbit anti-KLF4 antibody and anti-Lamin A/C monoclonal antibody were purchased from Abcam (Cambridge, MA).

    Techniques: Transfection, Plasmid Preparation, Activity Assay, Luciferase, Infection, Mutagenesis, Binding Assay, Immunoprecipitation

    Formononetin increases KLF4 expression and nuclear translocation in PMs and HASMCs. ( A, B ) After overexpression of KLF4 by infection with Ad-KLF4, PMs and HASMCs were exposed to oxLDL in presence or absence of formononetin. Expression of KLF4, SRA, CD36 and LOX-1 protein in PMs or KLF4, SRA in HASMCs was determined by Western blot (left) followed by quantification of band density correspondingly (right), n=5. ( C, D ) PMs and HASMCs were stained with Oil Red O after overexpression of KLF4, n=5. ( E, F ) PMs and HASMCs were treated with formononetin at the indicated concentrations overnight. And then expression of KLF4 protein in nuclear extracts was determined by Western blot, respectively, n=5. ( G, H ) Expression of KLF4 protein in PMs and HASMCs was determined by immunofluorescent staining, n=5. ( I, J ) Expression of KLF4 protein in macrophages and VSMCs within plaque was determined by co-immunofluorescent staining with KLF4 and CD68 or αSMA antibodies, n=5. Data are presented as mean ± SEM, * P

    Journal: Theranostics

    Article Title: Formononetin attenuates atherosclerosis via regulating interaction between KLF4 and SRA in apoE-/- mice

    doi: 10.7150/thno.38115

    Figure Lengend Snippet: Formononetin increases KLF4 expression and nuclear translocation in PMs and HASMCs. ( A, B ) After overexpression of KLF4 by infection with Ad-KLF4, PMs and HASMCs were exposed to oxLDL in presence or absence of formononetin. Expression of KLF4, SRA, CD36 and LOX-1 protein in PMs or KLF4, SRA in HASMCs was determined by Western blot (left) followed by quantification of band density correspondingly (right), n=5. ( C, D ) PMs and HASMCs were stained with Oil Red O after overexpression of KLF4, n=5. ( E, F ) PMs and HASMCs were treated with formononetin at the indicated concentrations overnight. And then expression of KLF4 protein in nuclear extracts was determined by Western blot, respectively, n=5. ( G, H ) Expression of KLF4 protein in PMs and HASMCs was determined by immunofluorescent staining, n=5. ( I, J ) Expression of KLF4 protein in macrophages and VSMCs within plaque was determined by co-immunofluorescent staining with KLF4 and CD68 or αSMA antibodies, n=5. Data are presented as mean ± SEM, * P

    Article Snippet: Rabbit anti-KLF4 antibody and anti-Lamin A/C monoclonal antibody were purchased from Abcam (Cambridge, MA).

    Techniques: Expressing, Translocation Assay, Over Expression, Infection, Western Blot, Staining

    Proteolytic regulation of KLF4 by estrogen signaling contributes to its overexpression in breast cancer. A , KLF4 mRNA level is down-regulated in breast cancer. Oncomine was used to analyze the previously published microarray data. B , KLF4 protein level

    Journal: The Journal of Biological Chemistry

    Article Title: Novel Insight into KLF4 Proteolytic Regulation in Estrogen Receptor Signaling and Breast Carcinogenesis *

    doi: 10.1074/jbc.M112.343566

    Figure Lengend Snippet: Proteolytic regulation of KLF4 by estrogen signaling contributes to its overexpression in breast cancer. A , KLF4 mRNA level is down-regulated in breast cancer. Oncomine was used to analyze the previously published microarray data. B , KLF4 protein level

    Article Snippet: For immunoprecipitation assay, cell lysate was incubated with anti-FLAG M2 gel (Sigma) or anti-KLF4 (Santa Cruz Biotechnology) antibody overnight at 4 °C on a rotator, followed by the addition of protein A/G plus agarose (Pierce) to the reaction containing anti-KLF4 antibody for 2 h at 4 °C.

    Techniques: Over Expression, Microarray

    The proteolytic regulation of KLF4 by VHL is involved in estrogen signaling and its mitogenic effect. A , stimulation with estrogen results in down-regulation of VHL, which in turn leads to accumulated KLF4. Hormone-stripped MCF-7 cells were treated with

    Journal: The Journal of Biological Chemistry

    Article Title: Novel Insight into KLF4 Proteolytic Regulation in Estrogen Receptor Signaling and Breast Carcinogenesis *

    doi: 10.1074/jbc.M112.343566

    Figure Lengend Snippet: The proteolytic regulation of KLF4 by VHL is involved in estrogen signaling and its mitogenic effect. A , stimulation with estrogen results in down-regulation of VHL, which in turn leads to accumulated KLF4. Hormone-stripped MCF-7 cells were treated with

    Article Snippet: For immunoprecipitation assay, cell lysate was incubated with anti-FLAG M2 gel (Sigma) or anti-KLF4 (Santa Cruz Biotechnology) antibody overnight at 4 °C on a rotator, followed by the addition of protein A/G plus agarose (Pierce) to the reaction containing anti-KLF4 antibody for 2 h at 4 °C.

    Techniques:

    Mapping the critical regions on KLF4 that mediate its degradation by VHL. A , generation of a series of KLF4 deletion mutants. B , evaluation of protein stability for a set of designed KLF4 mutants. KLF4 wild-type or deletion mutant was co-transfected with

    Journal: The Journal of Biological Chemistry

    Article Title: Novel Insight into KLF4 Proteolytic Regulation in Estrogen Receptor Signaling and Breast Carcinogenesis *

    doi: 10.1074/jbc.M112.343566

    Figure Lengend Snippet: Mapping the critical regions on KLF4 that mediate its degradation by VHL. A , generation of a series of KLF4 deletion mutants. B , evaluation of protein stability for a set of designed KLF4 mutants. KLF4 wild-type or deletion mutant was co-transfected with

    Article Snippet: For immunoprecipitation assay, cell lysate was incubated with anti-FLAG M2 gel (Sigma) or anti-KLF4 (Santa Cruz Biotechnology) antibody overnight at 4 °C on a rotator, followed by the addition of protein A/G plus agarose (Pierce) to the reaction containing anti-KLF4 antibody for 2 h at 4 °C.

    Techniques: Mutagenesis, Transfection

    Elevation of KLF4 protein levels by E2 is required to facilitate estrogen signaling and its mitogenic effect. A , depletion of E2 in the culture medium leads to a drop of KLF4 protein levels. MCF-7 cells were hormone-stripped for the indicated time and

    Journal: The Journal of Biological Chemistry

    Article Title: Novel Insight into KLF4 Proteolytic Regulation in Estrogen Receptor Signaling and Breast Carcinogenesis *

    doi: 10.1074/jbc.M112.343566

    Figure Lengend Snippet: Elevation of KLF4 protein levels by E2 is required to facilitate estrogen signaling and its mitogenic effect. A , depletion of E2 in the culture medium leads to a drop of KLF4 protein levels. MCF-7 cells were hormone-stripped for the indicated time and

    Article Snippet: For immunoprecipitation assay, cell lysate was incubated with anti-FLAG M2 gel (Sigma) or anti-KLF4 (Santa Cruz Biotechnology) antibody overnight at 4 °C on a rotator, followed by the addition of protein A/G plus agarose (Pierce) to the reaction containing anti-KLF4 antibody for 2 h at 4 °C.

    Techniques:

    Model for the regulation of KLF4 by VHL in the estrogen signal transduction and concomitant mitogenic effect.

    Journal: The Journal of Biological Chemistry

    Article Title: Novel Insight into KLF4 Proteolytic Regulation in Estrogen Receptor Signaling and Breast Carcinogenesis *

    doi: 10.1074/jbc.M112.343566

    Figure Lengend Snippet: Model for the regulation of KLF4 by VHL in the estrogen signal transduction and concomitant mitogenic effect.

    Article Snippet: For immunoprecipitation assay, cell lysate was incubated with anti-FLAG M2 gel (Sigma) or anti-KLF4 (Santa Cruz Biotechnology) antibody overnight at 4 °C on a rotator, followed by the addition of protein A/G plus agarose (Pierce) to the reaction containing anti-KLF4 antibody for 2 h at 4 °C.

    Techniques: Transduction

    Regulation of KLF4 by VHL is validated in human breast cancer tissue. A and B , immunohistochemistry analysis of KLF4 and VHL protein expression in human breast cancer. Sections from breast cancer and adjacent normal tissues were analyzed by immunostaining

    Journal: The Journal of Biological Chemistry

    Article Title: Novel Insight into KLF4 Proteolytic Regulation in Estrogen Receptor Signaling and Breast Carcinogenesis *

    doi: 10.1074/jbc.M112.343566

    Figure Lengend Snippet: Regulation of KLF4 by VHL is validated in human breast cancer tissue. A and B , immunohistochemistry analysis of KLF4 and VHL protein expression in human breast cancer. Sections from breast cancer and adjacent normal tissues were analyzed by immunostaining

    Article Snippet: For immunoprecipitation assay, cell lysate was incubated with anti-FLAG M2 gel (Sigma) or anti-KLF4 (Santa Cruz Biotechnology) antibody overnight at 4 °C on a rotator, followed by the addition of protein A/G plus agarose (Pierce) to the reaction containing anti-KLF4 antibody for 2 h at 4 °C.

    Techniques: Immunohistochemistry, Expressing, Immunostaining

    VHL E3 ligase plays critical role governing KLF4 steady-state turnover in breast cancer cells. A , evaluation of candidate E3 ubiquitin ligases involving in KLF4 protein turnover. MCF-7 cells were transfected with a series of siRNAs against various candidate

    Journal: The Journal of Biological Chemistry

    Article Title: Novel Insight into KLF4 Proteolytic Regulation in Estrogen Receptor Signaling and Breast Carcinogenesis *

    doi: 10.1074/jbc.M112.343566

    Figure Lengend Snippet: VHL E3 ligase plays critical role governing KLF4 steady-state turnover in breast cancer cells. A , evaluation of candidate E3 ubiquitin ligases involving in KLF4 protein turnover. MCF-7 cells were transfected with a series of siRNAs against various candidate

    Article Snippet: For immunoprecipitation assay, cell lysate was incubated with anti-FLAG M2 gel (Sigma) or anti-KLF4 (Santa Cruz Biotechnology) antibody overnight at 4 °C on a rotator, followed by the addition of protein A/G plus agarose (Pierce) to the reaction containing anti-KLF4 antibody for 2 h at 4 °C.

    Techniques: Transfection

    Negative regulation of STK33 by KLF4 in gastric cancer cells. BCG-823 cells were transfected with siKLF4#1 and siKLF4#2 or control siRNA for 48 hours, and MKN45, MGC-803 and SGC-7901 cells were transfected with pFlag-KLF4 or control vector for 48 hours. A, total RNA and protein lysates were harvested, and the expression of STK33 and KLF4 in the lysates was determined using real-time PCR and Western blot. B, immunohistochemical stains for KLF4 and STK33 from TMAs with consecutive gastric cancer tissue sections. Representative images of gastric cancer sections with STK33 and KLF4 staining are shown (100× magnification in the main images, 200× magnification in the inserts). C, assessment of the negative correlation between KLF4 and STK33 expression in gastric cancer specimens ( N = 45) using Pearson correlation coefficient analysis. Some of the dots on the graph represent more than one specimen. D, Boyden chamber analysis of effect of alerted expression of STK33 on KLF4-mediated invasion in gastric cancer cells. The experiments were performed independently three times. Ŝtatistically significant when compared with siNC group ( P

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Krüppel-like Factor 4 Suppresses Serine/Threonine Kinase 33 Activation and Metastasis of Gastric Cancer through Reversing Epithelial–Mesenchymal Transition

    doi: 10.1158/1078-0432.CCR-17-3346

    Figure Lengend Snippet: Negative regulation of STK33 by KLF4 in gastric cancer cells. BCG-823 cells were transfected with siKLF4#1 and siKLF4#2 or control siRNA for 48 hours, and MKN45, MGC-803 and SGC-7901 cells were transfected with pFlag-KLF4 or control vector for 48 hours. A, total RNA and protein lysates were harvested, and the expression of STK33 and KLF4 in the lysates was determined using real-time PCR and Western blot. B, immunohistochemical stains for KLF4 and STK33 from TMAs with consecutive gastric cancer tissue sections. Representative images of gastric cancer sections with STK33 and KLF4 staining are shown (100× magnification in the main images, 200× magnification in the inserts). C, assessment of the negative correlation between KLF4 and STK33 expression in gastric cancer specimens ( N = 45) using Pearson correlation coefficient analysis. Some of the dots on the graph represent more than one specimen. D, Boyden chamber analysis of effect of alerted expression of STK33 on KLF4-mediated invasion in gastric cancer cells. The experiments were performed independently three times. Ŝtatistically significant when compared with siNC group ( P

    Article Snippet: Standard Western blotting was carried out using whole-cell protein lysates; primary antibodies against STK33 (Cat. #ab206296, Abcam), KLF4 (Cat. #sc-166238, Santa Cruz), Flag (Sigma), Vimentin (Santa Cruz), E-cadherin (Proteintech), N-cadherin (Cat. #66219-I-LG, Proteintech), Snail (Cat. #ab53519, Abcam), Slug (Cat. #ab27568, Abcam), ZEB1 (Cat. # 3396,Cell Signaling Technology), ZEB2 (Cat. #271984, Santa Cruz), Twist (Cat. #ab50581, Abcam); and a secondary antibody (anti-rabbit IgG or anti-mouse IgG; Westang Biotechnology).

    Techniques: Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining

    Expression of Pluripotency-Associated Genes in Putative iPS Clones (A) (B) HCF- and BJ-derived iPS clones were analyzed for expression of pluripotency markers by immunostaining. HCF#1 and BJ#SA cells were positive for pluripotency markers SSEA4, TRA-1-60, TRA-1-81, OCT4, SOX2, KLF4 and NANOG, while no notable staining was observed for SSEA1. Cells were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI). Control (m) and Control (r); control cells treated with FITC-conjugated secondary antibodies against mouse IgG and rabbit IgG. Scale bars indicate 20 µm. (C) HCF- and BJ-derived iPS-like clones were analyzed for pluripotency-associated gene expression by RT-PCR. Total cellular RNA from parental BJ and HCF fibroblasts and no template (water) samples were included as controls. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcript was amplified as an internal RNA control.

    Journal: Gene therapy

    Article Title: Indolactam V/GLP-1-mediated Differentiation of Human iPS Cells into Glucose-Responsive Insulin-Secreting Progeny

    doi: 10.1038/gt.2010.145

    Figure Lengend Snippet: Expression of Pluripotency-Associated Genes in Putative iPS Clones (A) (B) HCF- and BJ-derived iPS clones were analyzed for expression of pluripotency markers by immunostaining. HCF#1 and BJ#SA cells were positive for pluripotency markers SSEA4, TRA-1-60, TRA-1-81, OCT4, SOX2, KLF4 and NANOG, while no notable staining was observed for SSEA1. Cells were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI). Control (m) and Control (r); control cells treated with FITC-conjugated secondary antibodies against mouse IgG and rabbit IgG. Scale bars indicate 20 µm. (C) HCF- and BJ-derived iPS-like clones were analyzed for pluripotency-associated gene expression by RT-PCR. Total cellular RNA from parental BJ and HCF fibroblasts and no template (water) samples were included as controls. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcript was amplified as an internal RNA control.

    Article Snippet: Primary and secondary antibodies used for characterization of iPS and derived cells were: SSEA-1, SSEA-4, TRA-1–60, TRA-1-81 (Millipore #SCR001), OCT4 (Cell Signaling Technology #2750), SOX2 (Cell Signaling Technology #2748), KLF4 (Abcam #ab26648), NANOG (Abcam #ab21624), mouse anti-SOX17 (R & D Systems #MAB1924), rabbit anti-HNF3 beta/FOXA2 (Millipore #07-633), rabbit anti-PDX1 (Santa Cruz Biotechnology #sc-25403), rabbit anti-NGN3 (Millipore #AB5684), rabbit anti-NEUROD1 (Abcam #16508), mouse anti-insulin (Sigma #I2018), rabbit anti-C-peptide (Cell Signaling Technology #4593), rabbit anti-Insulin (Cell Signaling Technology #4590), mouse anti-proinsulin C-peptide (Millipore #CBL94), mouse anti-glucagon (Abcam #ab10988), MafA (Santa Cruz Biotechnology #sc-66958), and rabbit anti-somatostatin (Dako #A0566).

    Techniques: Expressing, Derivative Assay, Clone Assay, Immunostaining, Staining, Reverse Transcription Polymerase Chain Reaction, Amplification

    Generation of Human iPS Clones from BJ and HCF Fibroblasts (A) Lentiviral vector-mediated delivery of OCT3/4, SOX2, KLF4 and c-MYC resulted in iPS-like colony formation. (i) SNL feeder cells, (ii) uninfected HCF fibroblasts, (iii) HCF-derived iPS-like colony at two weeks post-infection, (iv) iPS-like cells with high magnification. iPS cells exhibited morphology similar to human ES cells, characterized by large nuclei and scant cytoplasm, (v) uninfected BJ fibroblasts, (vi) BJ fibroblasts-derived iPS-like colony at two weeks after infection, (vii) image of a BJ-derived clone expanded on feeder cells, (viii) high magnification image of BJ-derived clone. (B) Feeder-free generation of human iPS cells allowed visualization of the early reprogramming events. (i) Uninfected BJ fibroblasts, (ii) an early stage iPS-like colony in vector-transduced BJ cells one week after infection, (iii) high magnification image of BJ fibroblast-derived iPS-like colony. (C) Morphology of iPS clones cultured under feeder-free conditions. BJ#SA was established on SNL feeder cells, while HCF#1 and BJ#1 were derived feeder-free. (D) HCF#1, BJ#SA and BJ#1 cultured under feeder-free conditions expressed high levels of alkaline phosphatase (AP).

    Journal: Gene therapy

    Article Title: Indolactam V/GLP-1-mediated Differentiation of Human iPS Cells into Glucose-Responsive Insulin-Secreting Progeny

    doi: 10.1038/gt.2010.145

    Figure Lengend Snippet: Generation of Human iPS Clones from BJ and HCF Fibroblasts (A) Lentiviral vector-mediated delivery of OCT3/4, SOX2, KLF4 and c-MYC resulted in iPS-like colony formation. (i) SNL feeder cells, (ii) uninfected HCF fibroblasts, (iii) HCF-derived iPS-like colony at two weeks post-infection, (iv) iPS-like cells with high magnification. iPS cells exhibited morphology similar to human ES cells, characterized by large nuclei and scant cytoplasm, (v) uninfected BJ fibroblasts, (vi) BJ fibroblasts-derived iPS-like colony at two weeks after infection, (vii) image of a BJ-derived clone expanded on feeder cells, (viii) high magnification image of BJ-derived clone. (B) Feeder-free generation of human iPS cells allowed visualization of the early reprogramming events. (i) Uninfected BJ fibroblasts, (ii) an early stage iPS-like colony in vector-transduced BJ cells one week after infection, (iii) high magnification image of BJ fibroblast-derived iPS-like colony. (C) Morphology of iPS clones cultured under feeder-free conditions. BJ#SA was established on SNL feeder cells, while HCF#1 and BJ#1 were derived feeder-free. (D) HCF#1, BJ#SA and BJ#1 cultured under feeder-free conditions expressed high levels of alkaline phosphatase (AP).

    Article Snippet: Primary and secondary antibodies used for characterization of iPS and derived cells were: SSEA-1, SSEA-4, TRA-1–60, TRA-1-81 (Millipore #SCR001), OCT4 (Cell Signaling Technology #2750), SOX2 (Cell Signaling Technology #2748), KLF4 (Abcam #ab26648), NANOG (Abcam #ab21624), mouse anti-SOX17 (R & D Systems #MAB1924), rabbit anti-HNF3 beta/FOXA2 (Millipore #07-633), rabbit anti-PDX1 (Santa Cruz Biotechnology #sc-25403), rabbit anti-NGN3 (Millipore #AB5684), rabbit anti-NEUROD1 (Abcam #16508), mouse anti-insulin (Sigma #I2018), rabbit anti-C-peptide (Cell Signaling Technology #4593), rabbit anti-Insulin (Cell Signaling Technology #4590), mouse anti-proinsulin C-peptide (Millipore #CBL94), mouse anti-glucagon (Abcam #ab10988), MafA (Santa Cruz Biotechnology #sc-66958), and rabbit anti-somatostatin (Dako #A0566).

    Techniques: Clone Assay, Plasmid Preparation, Derivative Assay, Infection, Cell Culture

    Model of mechanism by which Mule may control T-cell proliferation through ubiquitination and degradation of KLF4. ( a ) WT T cells at steady-state. In the absence of antigenic stimulation, Mule expression in T cells is low due to self-ubiquitination and degradation and its E3 ligase activity is insufficient to remove KLF4. KLF4 transactivates E2F2, which acts as a transcriptional repressor together with CDKI p21 and p27 to block entry into the cell cycle. ( b ) Antigen-stimulated WT T cells. In response to TCR engagement by antigen, Mule expression is rapidly increased and sustained due to inhibition of its self-ubiquitination and degradation. Mule ubiquitinates KLF4 and promotes its degradation such that insufficient KLF4 remains to successfully transactivate E2F2, p21 and p27. T cells can thus transcribe genes promoting cell cycle entry. ( c ) In antigen-stimulated Mule -deficient T cells, KLF4 cannot be degraded. The accumulating KLF4 protein transactivates E2F2, p21 and p27, leading to repressed expression of cell cycle genes. These T cells then fail to proliferate efficiently.

    Journal: Nature Communications

    Article Title: K48-linked KLF4 ubiquitination by E3 ligase Mule controls T-cell proliferation and cell cycle progression

    doi: 10.1038/ncomms14003

    Figure Lengend Snippet: Model of mechanism by which Mule may control T-cell proliferation through ubiquitination and degradation of KLF4. ( a ) WT T cells at steady-state. In the absence of antigenic stimulation, Mule expression in T cells is low due to self-ubiquitination and degradation and its E3 ligase activity is insufficient to remove KLF4. KLF4 transactivates E2F2, which acts as a transcriptional repressor together with CDKI p21 and p27 to block entry into the cell cycle. ( b ) Antigen-stimulated WT T cells. In response to TCR engagement by antigen, Mule expression is rapidly increased and sustained due to inhibition of its self-ubiquitination and degradation. Mule ubiquitinates KLF4 and promotes its degradation such that insufficient KLF4 remains to successfully transactivate E2F2, p21 and p27. T cells can thus transcribe genes promoting cell cycle entry. ( c ) In antigen-stimulated Mule -deficient T cells, KLF4 cannot be degraded. The accumulating KLF4 protein transactivates E2F2, p21 and p27, leading to repressed expression of cell cycle genes. These T cells then fail to proliferate efficiently.

    Article Snippet: Fractioned proteins were transferred to a nitrocellulose membrane by i-Blot according to the manufacturer's instructions (Invitrogen) and immunoblotted using Abs recognizing the following: ubiquitin (FK2; Enzo Life Sciences; 1:1,000); Lys-specific ubiquitin (Apu2; Millipore; 1:1,000); KLF4 (GeneTex; 1:1,000 ); Mule (Bethyl Laboratories Inc.; 1:1,000); p21 (Santa Cruz; 1:1,000); p27 (BD Biosciences; 1:1,000); p53 (Santa Cruz; 1:1,000); Ser18-p53 (R & D; 1:1,000); or phospho-Erk1/2, phospho-JNK1/2, IκBα, phospho-IκBα, p65 or phospho-IKKα/β (all from Cell Signaling; 1:1,000).

    Techniques: Expressing, Activity Assay, Blocking Assay, Inhibition

    Identification of KLF4 as a novel Mule substrate. ( a ) IB to detect ELF4 and KLF4 in purified control and TMKO CD4 + and CD8 + T cells. ( b ) IB to detect ubiquitinated KLF4 after incubation of recombinant GST-KLF4 with ubiquitin, E1, HBCH5b (E2) and Mule (E3) or Mulec4341A mutant protein (lacks E3 ligase activity). Left lane, negative control; middle lane, assay with WT Mule; right lane, assay with Mulec4341A protein. ( c ) Upper panel: Lysates of 293T cells overexpressing vector control or Flag-Mule were transiently transfected with HA-KLF4 plasmid, IP'd with anti-Flag Ab and IB'd with anti-Mule and anti-KLF4 Abs. Lower panel: Lysates in the upper panel were IP'd with anti-HA Ab and IB'd with anti-Flag Ab to detect Mule, and with anti-HA Ab to detect KLF4. Total cell lysate was IB'd in parallel as a control. ( d ) IB to detect Flag, Mule and KLF4 in lysates of 293T cells overexpressing vector control or Flag-Mule. ( e ) 293T cells stably expressing either empty vector pCI-neo (left) or human Mule cDNA (right) were transiently transfected with HA-KLF4 plasmid and His-ubiquitin. Cell lysates were IP'd with anti-HA beads, and the beads were subjected to IB with either anti-KLF4 Ab (left) or anti-K48-linked Ub Ab (right). ( f ) 293T cells stably expressing Mule cDNA were transiently transfected with HA-Klf4 plasmid together with WT Ub, K48-only Ub or K63-only Ub. Cell lysates were IP'd with anti-HA beads, and the beads were IB'd with either anti-Ub or anti-HA Ab. Total cell lysate was IB'd with anti-KLF4 Ab. Results are representative of two to three independent experiments.

    Journal: Nature Communications

    Article Title: K48-linked KLF4 ubiquitination by E3 ligase Mule controls T-cell proliferation and cell cycle progression

    doi: 10.1038/ncomms14003

    Figure Lengend Snippet: Identification of KLF4 as a novel Mule substrate. ( a ) IB to detect ELF4 and KLF4 in purified control and TMKO CD4 + and CD8 + T cells. ( b ) IB to detect ubiquitinated KLF4 after incubation of recombinant GST-KLF4 with ubiquitin, E1, HBCH5b (E2) and Mule (E3) or Mulec4341A mutant protein (lacks E3 ligase activity). Left lane, negative control; middle lane, assay with WT Mule; right lane, assay with Mulec4341A protein. ( c ) Upper panel: Lysates of 293T cells overexpressing vector control or Flag-Mule were transiently transfected with HA-KLF4 plasmid, IP'd with anti-Flag Ab and IB'd with anti-Mule and anti-KLF4 Abs. Lower panel: Lysates in the upper panel were IP'd with anti-HA Ab and IB'd with anti-Flag Ab to detect Mule, and with anti-HA Ab to detect KLF4. Total cell lysate was IB'd in parallel as a control. ( d ) IB to detect Flag, Mule and KLF4 in lysates of 293T cells overexpressing vector control or Flag-Mule. ( e ) 293T cells stably expressing either empty vector pCI-neo (left) or human Mule cDNA (right) were transiently transfected with HA-KLF4 plasmid and His-ubiquitin. Cell lysates were IP'd with anti-HA beads, and the beads were subjected to IB with either anti-KLF4 Ab (left) or anti-K48-linked Ub Ab (right). ( f ) 293T cells stably expressing Mule cDNA were transiently transfected with HA-Klf4 plasmid together with WT Ub, K48-only Ub or K63-only Ub. Cell lysates were IP'd with anti-HA beads, and the beads were IB'd with either anti-Ub or anti-HA Ab. Total cell lysate was IB'd with anti-KLF4 Ab. Results are representative of two to three independent experiments.

    Article Snippet: Fractioned proteins were transferred to a nitrocellulose membrane by i-Blot according to the manufacturer's instructions (Invitrogen) and immunoblotted using Abs recognizing the following: ubiquitin (FK2; Enzo Life Sciences; 1:1,000); Lys-specific ubiquitin (Apu2; Millipore; 1:1,000); KLF4 (GeneTex; 1:1,000 ); Mule (Bethyl Laboratories Inc.; 1:1,000); p21 (Santa Cruz; 1:1,000); p27 (BD Biosciences; 1:1,000); p53 (Santa Cruz; 1:1,000); Ser18-p53 (R & D; 1:1,000); or phospho-Erk1/2, phospho-JNK1/2, IκBα, phospho-IκBα, p65 or phospho-IKKα/β (all from Cell Signaling; 1:1,000).

    Techniques: Purification, Incubation, Recombinant, Mutagenesis, Activity Assay, Negative Control, Plasmid Preparation, Transfection, Stable Transfection, Expressing

    Activation of KLF4 in Mule- deficient T cells impairs cell cycle entry. ( a ) IB to detect KLF4, p21 and p27 proteins in purified control and TMKO T cells that were left untreated (0) or treated with anti-CD3/28 Abs for the indicated times. ( b ) IB to detect phospho-Rb in purified control and TMKO T cells that were left untreated (0) or treated with anti-CD3/28 Abs for the indicated times. ( c ) FCM analysis of E2F2 expression by gated purified control and TMKO CD4 + or CD8 + T cells that were fixed, permeabilized and subjected to intracellular staining with anti-E2F2 Ab. ( d ) Quantitation of RT–PCR analysis of E2F1 and E2F2 mRNA expression in purified, untreated control and TMKO CD4 + and CD8 + T cells. Data were normalized to β-actin mRNA and the relative change in gene expression was calculated using the comparative threshold cycle method (2ΔΔ Ct ). Results are the mean±s.d. ( n =3–4). ( e ) Quantitation of ChIP assays of the binding of KLF4 to the E2F2 enhancer in control (Ctl) and Mule -deficient MEFs, and in purified control and TMKO T cells. ( f ) Quantitation by EDU assay of the percentage of cycling cells in cultures of purified control and TMKO T cells that were treated for 24 h with anti-CD3/28 Abs at the indicated doses. Data are expressed as the percentage of S phase-positive T cells and are the mean±s.d. ( n =3). * P

    Journal: Nature Communications

    Article Title: K48-linked KLF4 ubiquitination by E3 ligase Mule controls T-cell proliferation and cell cycle progression

    doi: 10.1038/ncomms14003

    Figure Lengend Snippet: Activation of KLF4 in Mule- deficient T cells impairs cell cycle entry. ( a ) IB to detect KLF4, p21 and p27 proteins in purified control and TMKO T cells that were left untreated (0) or treated with anti-CD3/28 Abs for the indicated times. ( b ) IB to detect phospho-Rb in purified control and TMKO T cells that were left untreated (0) or treated with anti-CD3/28 Abs for the indicated times. ( c ) FCM analysis of E2F2 expression by gated purified control and TMKO CD4 + or CD8 + T cells that were fixed, permeabilized and subjected to intracellular staining with anti-E2F2 Ab. ( d ) Quantitation of RT–PCR analysis of E2F1 and E2F2 mRNA expression in purified, untreated control and TMKO CD4 + and CD8 + T cells. Data were normalized to β-actin mRNA and the relative change in gene expression was calculated using the comparative threshold cycle method (2ΔΔ Ct ). Results are the mean±s.d. ( n =3–4). ( e ) Quantitation of ChIP assays of the binding of KLF4 to the E2F2 enhancer in control (Ctl) and Mule -deficient MEFs, and in purified control and TMKO T cells. ( f ) Quantitation by EDU assay of the percentage of cycling cells in cultures of purified control and TMKO T cells that were treated for 24 h with anti-CD3/28 Abs at the indicated doses. Data are expressed as the percentage of S phase-positive T cells and are the mean±s.d. ( n =3). * P

    Article Snippet: Fractioned proteins were transferred to a nitrocellulose membrane by i-Blot according to the manufacturer's instructions (Invitrogen) and immunoblotted using Abs recognizing the following: ubiquitin (FK2; Enzo Life Sciences; 1:1,000); Lys-specific ubiquitin (Apu2; Millipore; 1:1,000); KLF4 (GeneTex; 1:1,000 ); Mule (Bethyl Laboratories Inc.; 1:1,000); p21 (Santa Cruz; 1:1,000); p27 (BD Biosciences; 1:1,000); p53 (Santa Cruz; 1:1,000); Ser18-p53 (R & D; 1:1,000); or phospho-Erk1/2, phospho-JNK1/2, IκBα, phospho-IκBα, p65 or phospho-IKKα/β (all from Cell Signaling; 1:1,000).

    Techniques: Activation Assay, Purification, Expressing, Staining, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay, CTL Assay, EdU Assay

    Reprogramming HFFs using synthetic modified mRNA. (A) Synthetic modified mRNAs are translated into proteins. Cells transfected with mRNA cocktail expressing reprogramming factors Klf4, cMyc, Oct4, LIN28, and Sox2. The 15-h time point was chosen for analysis

    Journal: Stem Cells and Development

    Article Title: Induced Pluripotent Stem Cell Differentiation and Three-Dimensional Tissue Formation Attenuate Clonal Epigenetic Differences in Trichohyalin

    doi: 10.1089/scd.2016.0156

    Figure Lengend Snippet: Reprogramming HFFs using synthetic modified mRNA. (A) Synthetic modified mRNAs are translated into proteins. Cells transfected with mRNA cocktail expressing reprogramming factors Klf4, cMyc, Oct4, LIN28, and Sox2. The 15-h time point was chosen for analysis

    Article Snippet: The following antibodies were used: rabbit anti-KRT14 (Covance), -Oct4 (Santa Cruz Biotechnology), -Sox2 (GeneTex), -LCE2B (Bioss), -p63 (Abcam), mouse anti-cMyc (Santa Cruz Biotechnology), -Klf4 (ATGen), -TCHH (ImmuQuest), and goat anti-LIN28 (R & D Systems).

    Techniques: Modification, Transfection, Expressing

    KLF4 suppressed HCC migration via regulation of MGLL. A. MGLL was knocked down in SNU449 and BEL7402 cells with or without KLF4 overexpression. Cell lysates were then subjected to western blotting analysis using the indicated antibodies. B-E. Effects of MGLL on migration were examined by transwell and wound healing assays. The results are representative of three independent experiments. *P

    Journal: American Journal of Cancer Research

    Article Title: KLF4 suppresses the migration of hepatocellular carcinoma by transcriptionally upregulating monoglyceride lipase

    doi:

    Figure Lengend Snippet: KLF4 suppressed HCC migration via regulation of MGLL. A. MGLL was knocked down in SNU449 and BEL7402 cells with or without KLF4 overexpression. Cell lysates were then subjected to western blotting analysis using the indicated antibodies. B-E. Effects of MGLL on migration were examined by transwell and wound healing assays. The results are representative of three independent experiments. *P

    Article Snippet: The following antibodies and reagents were used: MGLL (1:100, sigma, HPA011348), beta-actin (1:1000, Proteintech Group, #60008-1-Ig), KLF4 (1:500, Cell Signalling Technology, #12173), and SaveltTM (Hanbio Co., LTD 1:1000).

    Techniques: Migration, Over Expression, Western Blot

    KLF4 enhanced MGLL expression. A. Schematic illustration of pGL3-based reported constructs were used in luciferase assays to examine the transcriptional activity of MGLL. B, C. The promoters of MGLL named P1, P2, P3, and P4 were individually transfected into SNU449 and BEL7402 cells with or without KLF4 overexpression. Luciferase activity was measured. The results are representative of three independent experiments. *P

    Journal: American Journal of Cancer Research

    Article Title: KLF4 suppresses the migration of hepatocellular carcinoma by transcriptionally upregulating monoglyceride lipase

    doi:

    Figure Lengend Snippet: KLF4 enhanced MGLL expression. A. Schematic illustration of pGL3-based reported constructs were used in luciferase assays to examine the transcriptional activity of MGLL. B, C. The promoters of MGLL named P1, P2, P3, and P4 were individually transfected into SNU449 and BEL7402 cells with or without KLF4 overexpression. Luciferase activity was measured. The results are representative of three independent experiments. *P

    Article Snippet: The following antibodies and reagents were used: MGLL (1:100, sigma, HPA011348), beta-actin (1:1000, Proteintech Group, #60008-1-Ig), KLF4 (1:500, Cell Signalling Technology, #12173), and SaveltTM (Hanbio Co., LTD 1:1000).

    Techniques: Expressing, Construct, Luciferase, Activity Assay, Transfection, Over Expression

    KLF4 bound to the promoter of MGLL. A, B. The streptavidin-biotin pull down assay was performed to find out the specific proteins which bind to MGLL promoter. Nuclear extracts prepared from SNU449 and BEL7402 cells were incubated with biotin-labeled MGLL promoter probe (-1000 to 0) and streptavidin-agarose beads. The DNA-protein complexes were separated by SDS-PAGE, and protein bands were visualized by Coomassie Blue Staining. The arrow indicates the candidate MGLL promoter-binding protein. C. ChIP analysis showed the binding of KLF4 to the promoter of MGLL in SNU449 cells with or without KLF4 overexpression. An isotype-matched IgG was used as a negative control. D. ChIP analysis showed the binding of KLF4 to the promoter of MGLL in SNU449 and BEL7402 cells with or without KLF4 knockdown. An isotype-matched IgG was used as a negative control.

    Journal: American Journal of Cancer Research

    Article Title: KLF4 suppresses the migration of hepatocellular carcinoma by transcriptionally upregulating monoglyceride lipase

    doi:

    Figure Lengend Snippet: KLF4 bound to the promoter of MGLL. A, B. The streptavidin-biotin pull down assay was performed to find out the specific proteins which bind to MGLL promoter. Nuclear extracts prepared from SNU449 and BEL7402 cells were incubated with biotin-labeled MGLL promoter probe (-1000 to 0) and streptavidin-agarose beads. The DNA-protein complexes were separated by SDS-PAGE, and protein bands were visualized by Coomassie Blue Staining. The arrow indicates the candidate MGLL promoter-binding protein. C. ChIP analysis showed the binding of KLF4 to the promoter of MGLL in SNU449 cells with or without KLF4 overexpression. An isotype-matched IgG was used as a negative control. D. ChIP analysis showed the binding of KLF4 to the promoter of MGLL in SNU449 and BEL7402 cells with or without KLF4 knockdown. An isotype-matched IgG was used as a negative control.

    Article Snippet: The following antibodies and reagents were used: MGLL (1:100, sigma, HPA011348), beta-actin (1:1000, Proteintech Group, #60008-1-Ig), KLF4 (1:500, Cell Signalling Technology, #12173), and SaveltTM (Hanbio Co., LTD 1:1000).

    Techniques: Pull Down Assay, Incubation, Labeling, SDS Page, Staining, Binding Assay, Chromatin Immunoprecipitation, Over Expression, Negative Control

    Immunofluorescence identification of the generated iPS. The newly derived mRNA iPS colonies were stained for the specialized markers of pluripotency: Oct4, Sox2, Nanog, SSEA-1, Klf4 and c-Myc. Nuclei were counter-stained with DAPI; (magnification 200×).

    Journal: International Journal of Molecular Sciences

    Article Title: Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

    doi: 10.3390/ijms151221840

    Figure Lengend Snippet: Immunofluorescence identification of the generated iPS. The newly derived mRNA iPS colonies were stained for the specialized markers of pluripotency: Oct4, Sox2, Nanog, SSEA-1, Klf4 and c-Myc. Nuclei were counter-stained with DAPI; (magnification 200×).

    Article Snippet: Cells were incubated overnight at 4 °C with one of the following antibodies: anti-Oct4 (1:500; Abcam, Cambridge, MA, USA), anti-Sox2 (1:500; NB110-37235, Novus Biologicals, Littleton, CO, USA), anti-Nanog (1:500; Abcam, Cambridge, MA, USA), anti-c-Myc (1:250; bs-4963R, Bioss, Woburn, MA, USA), anti-Klf4 (1:250, bs-1064R, Bioss, Woburn, MA, USA).

    Techniques: Immunofluorescence, Generated, Derivative Assay, Staining

    Characterization of the mRNA induced pluripotent stem cells (iPSCs). ( a ) The morphological characteristics of iPSCs with round ES-like colonies were distinguished by tightly defined borders and a high nuclear/cytoplasm ratio (magnification 40×); ( b – d ) showed positive alkaline phosphatase activity of the mRNA iPS either grossly ( b , magnification 10×) or through microscopic observation ( c , magnification 100×) and ( d , magnification 200×); ( e ) Total RNA was isolated from three mRNA iPS clones and non-transfected MEF cells were used for RT-PCR to detect expression of pluripotency markers: Oct4 , Sox2 , Nanog , Rex1 , Klf4 and cMyc . Results showed that iPS colonies expressed all markers in contrast to MEF cells; ( f ) Detection of the methylation status of Nanog and Oct4 promoters in both MEF cells, iPS and ESCs by bisulfite sequencing revealed a high percentage of de-methylation in both promoters compared to the parent MEF cells. Open circles indicate the un-methylated state and dark, filled circles indicate the methylated state.

    Journal: International Journal of Molecular Sciences

    Article Title: Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

    doi: 10.3390/ijms151221840

    Figure Lengend Snippet: Characterization of the mRNA induced pluripotent stem cells (iPSCs). ( a ) The morphological characteristics of iPSCs with round ES-like colonies were distinguished by tightly defined borders and a high nuclear/cytoplasm ratio (magnification 40×); ( b – d ) showed positive alkaline phosphatase activity of the mRNA iPS either grossly ( b , magnification 10×) or through microscopic observation ( c , magnification 100×) and ( d , magnification 200×); ( e ) Total RNA was isolated from three mRNA iPS clones and non-transfected MEF cells were used for RT-PCR to detect expression of pluripotency markers: Oct4 , Sox2 , Nanog , Rex1 , Klf4 and cMyc . Results showed that iPS colonies expressed all markers in contrast to MEF cells; ( f ) Detection of the methylation status of Nanog and Oct4 promoters in both MEF cells, iPS and ESCs by bisulfite sequencing revealed a high percentage of de-methylation in both promoters compared to the parent MEF cells. Open circles indicate the un-methylated state and dark, filled circles indicate the methylated state.

    Article Snippet: Cells were incubated overnight at 4 °C with one of the following antibodies: anti-Oct4 (1:500; Abcam, Cambridge, MA, USA), anti-Sox2 (1:500; NB110-37235, Novus Biologicals, Littleton, CO, USA), anti-Nanog (1:500; Abcam, Cambridge, MA, USA), anti-c-Myc (1:250; bs-4963R, Bioss, Woburn, MA, USA), anti-Klf4 (1:250, bs-1064R, Bioss, Woburn, MA, USA).

    Techniques: Activity Assay, Isolation, Clone Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Methylation, Methylation Sequencing

    Changes in introduced factors and pluripotency markers during reprogramming. Quantification of pluripotency factors expression by qRT-PCR at different times (D1, D6, D9, D12 and D15) throughout the reprogramming timeline. ( a ) Total and endogenous expression levels of the transfected factors; Oct4 , Sox2 , c-Myc and Klf4 ; ( b ) the expression level of non-transfected factors ( Nanog and Rex-1 ); ( c ) Immunostaining of non-transfected factors at day 6 confirmed the results of the qRT-PCR concerning Nanog and SSEA-1 , as they showed nuclear localization of Nanog and surface expression of SSEA-1. Nuclei were counterstained with DAPI; (magnification 100×).

    Journal: International Journal of Molecular Sciences

    Article Title: Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

    doi: 10.3390/ijms151221840

    Figure Lengend Snippet: Changes in introduced factors and pluripotency markers during reprogramming. Quantification of pluripotency factors expression by qRT-PCR at different times (D1, D6, D9, D12 and D15) throughout the reprogramming timeline. ( a ) Total and endogenous expression levels of the transfected factors; Oct4 , Sox2 , c-Myc and Klf4 ; ( b ) the expression level of non-transfected factors ( Nanog and Rex-1 ); ( c ) Immunostaining of non-transfected factors at day 6 confirmed the results of the qRT-PCR concerning Nanog and SSEA-1 , as they showed nuclear localization of Nanog and surface expression of SSEA-1. Nuclei were counterstained with DAPI; (magnification 100×).

    Article Snippet: Cells were incubated overnight at 4 °C with one of the following antibodies: anti-Oct4 (1:500; Abcam, Cambridge, MA, USA), anti-Sox2 (1:500; NB110-37235, Novus Biologicals, Littleton, CO, USA), anti-Nanog (1:500; Abcam, Cambridge, MA, USA), anti-c-Myc (1:250; bs-4963R, Bioss, Woburn, MA, USA), anti-Klf4 (1:250, bs-1064R, Bioss, Woburn, MA, USA).

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Immunostaining

    Plasmid construction and mRNA synthesis. ( a ) Successful amplification of the four factors from selected organs using specific primers for each. Oct4 (O) and Sox2 (S) were amplified from testes while c-Myc (C) was amplified from small intestine. Klf4 (K) was correctly amplified from colon. The DNA marker (M) used was a 2000 bp marker; ( b ) The eukaryotic expression vector (pCDNA 3) was digested using the same enzymes used for cutting the genes. Lane ( 1 ) shows the result of double restriction of the plasmid using Eco RI and Xho I, while Lane ( 2 ) revealed the single cut using Xho I. Lane ( 3 ), shows the uncut circular plasmid. (M): DNA marker (5000 bp); ( c ) Confirmation of the successful cloning through digestion of the newly formed recombinant plasmids (pCDNA– Oct4 , pCDNA– Sox2 , pCDNA– cMyc and pCDNA– Klf4 ) using Eco RI and Xho I enzymes resulted in two bands that were similar to the expected size of each gene as showed in Lane ( 2 ), while Lane ( 1 ) shows the single cut of each recombinant plasmid (M): DNA marker (5000 bp).

    Journal: International Journal of Molecular Sciences

    Article Title: Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

    doi: 10.3390/ijms151221840

    Figure Lengend Snippet: Plasmid construction and mRNA synthesis. ( a ) Successful amplification of the four factors from selected organs using specific primers for each. Oct4 (O) and Sox2 (S) were amplified from testes while c-Myc (C) was amplified from small intestine. Klf4 (K) was correctly amplified from colon. The DNA marker (M) used was a 2000 bp marker; ( b ) The eukaryotic expression vector (pCDNA 3) was digested using the same enzymes used for cutting the genes. Lane ( 1 ) shows the result of double restriction of the plasmid using Eco RI and Xho I, while Lane ( 2 ) revealed the single cut using Xho I. Lane ( 3 ), shows the uncut circular plasmid. (M): DNA marker (5000 bp); ( c ) Confirmation of the successful cloning through digestion of the newly formed recombinant plasmids (pCDNA– Oct4 , pCDNA– Sox2 , pCDNA– cMyc and pCDNA– Klf4 ) using Eco RI and Xho I enzymes resulted in two bands that were similar to the expected size of each gene as showed in Lane ( 2 ), while Lane ( 1 ) shows the single cut of each recombinant plasmid (M): DNA marker (5000 bp).

    Article Snippet: Cells were incubated overnight at 4 °C with one of the following antibodies: anti-Oct4 (1:500; Abcam, Cambridge, MA, USA), anti-Sox2 (1:500; NB110-37235, Novus Biologicals, Littleton, CO, USA), anti-Nanog (1:500; Abcam, Cambridge, MA, USA), anti-c-Myc (1:250; bs-4963R, Bioss, Woburn, MA, USA), anti-Klf4 (1:250, bs-1064R, Bioss, Woburn, MA, USA).

    Techniques: Plasmid Preparation, Amplification, Marker, Expressing, Clone Assay, Recombinant

    DDX3 suppresses stemness gene signature. ( a ) Lower expression of DDX3 along with overexpression of stemness markers was observed in the poorly differentiated cell line. Cell lysates (50 μg) of HepG2, Hep3B, HuH-7 and SK-Hep-1 cells were analyzed by western blotting with antibodies against DDX3, Nanog, Oct4, c-Myc, Sox2, KLF4, Bmi1, CK19 and β-actin. ( b ) Knockdown of DDX3 led to up-regulation of stemness markers. Cell lysates (50 μg) of stable shLuc, shDDX3 #2 and shDDX3 #3 HepG2 cells were analyzed by western blotting with antibodies described in ( a ). ( c ) DDX3 overexpression suppressed stemness markers. Plasmid pcDNA3-SRα/FLAG or pcDNA3-SRα/FLAG-DDX3 was transfected into SK-Hep-1 cells. At 48 h post transfection, cell lysates were prepared and subjected to immunoblotting with antibodies described in ( a ) and anti-FLAG antibody. β-actin was used as internal control in ( a – c ). ( d ) DDX3 knockdown was correlated with up-regulation of hepatic CSC surface markers. mRNA expressions of DDX3, CD133, CD13, EpCAM, CD90 and GAPDH in shLuc, shDDX3 #2 and shDDX3 #3 cells were detected by qRT-PCR. GAPDH was used as internal control. Fold change of each mRNA transcript in shDDX3 #2 and shDDX3 #3 cells was relative to that of shLuc cells. ( e ) DDX3 overexpression resulted in suppression of hepatic CSC surface markers. SK-Hep-1 cells were transfected with plasmid pcDNA3-SRα/FLAG or pcDNA3-SRα/FLAG-DDX3 as described in ( c ). At 48 h post transfection, total RNA was extracted and subjected to qRT-PCR analysis. GAPDH was used as internal control. Fold change of each mRNA transcript in FLAG-DDX3-expressing cells was relative to that of vector control cells. All experiments were performed at least three times, and the error bar indicates ± 1 s.d. of the mean. Statistical analyses were carried out using t test (* p

    Journal: Scientific Reports

    Article Title: DDX3 Represses Stemness by Epigenetically Modulating Tumor-suppressive miRNAs in Hepatocellular Carcinoma

    doi: 10.1038/srep28637

    Figure Lengend Snippet: DDX3 suppresses stemness gene signature. ( a ) Lower expression of DDX3 along with overexpression of stemness markers was observed in the poorly differentiated cell line. Cell lysates (50 μg) of HepG2, Hep3B, HuH-7 and SK-Hep-1 cells were analyzed by western blotting with antibodies against DDX3, Nanog, Oct4, c-Myc, Sox2, KLF4, Bmi1, CK19 and β-actin. ( b ) Knockdown of DDX3 led to up-regulation of stemness markers. Cell lysates (50 μg) of stable shLuc, shDDX3 #2 and shDDX3 #3 HepG2 cells were analyzed by western blotting with antibodies described in ( a ). ( c ) DDX3 overexpression suppressed stemness markers. Plasmid pcDNA3-SRα/FLAG or pcDNA3-SRα/FLAG-DDX3 was transfected into SK-Hep-1 cells. At 48 h post transfection, cell lysates were prepared and subjected to immunoblotting with antibodies described in ( a ) and anti-FLAG antibody. β-actin was used as internal control in ( a – c ). ( d ) DDX3 knockdown was correlated with up-regulation of hepatic CSC surface markers. mRNA expressions of DDX3, CD133, CD13, EpCAM, CD90 and GAPDH in shLuc, shDDX3 #2 and shDDX3 #3 cells were detected by qRT-PCR. GAPDH was used as internal control. Fold change of each mRNA transcript in shDDX3 #2 and shDDX3 #3 cells was relative to that of shLuc cells. ( e ) DDX3 overexpression resulted in suppression of hepatic CSC surface markers. SK-Hep-1 cells were transfected with plasmid pcDNA3-SRα/FLAG or pcDNA3-SRα/FLAG-DDX3 as described in ( c ). At 48 h post transfection, total RNA was extracted and subjected to qRT-PCR analysis. GAPDH was used as internal control. Fold change of each mRNA transcript in FLAG-DDX3-expressing cells was relative to that of vector control cells. All experiments were performed at least three times, and the error bar indicates ± 1 s.d. of the mean. Statistical analyses were carried out using t test (* p

    Article Snippet: Anti-Sox2 (GTX627404), anti-KLF4 (GTX101508), anti-DNMT3A (GTX116011), anti-DNMT3B (GTX62171) and anti-DNMT1 (GTX62550) antibodies were purchased from GeneTex (Irvine, CA, USA).

    Techniques: Expressing, Over Expression, Western Blot, Plasmid Preparation, Transfection, Quantitative RT-PCR

    KLF4 is a direct target of miR-543 and KLF4 expression is inversely correlated with miR-543 expression in CRC tissues (A) Western blot analysis of KLF4 protein expression after transfection in HCT116 and SW-480 cells. (B) Luciferase activities of wild-type and the mutant pmirGLO- KLF4 -3′-UTR reporter in HCT116 and SW-480 cells. (C) The predicted miR-543 binding site on the KLF4 mRNA 3′-UTR and the corresponding mutations in 3′-UTR of KLF4 . (D, E) KLF4 expression on protein level (D) and mRNA level (E) was determined in CRC tissues (T) and adjacent nontumorous tissues (ANT). (F) Spearman's correlation analysis was performed to detect the association between the expression level of miR-543 and KLF4 in GC tissues. (G, H) KLF4 expression on protein level (G) and mRNA level (H) was determined in three CRC cell lines (HCT116, SW-480 and HT29) and nontumorous mucosa. Error bars represent mean ± SD from three independent experiments. * p

    Journal: Oncotarget

    Article Title: miR-543 promotes colorectal cancer proliferation and metastasis by targeting KLF4

    doi: 10.18632/oncotarget.19495

    Figure Lengend Snippet: KLF4 is a direct target of miR-543 and KLF4 expression is inversely correlated with miR-543 expression in CRC tissues (A) Western blot analysis of KLF4 protein expression after transfection in HCT116 and SW-480 cells. (B) Luciferase activities of wild-type and the mutant pmirGLO- KLF4 -3′-UTR reporter in HCT116 and SW-480 cells. (C) The predicted miR-543 binding site on the KLF4 mRNA 3′-UTR and the corresponding mutations in 3′-UTR of KLF4 . (D, E) KLF4 expression on protein level (D) and mRNA level (E) was determined in CRC tissues (T) and adjacent nontumorous tissues (ANT). (F) Spearman's correlation analysis was performed to detect the association between the expression level of miR-543 and KLF4 in GC tissues. (G, H) KLF4 expression on protein level (G) and mRNA level (H) was determined in three CRC cell lines (HCT116, SW-480 and HT29) and nontumorous mucosa. Error bars represent mean ± SD from three independent experiments. * p

    Article Snippet: The following commercial antibodies were used in this study: KLF4 (1:1000, Proteintech, Wuhan, China), E-cadherin (1:1000, Santa Cruz, CA, USA), vimentin (1:1000, Santa Cruz) β-actin (1:1000; Cell Signaling, Danvers, MA, USA).

    Techniques: Expressing, Western Blot, Transfection, Luciferase, Mutagenesis, Binding Assay

    Downregulation of KLF4 rescues miR-543's oncogenic effect on CRC cell proliferation, apoptosis, migration, and invasion in HCT116 cells (A) KLF4 protein expression was detected in HCT116 cells co-transfected with miR-543 inhibitor/miR-NC and KLF4 siRNA or si-NC. (B–E) Cell proliferation, apoptosis, migration and invasion were assessed in HCT116 cells co-transfected with miR-543 inhibitor/miR-NC and KLF4 siRNA or si-NC. * p

    Journal: Oncotarget

    Article Title: miR-543 promotes colorectal cancer proliferation and metastasis by targeting KLF4

    doi: 10.18632/oncotarget.19495

    Figure Lengend Snippet: Downregulation of KLF4 rescues miR-543's oncogenic effect on CRC cell proliferation, apoptosis, migration, and invasion in HCT116 cells (A) KLF4 protein expression was detected in HCT116 cells co-transfected with miR-543 inhibitor/miR-NC and KLF4 siRNA or si-NC. (B–E) Cell proliferation, apoptosis, migration and invasion were assessed in HCT116 cells co-transfected with miR-543 inhibitor/miR-NC and KLF4 siRNA or si-NC. * p

    Article Snippet: The following commercial antibodies were used in this study: KLF4 (1:1000, Proteintech, Wuhan, China), E-cadherin (1:1000, Santa Cruz, CA, USA), vimentin (1:1000, Santa Cruz) β-actin (1:1000; Cell Signaling, Danvers, MA, USA).

    Techniques: Migration, Expressing, Transfection

    Active p38/MAPK pathway is necessary for FBXO32-mediated KLF4 degradation. ( a ) p38 inhibitor leads to KLF4 accumulation. MCF7 cells plated in six-well plate were treated with DMSO or indicated inhibitors for 6 h. The protein level of KLF4 was detected by immunoblot. ( b ) KLF4 protein half-life is elongated under p38 inhibition. MCF7 cells plated in six-well plate were treated with DMSO or p38 inhibitors (SB203580 and SB239063) for 6 h. Then CHX was added 0, 2, 4 or 6 h before harvest. KLF4 protein level was tested by immunoblotting. ( c ) p38 inhibitor abolishes FBXO32-mediated KLF4 degradation. HEK293T cells were transfected with His-tagged KLF4 and Myc-tagged FBXO32. Twenty-four hours after transfection, the cells were treated with DMSO or p38 inhibitor for 6 h followed by treatment with CHX for indicated time. Western blot was performed to examine the protein level of KLF4. ( d , f ) p38 inhibitor disrupts KLF4 and FBXO32 interaction. HEK293T cells were transfected with Flag-tagged KLF4 and Myc-tagged FBXO32 or empty vector. Twenty-four hours after transfection, the cells were treated with DMSO or SB203580 for 6 h followed by 4-h treatment with MG132. The cell lysates were precipitated with anti-Flag M2 affinity gel and the precipitates were analyzed by western blot ( d ). MCF7 cells cultured in 10 cm dishes were treated with either DMSO or SB203580 for 6 h. The cell lysates were subjected to co-IP analysis by KLF4 antibody. The immunoprecipitates were blotted by FBXO32 antibody ( f ). ( e, g ) p38 inhibitor decreased FBXO32-mediated KLF4 ubiquitination. Indicated plasmids were co-transfected into HEK293T cells. Twenty-four hours after transfection, the cells were treated with DMSO or SB203580 for 6 h followed by 6- h treatment with MG132. The cells were subjected to ubiquitination assay and the ubiquitinated KLF4 were detected by immunoblotting with anti-HA antibody ( e ). FBXO32 overexpressed MCF7 cells cultured in 10 cm dishes were treated with either DMSO or SB203580 for 6 h. The cell lysates were subjected to ubiquitination analysis. Ubiquitinated KLF4 proteins were revealed by immunoblotting with ubiquitin antibody ( g ).

    Journal: Oncogene

    Article Title: FBXO32 suppresses breast cancer tumorigenesis through targeting KLF4 to proteasomal degradation

    doi: 10.1038/onc.2016.479

    Figure Lengend Snippet: Active p38/MAPK pathway is necessary for FBXO32-mediated KLF4 degradation. ( a ) p38 inhibitor leads to KLF4 accumulation. MCF7 cells plated in six-well plate were treated with DMSO or indicated inhibitors for 6 h. The protein level of KLF4 was detected by immunoblot. ( b ) KLF4 protein half-life is elongated under p38 inhibition. MCF7 cells plated in six-well plate were treated with DMSO or p38 inhibitors (SB203580 and SB239063) for 6 h. Then CHX was added 0, 2, 4 or 6 h before harvest. KLF4 protein level was tested by immunoblotting. ( c ) p38 inhibitor abolishes FBXO32-mediated KLF4 degradation. HEK293T cells were transfected with His-tagged KLF4 and Myc-tagged FBXO32. Twenty-four hours after transfection, the cells were treated with DMSO or p38 inhibitor for 6 h followed by treatment with CHX for indicated time. Western blot was performed to examine the protein level of KLF4. ( d , f ) p38 inhibitor disrupts KLF4 and FBXO32 interaction. HEK293T cells were transfected with Flag-tagged KLF4 and Myc-tagged FBXO32 or empty vector. Twenty-four hours after transfection, the cells were treated with DMSO or SB203580 for 6 h followed by 4-h treatment with MG132. The cell lysates were precipitated with anti-Flag M2 affinity gel and the precipitates were analyzed by western blot ( d ). MCF7 cells cultured in 10 cm dishes were treated with either DMSO or SB203580 for 6 h. The cell lysates were subjected to co-IP analysis by KLF4 antibody. The immunoprecipitates were blotted by FBXO32 antibody ( f ). ( e, g ) p38 inhibitor decreased FBXO32-mediated KLF4 ubiquitination. Indicated plasmids were co-transfected into HEK293T cells. Twenty-four hours after transfection, the cells were treated with DMSO or SB203580 for 6 h followed by 6- h treatment with MG132. The cells were subjected to ubiquitination assay and the ubiquitinated KLF4 were detected by immunoblotting with anti-HA antibody ( e ). FBXO32 overexpressed MCF7 cells cultured in 10 cm dishes were treated with either DMSO or SB203580 for 6 h. The cell lysates were subjected to ubiquitination analysis. Ubiquitinated KLF4 proteins were revealed by immunoblotting with ubiquitin antibody ( g ).

    Article Snippet: The following antibodies were used in immunohistochemistry and immunofluorescent double staining: anti-FBXO32 (LS-B9748, Lifespan Biosciences, Seattle, WA, USA) and anti-KLF4 (NBP2-24749, Novus).

    Techniques: Inhibition, Transfection, Western Blot, Plasmid Preparation, Cell Culture, Co-Immunoprecipitation Assay, Ubiquitin Assay

    FBXO32 interacts with KLF4 and mediates KLF4 ubiquitination and degradation. ( a ) Exogenous FBXO32 interacts with KLF4. HEK293T cells were transfected with Flag-tagged KLF4 and either empty vector or the Myc-tagged FBXO32. Twenty-four hours after transfection, the cells were treated with 20 μM MG132 for 4 h. The co-IP experiment was performed using anti-Flag M2 affinity gel. The immunoprecipitates were analyzed by western blot with anti-Myc antibody. ( b ) FBXO32 increases exogenous KLF4 ubiquitination. Indicated plasmids were transfected into HEK293T cells. Twenty-four hours after transfection, the cells were treated with 20 μM MG132 for 6 h to accumulate the ubiquitinated KLF4. Anti-Flag M2 affinity gel was used to immunoprecipitate tagged KLF4. The ubiquitin was probed by HA-specific antibody to reflect the ubiquitination of KLF4. ( c ) MG132 restores FBXO32-mediated KLF4 decreasing. HA-tagged KLF4 is transfected with either the empty vector or the Myc-tagged FBXO32 in HEK293T cells. Twenty-four hours after transfection, CHX and MG132 were added as indicated for 4 h. The cell lysates were subjected to western blot to evaluate the protein level of KLF4. ( d ) FBXO32 knockdown leads to KLF4 protein accumulation. siRNAs specifically targeting FBXO32 are transfected into MCF7 and MCF10A cells, the expression of KLF4 and FBXO32 was tested by western blot. ( e ) FBXO32 overexpression decreases the protein level of KLF4. MCF7 and MCF10A cells stably expressed FBXO32 were established. The cell lysates with equal amount of total protein were used to test the protein levels of FBXO32 and KLF4 via immunoblotting. ( f ) Endogenous FBXO32 interacts with KLF4. MCF7 or MCF10A cells cultured in 10 cm dish were treated with MG132 for 4 h and harvested in 1 ml of cell lysis buffer. IP was performed with anti-KLF4 primary antibody. The immunoprecipitates were analyzed by western blot and detected with anti-FBXO32 antibody. ( g , h ) Effect of FBXO32 on the ubiquitination of endogenous KLF4. Control and FBXO32 overexpressed MCF7 cells ( g ) or control and FBXO32 knockdown MCF10A cells ( h ) were treated with MG132 for 6 h and subjected to ubiquitination assay. Immunoprecipitated KLF4 proteins were analyzed with western blot and detected with ubiquitin antibody.

    Journal: Oncogene

    Article Title: FBXO32 suppresses breast cancer tumorigenesis through targeting KLF4 to proteasomal degradation

    doi: 10.1038/onc.2016.479

    Figure Lengend Snippet: FBXO32 interacts with KLF4 and mediates KLF4 ubiquitination and degradation. ( a ) Exogenous FBXO32 interacts with KLF4. HEK293T cells were transfected with Flag-tagged KLF4 and either empty vector or the Myc-tagged FBXO32. Twenty-four hours after transfection, the cells were treated with 20 μM MG132 for 4 h. The co-IP experiment was performed using anti-Flag M2 affinity gel. The immunoprecipitates were analyzed by western blot with anti-Myc antibody. ( b ) FBXO32 increases exogenous KLF4 ubiquitination. Indicated plasmids were transfected into HEK293T cells. Twenty-four hours after transfection, the cells were treated with 20 μM MG132 for 6 h to accumulate the ubiquitinated KLF4. Anti-Flag M2 affinity gel was used to immunoprecipitate tagged KLF4. The ubiquitin was probed by HA-specific antibody to reflect the ubiquitination of KLF4. ( c ) MG132 restores FBXO32-mediated KLF4 decreasing. HA-tagged KLF4 is transfected with either the empty vector or the Myc-tagged FBXO32 in HEK293T cells. Twenty-four hours after transfection, CHX and MG132 were added as indicated for 4 h. The cell lysates were subjected to western blot to evaluate the protein level of KLF4. ( d ) FBXO32 knockdown leads to KLF4 protein accumulation. siRNAs specifically targeting FBXO32 are transfected into MCF7 and MCF10A cells, the expression of KLF4 and FBXO32 was tested by western blot. ( e ) FBXO32 overexpression decreases the protein level of KLF4. MCF7 and MCF10A cells stably expressed FBXO32 were established. The cell lysates with equal amount of total protein were used to test the protein levels of FBXO32 and KLF4 via immunoblotting. ( f ) Endogenous FBXO32 interacts with KLF4. MCF7 or MCF10A cells cultured in 10 cm dish were treated with MG132 for 4 h and harvested in 1 ml of cell lysis buffer. IP was performed with anti-KLF4 primary antibody. The immunoprecipitates were analyzed by western blot and detected with anti-FBXO32 antibody. ( g , h ) Effect of FBXO32 on the ubiquitination of endogenous KLF4. Control and FBXO32 overexpressed MCF7 cells ( g ) or control and FBXO32 knockdown MCF10A cells ( h ) were treated with MG132 for 6 h and subjected to ubiquitination assay. Immunoprecipitated KLF4 proteins were analyzed with western blot and detected with ubiquitin antibody.

    Article Snippet: The following antibodies were used in immunohistochemistry and immunofluorescent double staining: anti-FBXO32 (LS-B9748, Lifespan Biosciences, Seattle, WA, USA) and anti-KLF4 (NBP2-24749, Novus).

    Techniques: Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot, Expressing, Over Expression, Stable Transfection, Cell Culture, Lysis, Ubiquitin Assay, Immunoprecipitation

    The C-terminus of FBXO32 is responsible for recognizing and interacting with KLF4 and the F-box domain is critical for FBXO32 E3 ligase function. ( a ) Schematic of human FBXO32 with previously identified domains as well as deletion mutants generated in the present work. ( b ) The F-box domain is critical for the E3 ligase activity of FBXO32. HA-tagged KLF4 and GST-tagged FBXO32 were transfected into HEK293T cells. Twenty-four hours after transfection, the cells were treated with CHX for indicated time. The cell lysates were analyzed by western blot for KLF4 protein level. ( c ) Deletion of F-box abrogated FBXO32-mediated KLF4 ubiquitination. Flag-tagged KLF4 and GST-tagged wild-type or F-box deletion mutant FBXO32 were co-transfected into HEK293T cells. The cells were treated with MG132 and subjected to ubiquitination assay as described. ( d ) FBXO32 interacts with KLF4 via its C-terminus. Indicated plasmids were transfected into HEK293T cells. Twenty-four hours later, the cells were treated with MG132 and subjected to GST pulldown. The precipitates were analyzed by western blot.

    Journal: Oncogene

    Article Title: FBXO32 suppresses breast cancer tumorigenesis through targeting KLF4 to proteasomal degradation

    doi: 10.1038/onc.2016.479

    Figure Lengend Snippet: The C-terminus of FBXO32 is responsible for recognizing and interacting with KLF4 and the F-box domain is critical for FBXO32 E3 ligase function. ( a ) Schematic of human FBXO32 with previously identified domains as well as deletion mutants generated in the present work. ( b ) The F-box domain is critical for the E3 ligase activity of FBXO32. HA-tagged KLF4 and GST-tagged FBXO32 were transfected into HEK293T cells. Twenty-four hours after transfection, the cells were treated with CHX for indicated time. The cell lysates were analyzed by western blot for KLF4 protein level. ( c ) Deletion of F-box abrogated FBXO32-mediated KLF4 ubiquitination. Flag-tagged KLF4 and GST-tagged wild-type or F-box deletion mutant FBXO32 were co-transfected into HEK293T cells. The cells were treated with MG132 and subjected to ubiquitination assay as described. ( d ) FBXO32 interacts with KLF4 via its C-terminus. Indicated plasmids were transfected into HEK293T cells. Twenty-four hours later, the cells were treated with MG132 and subjected to GST pulldown. The precipitates were analyzed by western blot.

    Article Snippet: The following antibodies were used in immunohistochemistry and immunofluorescent double staining: anti-FBXO32 (LS-B9748, Lifespan Biosciences, Seattle, WA, USA) and anti-KLF4 (NBP2-24749, Novus).

    Techniques: Generated, Activity Assay, Transfection, Western Blot, Mutagenesis, Ubiquitin Assay

    The KLF4 N-terminus is critical for interaction with FBXO32. ( a ) Schematic of human KLF4 with functional domain, as well as deletion mutants generated in this study. AD, transactivation domain; ID, transrepression domain; NLS, nuclear localization signal; Z, zinc-finger domain. ( b ) Deletion of amino acids 1–60 stabilizes KLF4. Flag-tagged wild-type or deletion mutants of KLF4 were transfected into HEK293T cells together with empty vector or Myc-tagged FBXO32. Twenty-four hours after transfection, the protein level of wild-type KLF4 or deletion mutants were tested by immunoblotting with antibody against Flag. ( c ) Deletion of amino acids 1–60 eliminates KLF4 and FBXO32 interaction. Indicated plasmids were transfected into HEK293T cells. Twenty-four hours after transfection, the cells were treated with MG132 for 4 h. Co-IP was performed with anti-Flag M2 affinity gel.

    Journal: Oncogene

    Article Title: FBXO32 suppresses breast cancer tumorigenesis through targeting KLF4 to proteasomal degradation

    doi: 10.1038/onc.2016.479

    Figure Lengend Snippet: The KLF4 N-terminus is critical for interaction with FBXO32. ( a ) Schematic of human KLF4 with functional domain, as well as deletion mutants generated in this study. AD, transactivation domain; ID, transrepression domain; NLS, nuclear localization signal; Z, zinc-finger domain. ( b ) Deletion of amino acids 1–60 stabilizes KLF4. Flag-tagged wild-type or deletion mutants of KLF4 were transfected into HEK293T cells together with empty vector or Myc-tagged FBXO32. Twenty-four hours after transfection, the protein level of wild-type KLF4 or deletion mutants were tested by immunoblotting with antibody against Flag. ( c ) Deletion of amino acids 1–60 eliminates KLF4 and FBXO32 interaction. Indicated plasmids were transfected into HEK293T cells. Twenty-four hours after transfection, the cells were treated with MG132 for 4 h. Co-IP was performed with anti-Flag M2 affinity gel.

    Article Snippet: The following antibodies were used in immunohistochemistry and immunofluorescent double staining: anti-FBXO32 (LS-B9748, Lifespan Biosciences, Seattle, WA, USA) and anti-KLF4 (NBP2-24749, Novus).

    Techniques: Functional Assay, Generated, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay

    FBXO32 facilitates tumorigenesis both in vitro and in vivo . ( a ) FBXO32/KLF4 double knockdown MCFCA1a cells. MCFCA1a cells with FBXO32 stable knockdown and FBXO32/KLF4 double knockdown were developed using a lentivirus system. Expression of FBXO32 and KLF4 were analyzed by western blot. ( b ) FBXO32 inhibits tumorigenesis through mediating proteasomal degradation of KLF4 in MCFCA1a cells. In all, 30 000 MCFCA1a cells were plated in 60-mm dish to grow in soft agar. Three weeks later, the cells were dyed with crystal violet and the colony numbers were counted. ( c ) FBXO32 inhibits tumorigenesis through mediating proteasomal degradation of KLF4 in a mouse model. In total, 4 × 10 5 MCFCA1a cells were injected orthotopically into BALB/c nude mice ( n = 8). The mice were killed 3 weeks later. The xenografts were weighed and image of primary tumors was presented. ( d ) Tissue microarray sections were stained by KLF4 and FBXO32 antibodies. Representative staining of KLF4 and FBXO32 (20 ×) was shown and the tissue staining was quantified and analyzed. ( e ) Immunofluorescent double staining of human breast cancer tissues. Tissue microarray section was immunoblotted by KLF4 and FBXO32 antibodies and labeled with second antibody conjugated with FITC and CY3. Immunofluorescence labeled tissue samples were then scanned by laser confocal microscopy. Representative staining were presented. ** P

    Journal: Oncogene

    Article Title: FBXO32 suppresses breast cancer tumorigenesis through targeting KLF4 to proteasomal degradation

    doi: 10.1038/onc.2016.479

    Figure Lengend Snippet: FBXO32 facilitates tumorigenesis both in vitro and in vivo . ( a ) FBXO32/KLF4 double knockdown MCFCA1a cells. MCFCA1a cells with FBXO32 stable knockdown and FBXO32/KLF4 double knockdown were developed using a lentivirus system. Expression of FBXO32 and KLF4 were analyzed by western blot. ( b ) FBXO32 inhibits tumorigenesis through mediating proteasomal degradation of KLF4 in MCFCA1a cells. In all, 30 000 MCFCA1a cells were plated in 60-mm dish to grow in soft agar. Three weeks later, the cells were dyed with crystal violet and the colony numbers were counted. ( c ) FBXO32 inhibits tumorigenesis through mediating proteasomal degradation of KLF4 in a mouse model. In total, 4 × 10 5 MCFCA1a cells were injected orthotopically into BALB/c nude mice ( n = 8). The mice were killed 3 weeks later. The xenografts were weighed and image of primary tumors was presented. ( d ) Tissue microarray sections were stained by KLF4 and FBXO32 antibodies. Representative staining of KLF4 and FBXO32 (20 ×) was shown and the tissue staining was quantified and analyzed. ( e ) Immunofluorescent double staining of human breast cancer tissues. Tissue microarray section was immunoblotted by KLF4 and FBXO32 antibodies and labeled with second antibody conjugated with FITC and CY3. Immunofluorescence labeled tissue samples were then scanned by laser confocal microscopy. Representative staining were presented. ** P

    Article Snippet: The following antibodies were used in immunohistochemistry and immunofluorescent double staining: anti-FBXO32 (LS-B9748, Lifespan Biosciences, Seattle, WA, USA) and anti-KLF4 (NBP2-24749, Novus).

    Techniques: In Vitro, In Vivo, Expressing, Western Blot, Injection, Mouse Assay, Microarray, Staining, Double Staining, Labeling, Immunofluorescence, Confocal Microscopy

    A genome-wide functional screen for E3 ubiquitin ligase(s) targeting KLF4 protein for degradation. ( a , b ) KLF4 protein has a short half-life and is degraded via ubiquitin–proteasome pathway. MCF7 cells were plated in six-well plates. Twenty-four hours later, the cells were treated with 50 μM CHX ( a ) or 20 μM MG132 ( b ) for the indicated time. KLF4 level was analyzed by western blot. ( c ) Exogenously expressed KLF4 is degraded through proteasomal pathway. HEK293T cells were transfected with Flag-tagged KLF4, treated with 50 μM CHX for indicated time with or without 20 μM MG132. The exogenous KLF4 was probed by immunoblot with Flag antibody. ( d ) Experimental procedure flow chart for identification of the E3 ubiquitin ligase(s) targeting KLF4 protein. ( e ) A second round of siRNA screening was carried out to confirm the candidates. KLF4 level was probed by immunoblot after siRNA knockdown of 14 candidates. Quantification was made by Image J (NIH, Bethesda, MD, USA). ( f ) FBXO32 facilitates KLF4 degradation. HA-tagged KLF4 is co-transfected with five E3 ligase candidates individually. Twenty-four hours after transfection, the cells were treated with CHX for indicated time. KLF4 and E3 ligase candidates were analyzed by western blot with antibodies against HA and myc. Quantification was made by Image J (NIH) and the statistics were done with GraphPad Prism.

    Journal: Oncogene

    Article Title: FBXO32 suppresses breast cancer tumorigenesis through targeting KLF4 to proteasomal degradation

    doi: 10.1038/onc.2016.479

    Figure Lengend Snippet: A genome-wide functional screen for E3 ubiquitin ligase(s) targeting KLF4 protein for degradation. ( a , b ) KLF4 protein has a short half-life and is degraded via ubiquitin–proteasome pathway. MCF7 cells were plated in six-well plates. Twenty-four hours later, the cells were treated with 50 μM CHX ( a ) or 20 μM MG132 ( b ) for the indicated time. KLF4 level was analyzed by western blot. ( c ) Exogenously expressed KLF4 is degraded through proteasomal pathway. HEK293T cells were transfected with Flag-tagged KLF4, treated with 50 μM CHX for indicated time with or without 20 μM MG132. The exogenous KLF4 was probed by immunoblot with Flag antibody. ( d ) Experimental procedure flow chart for identification of the E3 ubiquitin ligase(s) targeting KLF4 protein. ( e ) A second round of siRNA screening was carried out to confirm the candidates. KLF4 level was probed by immunoblot after siRNA knockdown of 14 candidates. Quantification was made by Image J (NIH, Bethesda, MD, USA). ( f ) FBXO32 facilitates KLF4 degradation. HA-tagged KLF4 is co-transfected with five E3 ligase candidates individually. Twenty-four hours after transfection, the cells were treated with CHX for indicated time. KLF4 and E3 ligase candidates were analyzed by western blot with antibodies against HA and myc. Quantification was made by Image J (NIH) and the statistics were done with GraphPad Prism.

    Article Snippet: The following antibodies were used in immunohistochemistry and immunofluorescent double staining: anti-FBXO32 (LS-B9748, Lifespan Biosciences, Seattle, WA, USA) and anti-KLF4 (NBP2-24749, Novus).

    Techniques: Genome Wide, Functional Assay, Western Blot, Transfection, Flow Cytometry

    Erk5 Signaling Stabilizes Naive Pluripotency and Suppresses Transition to Primed (A) Erk5 −/− mESCs were transfected with empty vector or Erk5 cDNA and Nanog, Oct4, Klf2, Rex1, and Esrrb mRNA levels determined by qRT-PCR following 3-day LIF withdrawal. Data represent average ± SD (n = 3). (B) Erk5 −/− mESCs were transfected with empty vector or Erk5 cDNA. Fgf5 and Brachyury mRNA levels were determined by qRT-PCR following 3 days in the presence or absence of LIF. Data represent average ± SD (n = 3). (C) Erk5 −/− mESCs were transfected with empty vector or Erk5 constructs and Klf2 protein expression determined by immunoblotting and normalized. Data are presented as average ± SD (n = 3). (D) Erk5 −/− mESCs were transfected with empty vector or Erk5 constructs and stimulated with H 2 O 2 , and Erk5 kinase activity was determined. Erk5 and Erk1/2 expression levels were determined by immunoblotting. Data represent average ± SD (n = 3). Intervening lanes were removed, indicated by a dotted line. (E) Erk5 +/+ mESCs were transfected with empty vector or Mek5DD cDNA. Nanog, Oct4, Klf2, Rex1, and Esrrb mRNA levels were then determined by qRT-PCR following 3 days of LIF withdrawal. Data represent average ± SD (n = 3). (F) Erk5 +/+ mESCs were transfected with empty vector or Mek5DD, and Fgf5 and Brachyury mRNA levels were determined by qRT-PCR after 4 or 5 days in the presence or absence of LIF, respectively. Data represent average ± SD (n = 3). (G) Stat3 pTyr705, total Stat3, Erk1/2 pThr202/Tyr204, and total Erk1/2 levels in Erk5 +/+ or Erk5 −/− mESC clones were determined by immunoblotting (n = 3). (H) Erk5 +/+ and Erk5 −/− mESCs were treated with 1 μM AZD4547 or ruxolitinib. Nanog, Dnmt3b, Klf2, Klf4, and Erk1/2 levels were then determined by immunoblotting. Intervening lanes were removed, as indicated by a dotted line (n = 3). See also Figure S2 .

    Journal: Cell Reports

    Article Title: Erk5 Is a Key Regulator of Naive-Primed Transition and Embryonic Stem Cell Identity

    doi: 10.1016/j.celrep.2016.07.033

    Figure Lengend Snippet: Erk5 Signaling Stabilizes Naive Pluripotency and Suppresses Transition to Primed (A) Erk5 −/− mESCs were transfected with empty vector or Erk5 cDNA and Nanog, Oct4, Klf2, Rex1, and Esrrb mRNA levels determined by qRT-PCR following 3-day LIF withdrawal. Data represent average ± SD (n = 3). (B) Erk5 −/− mESCs were transfected with empty vector or Erk5 cDNA. Fgf5 and Brachyury mRNA levels were determined by qRT-PCR following 3 days in the presence or absence of LIF. Data represent average ± SD (n = 3). (C) Erk5 −/− mESCs were transfected with empty vector or Erk5 constructs and Klf2 protein expression determined by immunoblotting and normalized. Data are presented as average ± SD (n = 3). (D) Erk5 −/− mESCs were transfected with empty vector or Erk5 constructs and stimulated with H 2 O 2 , and Erk5 kinase activity was determined. Erk5 and Erk1/2 expression levels were determined by immunoblotting. Data represent average ± SD (n = 3). Intervening lanes were removed, indicated by a dotted line. (E) Erk5 +/+ mESCs were transfected with empty vector or Mek5DD cDNA. Nanog, Oct4, Klf2, Rex1, and Esrrb mRNA levels were then determined by qRT-PCR following 3 days of LIF withdrawal. Data represent average ± SD (n = 3). (F) Erk5 +/+ mESCs were transfected with empty vector or Mek5DD, and Fgf5 and Brachyury mRNA levels were determined by qRT-PCR after 4 or 5 days in the presence or absence of LIF, respectively. Data represent average ± SD (n = 3). (G) Stat3 pTyr705, total Stat3, Erk1/2 pThr202/Tyr204, and total Erk1/2 levels in Erk5 +/+ or Erk5 −/− mESC clones were determined by immunoblotting (n = 3). (H) Erk5 +/+ and Erk5 −/− mESCs were treated with 1 μM AZD4547 or ruxolitinib. Nanog, Dnmt3b, Klf2, Klf4, and Erk1/2 levels were then determined by immunoblotting. Intervening lanes were removed, as indicated by a dotted line (n = 3). See also Figure S2 .

    Article Snippet: Antibodies and Chemicals Antibodies used were Nanog (ReproCell Inc.), Dnmt3b (Imgenex), Klf4 (R & D Systems), Erk1/2 and Oct4 (Santa Cruz Biotechnology), phospho-p44/42 MAPK (Erk1/2 Thr202/Tyr204), Stat3α, phospho-Stat3 (Tyr705) and c-Myc (Cell Signaling Technology), Erk5 (Division of Signal Transduction Therapy, Dundee, UK), Klf2 (Millipore), CD309 (Flk1) APC, Clone Avas 12a1, and CD140a (PDGFRα) PE, Clone APA5 (eBioscience).

    Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Construct, Expressing, Activity Assay, Clone Assay

    Systematic Identification of Kinase Inhibitors that Modulate Naive-Primed Pluripotent Transition (A) mESCs cultured in LIF/FBS transitioning between naive (green) and primed (red) pluripotent states. (B) mESCs were treated with the indicated concentrations of Jak inhibitors (ruxolitinib and tofacitinib), Fgfr inhibitors (PD173074/AZD4547), or Mek1/2 inhibitors (PD0325901/PD184352). Klf4, Nanog, Dnmt3b, and Erk1/2 levels were determined by immunoblotting. (C) 228 potent and selective kinase inhibitors were screened at 1 μM for effects on pluripotency signature. Nanog and Dnmt3b expression was determined for each inhibitor and images overlaid. Selected positive control inhibitors are highlighted. (D) The Nanog:Dnmt3b ratio for each kinase inhibitor was determined and inhibitors ranked accordingly. Inhibitors found to alter Nanog:Dnmt3b beyond a 2-fold threshold were identified as drivers of naive or primed pluripotency. Selected positive control inhibitors are highlighted. See also Tables S1 and S2 .

    Journal: Cell Reports

    Article Title: Erk5 Is a Key Regulator of Naive-Primed Transition and Embryonic Stem Cell Identity

    doi: 10.1016/j.celrep.2016.07.033

    Figure Lengend Snippet: Systematic Identification of Kinase Inhibitors that Modulate Naive-Primed Pluripotent Transition (A) mESCs cultured in LIF/FBS transitioning between naive (green) and primed (red) pluripotent states. (B) mESCs were treated with the indicated concentrations of Jak inhibitors (ruxolitinib and tofacitinib), Fgfr inhibitors (PD173074/AZD4547), or Mek1/2 inhibitors (PD0325901/PD184352). Klf4, Nanog, Dnmt3b, and Erk1/2 levels were determined by immunoblotting. (C) 228 potent and selective kinase inhibitors were screened at 1 μM for effects on pluripotency signature. Nanog and Dnmt3b expression was determined for each inhibitor and images overlaid. Selected positive control inhibitors are highlighted. (D) The Nanog:Dnmt3b ratio for each kinase inhibitor was determined and inhibitors ranked accordingly. Inhibitors found to alter Nanog:Dnmt3b beyond a 2-fold threshold were identified as drivers of naive or primed pluripotency. Selected positive control inhibitors are highlighted. See also Tables S1 and S2 .

    Article Snippet: Antibodies and Chemicals Antibodies used were Nanog (ReproCell Inc.), Dnmt3b (Imgenex), Klf4 (R & D Systems), Erk1/2 and Oct4 (Santa Cruz Biotechnology), phospho-p44/42 MAPK (Erk1/2 Thr202/Tyr204), Stat3α, phospho-Stat3 (Tyr705) and c-Myc (Cell Signaling Technology), Erk5 (Division of Signal Transduction Therapy, Dundee, UK), Klf2 (Millipore), CD309 (Flk1) APC, Clone Avas 12a1, and CD140a (PDGFRα) PE, Clone APA5 (eBioscience).

    Techniques: Cell Culture, Expressing, Positive Control

    TXL increases the expression of tight junction proteins and KLF4 in hypoxic aorta. A, Endothelial cells were treated with the indicated stimuli for 24 hours. Western blot analysis of VE-cadherin, beta-catenin, occludin, claudin-1, ZO-1, and KLF4 expression is shown (left panel). Densitometric scanning (right panel). Values are the mean ± SD from 3 independent experiments. * P

    Journal: Journal of Cardiovascular Pharmacology

    Article Title: Tongxinluo (TXL), a Traditional Chinese Medicinal Compound, Improves Endothelial Function After Chronic Hypoxia Both In Vivo and In Vitro

    doi: 10.1097/FJC.0000000000000226

    Figure Lengend Snippet: TXL increases the expression of tight junction proteins and KLF4 in hypoxic aorta. A, Endothelial cells were treated with the indicated stimuli for 24 hours. Western blot analysis of VE-cadherin, beta-catenin, occludin, claudin-1, ZO-1, and KLF4 expression is shown (left panel). Densitometric scanning (right panel). Values are the mean ± SD from 3 independent experiments. * P

    Article Snippet: Membranes were blocked with 5% milk in Tris-HCl–Tween buffer solution for 2 hours at 37°C and incubated overnight at 4°C with specific antibodies [anti–HIF-1α (1:1000; Abcam), anti–VE-cadherin (1:1000; Abcam), anti–beta-catenin (1:10,000; Abgent), antioccludin (1:100,000; Epitomics), anti–claudin-1 (1:500; Novus), and anti-KLF4 (1:1000; Epitomics)].

    Techniques: Expressing, Western Blot

    KLF4 may mediate TXL-induced expression of the tight junction proteins occludin and claudin-1. A, Endothelial cells were subjected to various stimuli for 24 hours. Western blot analysis was used to detect the expression of KLF4, VE-cadherin, beta-catenin, occludin, ZO-1, and claudin-1. Densitometric scanning (right panel). Values are the mean ± SD from 3 independent experiments. * P

    Journal: Journal of Cardiovascular Pharmacology

    Article Title: Tongxinluo (TXL), a Traditional Chinese Medicinal Compound, Improves Endothelial Function After Chronic Hypoxia Both In Vivo and In Vitro

    doi: 10.1097/FJC.0000000000000226

    Figure Lengend Snippet: KLF4 may mediate TXL-induced expression of the tight junction proteins occludin and claudin-1. A, Endothelial cells were subjected to various stimuli for 24 hours. Western blot analysis was used to detect the expression of KLF4, VE-cadherin, beta-catenin, occludin, ZO-1, and claudin-1. Densitometric scanning (right panel). Values are the mean ± SD from 3 independent experiments. * P

    Article Snippet: Membranes were blocked with 5% milk in Tris-HCl–Tween buffer solution for 2 hours at 37°C and incubated overnight at 4°C with specific antibodies [anti–HIF-1α (1:1000; Abcam), anti–VE-cadherin (1:1000; Abcam), anti–beta-catenin (1:10,000; Abgent), antioccludin (1:100,000; Epitomics), anti–claudin-1 (1:500; Novus), and anti-KLF4 (1:1000; Epitomics)].

    Techniques: Expressing, Western Blot

    Expression and Localization of pluripotent associated factors in control and CDX2-KD blastocysts. ( A , A′ and B , B′ ) Immunofluorescent image of OCT4 at cell nucleus was fully positive in control and CDX2-KD blastocysts. ( C , C′ and D , D′ ) Immunofluorescent image of SOX2 at cell nucleus was positive in ICM and negative in TE in control and CDX2-KD blastocysts. ICM staining was shown in inserted picture. ( E , E′ and F , F′ ) Immunofluorescent image of KLF4 at cell nucleus was fully positive in control and CDX2-KD blastocysts. ( G , G′ and H , H′ ) Immunofluorescent image of CDX2 at cell nucleus was fully positive in control blastocyst, and was full negative in CDX2-KD blastocyst. Bar = 100 μm.

    Journal: Scientific Reports

    Article Title: Establishment of bovine embryonic stem cells after knockdown of CDX2

    doi: 10.1038/srep28343

    Figure Lengend Snippet: Expression and Localization of pluripotent associated factors in control and CDX2-KD blastocysts. ( A , A′ and B , B′ ) Immunofluorescent image of OCT4 at cell nucleus was fully positive in control and CDX2-KD blastocysts. ( C , C′ and D , D′ ) Immunofluorescent image of SOX2 at cell nucleus was positive in ICM and negative in TE in control and CDX2-KD blastocysts. ICM staining was shown in inserted picture. ( E , E′ and F , F′ ) Immunofluorescent image of KLF4 at cell nucleus was fully positive in control and CDX2-KD blastocysts. ( G , G′ and H , H′ ) Immunofluorescent image of CDX2 at cell nucleus was fully positive in control blastocyst, and was full negative in CDX2-KD blastocyst. Bar = 100 μm.

    Article Snippet: Cells were incubated in primary antibody overnight at 4 °C and secondary antibody at 37 °C for 1 h. Primary antibodies were used that were anti-OCT-3/4 (Santa Cruz), anti-NANOG (Abcam), anti-SOX2 (Cell signaling), anti-SSEA1 (Santa Cruz), anti-SSEA4 (Santa Cruz), anti-E-CADHERIN (BD Bioscience), anti-KLF4 (Stemgent), anti-CDX2 (Biogenex).

    Techniques: Expressing, Staining

    KLF4 and A2bAR are correlated significantly in human adipose tissue. Subcutaneous ( A , n = 73) and visceral ( B , n = 19) adipose tissues were obtained from obese individuals. As described under “Experimental Procedures,” mRNA was extracted

    Journal: The Journal of Biological Chemistry

    Article Title: An Adenosine Receptor-Krüppel-like Factor 4 Protein Axis Inhibits Adipogenesis *

    doi: 10.1074/jbc.M114.566406

    Figure Lengend Snippet: KLF4 and A2bAR are correlated significantly in human adipose tissue. Subcutaneous ( A , n = 73) and visceral ( B , n = 19) adipose tissues were obtained from obese individuals. As described under “Experimental Procedures,” mRNA was extracted

    Article Snippet: The following antibodies were used at the following dilutions: PPARγ (Cell Signaling Technology, catalog no. 81B8, 1:1000); aP2/FABP4 (Cell Signaling Technology, catalog no. D25B3, 1:5000); C/EBP-α (Cell Signaling Technology, catalog no. 2295, 1:1000); KLF4 (MBL International, catalog no. PM057, 1:5000); α-tubulin (Sigma, catalog no. T6199, 1:10,000); β-actin (Sigma, catalog no. A5441, 1:10,000); anti-rabbit IgG, HRP-linked (Cell Signaling Technology, catalog no. 7074, 1:5000); and anti-mouse IgG, HRP-linked (Cell Signaling Technology, catalog no. 7076; 1:5000).

    Techniques:

    KLF4 is required for the action of BAY 60-6583. SVCs were isolated from the subcutaneous adipose tissue of WT mice. Cells received scrambled siRNA ( Scrambled ) or siRNA targeting KLF4 ( KLF4 siRNA ) and were treated with DMSO ( Vehicle ) or 1 μ m BAY

    Journal: The Journal of Biological Chemistry

    Article Title: An Adenosine Receptor-Krüppel-like Factor 4 Protein Axis Inhibits Adipogenesis *

    doi: 10.1074/jbc.M114.566406

    Figure Lengend Snippet: KLF4 is required for the action of BAY 60-6583. SVCs were isolated from the subcutaneous adipose tissue of WT mice. Cells received scrambled siRNA ( Scrambled ) or siRNA targeting KLF4 ( KLF4 siRNA ) and were treated with DMSO ( Vehicle ) or 1 μ m BAY

    Article Snippet: The following antibodies were used at the following dilutions: PPARγ (Cell Signaling Technology, catalog no. 81B8, 1:1000); aP2/FABP4 (Cell Signaling Technology, catalog no. D25B3, 1:5000); C/EBP-α (Cell Signaling Technology, catalog no. 2295, 1:1000); KLF4 (MBL International, catalog no. PM057, 1:5000); α-tubulin (Sigma, catalog no. T6199, 1:10,000); β-actin (Sigma, catalog no. A5441, 1:10,000); anti-rabbit IgG, HRP-linked (Cell Signaling Technology, catalog no. 7074, 1:5000); and anti-mouse IgG, HRP-linked (Cell Signaling Technology, catalog no. 7076; 1:5000).

    Techniques: Isolation, Mouse Assay

    Klf4 directly regulates the expression of crucial EMT genes. ( A ) Venn-diagrams depicting the commonly regulated genes between the data obtained by Klf4 chromatin immunoprecipitation-deep sequencing (ChIP-Seq), gene expression profiling of TGFβ-induced EMT (EMT Up and Dn, respectively), and gene expression profiling of siRNA-mediated Klf4 depletion in the presence of TGFβ for 2 days (Klf4 Up and Dn, respectively), all performed in NMuMG cells. The numbers of genes directly up-regulated (left diagram; 30 genes) or down-regulated (right diagram; 56) during EMT are shown (see Table S1 for the gene names). ( B ) Chromatin immunoprecipitation with antibody against Klf4 followed by quantitative PCR (ChIP-qPCR) to demonstrate the occupancy of Klf4 at the promoters of the N-cadherin ( Cdh2 ), vimentin ( Vim ) and β-catenin ( Ctnnb1 ) genes. The qPCR data were normalized to ChIP-qPCR of an intergenic region. ( C ) Wiggle-tracks to show the binding of Klf4 at the promoters of the N-cadherin ( Cdh2 ), vimentin ( Vim ), β-catenin ( Ctnnb1 ), Jnk1 ( Mapk8 ), vascular endothelial growth factor A ( Vegfa ) and endothelin1 ( Edn1 ) genes by using UCSC genome browser. These files were generated from Klf4 ChIP-Seq data of NMuMG cells. Statistical values were calculated using an unpaired/paired, two-tailed t-test and experiments were performed at least three times. * = p≤0.05; ** = p≤0.01.

    Journal: PLoS ONE

    Article Title: Klf4 Is a Transcriptional Regulator of Genes Critical for EMT, Including Jnk1 (Mapk8)

    doi: 10.1371/journal.pone.0057329

    Figure Lengend Snippet: Klf4 directly regulates the expression of crucial EMT genes. ( A ) Venn-diagrams depicting the commonly regulated genes between the data obtained by Klf4 chromatin immunoprecipitation-deep sequencing (ChIP-Seq), gene expression profiling of TGFβ-induced EMT (EMT Up and Dn, respectively), and gene expression profiling of siRNA-mediated Klf4 depletion in the presence of TGFβ for 2 days (Klf4 Up and Dn, respectively), all performed in NMuMG cells. The numbers of genes directly up-regulated (left diagram; 30 genes) or down-regulated (right diagram; 56) during EMT are shown (see Table S1 for the gene names). ( B ) Chromatin immunoprecipitation with antibody against Klf4 followed by quantitative PCR (ChIP-qPCR) to demonstrate the occupancy of Klf4 at the promoters of the N-cadherin ( Cdh2 ), vimentin ( Vim ) and β-catenin ( Ctnnb1 ) genes. The qPCR data were normalized to ChIP-qPCR of an intergenic region. ( C ) Wiggle-tracks to show the binding of Klf4 at the promoters of the N-cadherin ( Cdh2 ), vimentin ( Vim ), β-catenin ( Ctnnb1 ), Jnk1 ( Mapk8 ), vascular endothelial growth factor A ( Vegfa ) and endothelin1 ( Edn1 ) genes by using UCSC genome browser. These files were generated from Klf4 ChIP-Seq data of NMuMG cells. Statistical values were calculated using an unpaired/paired, two-tailed t-test and experiments were performed at least three times. * = p≤0.05; ** = p≤0.01.

    Article Snippet: Immunoblotting Immun oblotting was performed as described previously using following antibodies: Klf4 (09–821, Millipore), E-Cadherin (610182, Transduction Laboratories), N-Cadherin (M142, Takara), ZO-1 (617300, Zymed), Fibronectin (F-3648, Sigma), ZO-1 (617300, Zymed), Vimentin (V2258, Sigma), Vinculin (SC-7649, Santa Cruz Biotechnology), Jnk1/3 (SC-474, Santa Cruz), GAPDH (G8795, Sigma or ab9485, Abcam for E9 and Py2T lysates, respectively) and Actin (SC-1616, Santa Cruz).

    Techniques: Expressing, Chromatin Immunoprecipitation, Sequencing, Real-time Polymerase Chain Reaction, Binding Assay, Generated, Two Tailed Test

    Klf4 directly represses Jnk1 ( Mapk8 ) gene expression, and Jnk1 is required for TGFβ-induced EMT and loss of Klf4-induced cell migration and apoptosis in NMuMG cells. ( A ) siRNA-mediated depletion of Klf4 expression increases Jnk1 expression as determined by quantitative RT-PCR. NMuMG cells were treated or not with TGFβ for 2 days. ( B ) Ablation of Klf4 function induces Jnk1 expression. Immunoblotting analysis of the expression of Jnk1 during TGFβ-induced EMT in NMuMG cells transfected with control siRNA (siControl) or with siRNA against Klf4 (siKlf4). Immunoblotting for actin was used as loading control. ( C ) Klf4 directly binds the Jnk1 ( Mapk8 ) gene promoter in NMuMG cells as determined by ChIP using an antibody against Klf4 followed by qPCR using primers specific for the promoter region of the Jnk1 ( Mapk8 ) gene. The qPCR data were normalized to ChIP-qPCR of an intergenic region. ( D ) An AP1 activity reporter assay was performed to assess Jnk1/AP-1-mediated transcriptional activity after Klf4 depletion (siKlf4) in NMuMG cells with or without TGFβ for 2 days. Firefly luciferase activity was normalized to co-transfected Renilla luciferase activity (relative luminescence). ( E ) Ablation of Jnk1 expression prevents EMT. Phase contrast microscopy of NMuMG cells transfected with control siRNA (siControl) and siRNA against Jnk1 (siJnk1) before and after TGFβ treatment for 2 days. Size bar, 100 µm. ( F ) Jnk1 depletion represses EMT. Immunoblotting analysis of the expression levels of Jnk1, the epithelial markers E-cadherin (the correct band is marked with an asterisk) and ZO-1 and the mesenchymal marker N-cadherin in NMuMG cells treated with either control (siControl) or Jnk1-specific siRNA (siJnk1) during TGFβ-induced EMT. Immunoblotting for Jnk1 was used to assess the siRNA-mediated knock-down efficiency, and immunblotting for actin was used as a loading control. ( G ) Down-regulation of Jnk1 expression prevents EMT. The localization and expression levels of EMT markers as indicated was assessed by immunofluorescence microscopy after 7 days of TGFβ treatment in siControl and siJnk1-transfected NMuMG cells. Size bar, 50 µm. ( H ) Jnk1 is required for cell migration of NMuMG cells induced by the loss of Klf4 expression, as determined by scratch wound closure assays of control (siControl), Klf4-depleted (siKlf4), Jnk1-depleted (siJnk1), or siKlf4 and siJnk1 double-depleted (siKlf4+siJnk1) NMuMG cells. ( I ) Jnk1 is required for apoptosis of NMuMG cells induced by the loss of Klf4 expression and TGFβ treatment for 2 days, as determined by Annexin V staining and flow cytometry of control (siControl), Klf4-depleted (siKlf4), Jnk1-depleted (siJnk1), or siKlf4 and siJnk1 double-depleted (siKlf4+siJnk1) NMuMG cells. Statistical values were calculated using an unpaired/paired, two-tailed t-test and experiments were performed at least three times. * = p≤0.05; ** = p≤0.01; *** = p≤0.001.

    Journal: PLoS ONE

    Article Title: Klf4 Is a Transcriptional Regulator of Genes Critical for EMT, Including Jnk1 (Mapk8)

    doi: 10.1371/journal.pone.0057329

    Figure Lengend Snippet: Klf4 directly represses Jnk1 ( Mapk8 ) gene expression, and Jnk1 is required for TGFβ-induced EMT and loss of Klf4-induced cell migration and apoptosis in NMuMG cells. ( A ) siRNA-mediated depletion of Klf4 expression increases Jnk1 expression as determined by quantitative RT-PCR. NMuMG cells were treated or not with TGFβ for 2 days. ( B ) Ablation of Klf4 function induces Jnk1 expression. Immunoblotting analysis of the expression of Jnk1 during TGFβ-induced EMT in NMuMG cells transfected with control siRNA (siControl) or with siRNA against Klf4 (siKlf4). Immunoblotting for actin was used as loading control. ( C ) Klf4 directly binds the Jnk1 ( Mapk8 ) gene promoter in NMuMG cells as determined by ChIP using an antibody against Klf4 followed by qPCR using primers specific for the promoter region of the Jnk1 ( Mapk8 ) gene. The qPCR data were normalized to ChIP-qPCR of an intergenic region. ( D ) An AP1 activity reporter assay was performed to assess Jnk1/AP-1-mediated transcriptional activity after Klf4 depletion (siKlf4) in NMuMG cells with or without TGFβ for 2 days. Firefly luciferase activity was normalized to co-transfected Renilla luciferase activity (relative luminescence). ( E ) Ablation of Jnk1 expression prevents EMT. Phase contrast microscopy of NMuMG cells transfected with control siRNA (siControl) and siRNA against Jnk1 (siJnk1) before and after TGFβ treatment for 2 days. Size bar, 100 µm. ( F ) Jnk1 depletion represses EMT. Immunoblotting analysis of the expression levels of Jnk1, the epithelial markers E-cadherin (the correct band is marked with an asterisk) and ZO-1 and the mesenchymal marker N-cadherin in NMuMG cells treated with either control (siControl) or Jnk1-specific siRNA (siJnk1) during TGFβ-induced EMT. Immunoblotting for Jnk1 was used to assess the siRNA-mediated knock-down efficiency, and immunblotting for actin was used as a loading control. ( G ) Down-regulation of Jnk1 expression prevents EMT. The localization and expression levels of EMT markers as indicated was assessed by immunofluorescence microscopy after 7 days of TGFβ treatment in siControl and siJnk1-transfected NMuMG cells. Size bar, 50 µm. ( H ) Jnk1 is required for cell migration of NMuMG cells induced by the loss of Klf4 expression, as determined by scratch wound closure assays of control (siControl), Klf4-depleted (siKlf4), Jnk1-depleted (siJnk1), or siKlf4 and siJnk1 double-depleted (siKlf4+siJnk1) NMuMG cells. ( I ) Jnk1 is required for apoptosis of NMuMG cells induced by the loss of Klf4 expression and TGFβ treatment for 2 days, as determined by Annexin V staining and flow cytometry of control (siControl), Klf4-depleted (siKlf4), Jnk1-depleted (siJnk1), or siKlf4 and siJnk1 double-depleted (siKlf4+siJnk1) NMuMG cells. Statistical values were calculated using an unpaired/paired, two-tailed t-test and experiments were performed at least three times. * = p≤0.05; ** = p≤0.01; *** = p≤0.001.

    Article Snippet: Immunoblotting Immun oblotting was performed as described previously using following antibodies: Klf4 (09–821, Millipore), E-Cadherin (610182, Transduction Laboratories), N-Cadherin (M142, Takara), ZO-1 (617300, Zymed), Fibronectin (F-3648, Sigma), ZO-1 (617300, Zymed), Vimentin (V2258, Sigma), Vinculin (SC-7649, Santa Cruz Biotechnology), Jnk1/3 (SC-474, Santa Cruz), GAPDH (G8795, Sigma or ab9485, Abcam for E9 and Py2T lysates, respectively) and Actin (SC-1616, Santa Cruz).

    Techniques: Expressing, Migration, Quantitative RT-PCR, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Activity Assay, Reporter Assay, Luciferase, Microscopy, Marker, Immunofluorescence, Staining, Flow Cytometry, Cytometry, Two Tailed Test

    Klf4 expression is down-regulated during EMT. ( A–C ) Klf4 mRNA levels were quantified by quantitative RT-PCR during TGFβ-induced EMT in NMuMG cells (A), Py2T cells (B) and EpRas cells (C). ( D ) Klf4 levels were quantified by quantitative RT-PCR in SKBR3 cells after EGF-induced EMT. SKBR3 cells were treated with 25 ng/ml EGF for 4 days. Shown are the fold-changes as compared to day 0 (no EGF). ( E ) Quantitative RT-PCR analysis of Klf4 mRNA levels in MCF7 cells either stably expressing a control shRNA (shControl) or an shRNA against E-cadherin (shEcad). Statistical values were calculated using a paired, two-tailed t-test and experiments were performed at least three times. * = p≤0.05; ** = p≤0.01; *** = p≤0.001.

    Journal: PLoS ONE

    Article Title: Klf4 Is a Transcriptional Regulator of Genes Critical for EMT, Including Jnk1 (Mapk8)

    doi: 10.1371/journal.pone.0057329

    Figure Lengend Snippet: Klf4 expression is down-regulated during EMT. ( A–C ) Klf4 mRNA levels were quantified by quantitative RT-PCR during TGFβ-induced EMT in NMuMG cells (A), Py2T cells (B) and EpRas cells (C). ( D ) Klf4 levels were quantified by quantitative RT-PCR in SKBR3 cells after EGF-induced EMT. SKBR3 cells were treated with 25 ng/ml EGF for 4 days. Shown are the fold-changes as compared to day 0 (no EGF). ( E ) Quantitative RT-PCR analysis of Klf4 mRNA levels in MCF7 cells either stably expressing a control shRNA (shControl) or an shRNA against E-cadherin (shEcad). Statistical values were calculated using a paired, two-tailed t-test and experiments were performed at least three times. * = p≤0.05; ** = p≤0.01; *** = p≤0.001.

    Article Snippet: Immunoblotting Immun oblotting was performed as described previously using following antibodies: Klf4 (09–821, Millipore), E-Cadherin (610182, Transduction Laboratories), N-Cadherin (M142, Takara), ZO-1 (617300, Zymed), Fibronectin (F-3648, Sigma), ZO-1 (617300, Zymed), Vimentin (V2258, Sigma), Vinculin (SC-7649, Santa Cruz Biotechnology), Jnk1/3 (SC-474, Santa Cruz), GAPDH (G8795, Sigma or ab9485, Abcam for E9 and Py2T lysates, respectively) and Actin (SC-1616, Santa Cruz).

    Techniques: Expressing, Quantitative RT-PCR, Stable Transfection, shRNA, Two Tailed Test

    Klf4 inhibits cell migration and promotes cell survival and proliferation during TGFβ-induced EMT. ( A ) siRNA-mediated ablation of Klf4 results in an increase in NMuMG cell migration even in the absence of TGFβ as determined by scratch wound closure assays (see pictures in Figure S3A ). ( B ) Stable shRNA-mediated depletion in NMuMG cells using three different shRNA sequences specific for Klf4 results in moderately increased cell migration as determined by scratch wound closure assays (see pictures in Figure S3B ). ( C ) Stable shRNA-mediated depletion of Klf4 expression in NMuMG cells results in increased cell migration as determined by trans-well migration assays using 20% FBS as a chemo-attractant in the absence of TGFβ. ( D ) Stable shRNA-mediated depletion of Klf4 expression in NMuMG cells previously treated for 20 days with TGFβ results in increased cell migration as determined by trans-well migration assays using 20% FBS as a chemo-attractant. ( E ) siRNA-mediated ablation of Klf4 expression during TGFβ treatment of NMuMG cells results in a significant decrease in cell numbers as compared to cells transfected with control siRNA, while the depletion of Klf4 has no effect on cell number in the absence of TGFβ. ( F ) NMuMG cells transfected with either control siRNA (siControl) or with siRNA against Klf4 (siKlf4) were treated with TGFβ for the days indicated. Cells were stained with propidium iodide (PI), and the percentages of cells in G0/G1 and S-G2/M phases of the cell cycle were determined by flow cytometry analysis. ( G ) NMuMG cells transfected with either control siRNA (siControl) or with siRNA against Klf4 (siKlf4) were treated with TGFβ for the days indicated, and the rates of apoptosis were determined by Annexin-V staining and flow cytometry. Note that in the 15 days experiment, cells were first treated with TGFβ for 13 days and then transfected with siRNA constructs. Statistical values were calculated using an unpaired/paired, two-tailed t-test and experiments were performed at least three times. * = p≤0.05; ** = p≤0.01; *** = p≤0.001.

    Journal: PLoS ONE

    Article Title: Klf4 Is a Transcriptional Regulator of Genes Critical for EMT, Including Jnk1 (Mapk8)

    doi: 10.1371/journal.pone.0057329

    Figure Lengend Snippet: Klf4 inhibits cell migration and promotes cell survival and proliferation during TGFβ-induced EMT. ( A ) siRNA-mediated ablation of Klf4 results in an increase in NMuMG cell migration even in the absence of TGFβ as determined by scratch wound closure assays (see pictures in Figure S3A ). ( B ) Stable shRNA-mediated depletion in NMuMG cells using three different shRNA sequences specific for Klf4 results in moderately increased cell migration as determined by scratch wound closure assays (see pictures in Figure S3B ). ( C ) Stable shRNA-mediated depletion of Klf4 expression in NMuMG cells results in increased cell migration as determined by trans-well migration assays using 20% FBS as a chemo-attractant in the absence of TGFβ. ( D ) Stable shRNA-mediated depletion of Klf4 expression in NMuMG cells previously treated for 20 days with TGFβ results in increased cell migration as determined by trans-well migration assays using 20% FBS as a chemo-attractant. ( E ) siRNA-mediated ablation of Klf4 expression during TGFβ treatment of NMuMG cells results in a significant decrease in cell numbers as compared to cells transfected with control siRNA, while the depletion of Klf4 has no effect on cell number in the absence of TGFβ. ( F ) NMuMG cells transfected with either control siRNA (siControl) or with siRNA against Klf4 (siKlf4) were treated with TGFβ for the days indicated. Cells were stained with propidium iodide (PI), and the percentages of cells in G0/G1 and S-G2/M phases of the cell cycle were determined by flow cytometry analysis. ( G ) NMuMG cells transfected with either control siRNA (siControl) or with siRNA against Klf4 (siKlf4) were treated with TGFβ for the days indicated, and the rates of apoptosis were determined by Annexin-V staining and flow cytometry. Note that in the 15 days experiment, cells were first treated with TGFβ for 13 days and then transfected with siRNA constructs. Statistical values were calculated using an unpaired/paired, two-tailed t-test and experiments were performed at least three times. * = p≤0.05; ** = p≤0.01; *** = p≤0.001.

    Article Snippet: Immunoblotting Immun oblotting was performed as described previously using following antibodies: Klf4 (09–821, Millipore), E-Cadherin (610182, Transduction Laboratories), N-Cadherin (M142, Takara), ZO-1 (617300, Zymed), Fibronectin (F-3648, Sigma), ZO-1 (617300, Zymed), Vimentin (V2258, Sigma), Vinculin (SC-7649, Santa Cruz Biotechnology), Jnk1/3 (SC-474, Santa Cruz), GAPDH (G8795, Sigma or ab9485, Abcam for E9 and Py2T lysates, respectively) and Actin (SC-1616, Santa Cruz).

    Techniques: Migration, shRNA, Expressing, Transfection, Staining, Flow Cytometry, Cytometry, Construct, Two Tailed Test

    Klf4 maintains epithelial morphology and prevents EMT. ( A ) NMuMG cells were transfected with either control siRNA (siControl) or Klf4-specific siRNA (siKlf4) in the absence as well as in the presence of TGFβ for 2 or 15 days, as indicated. The levels of Klf4 mRNA were determined by quantitative RT-PCR and the morphology of the cells evaluated by phase contrast microscopy. Filopodia and membrane protrusions are marked with closed arrowheads. Size bar, 100 µm. ( B ) Immunoblotting analysis of the expression of the epithelial marker ZO-1 and the mesenchymal markers N-cadherin and Vimentin during TGFβ-induced EMT in control NMuMG cells (siControl) and Klf4 depleted cells (siKlf4). ( C ) Immunofluorescence microscopy analysis of changes in the localization and expression levels of EMT markers during EMT. NMuMG cells transfected with either control siRNA (siControl) or with siRNA against Klf4 (siKlf4) were left untreated or treated with TGFβ for 2 days and stained with antibodies against the epithelial markers E-cadherin and ZO-1, against the mesenchymal markers N-cadherin and vimentin, against paxillin to detect focal adhesion plaques, and with phalloidin to visualize the actin cytoskeleton. Size bar, 50 µm. Statistical values were calculated by using a paired, two-tailed t-test and experiments were performed at least three times. *** = p≤0.001.

    Journal: PLoS ONE

    Article Title: Klf4 Is a Transcriptional Regulator of Genes Critical for EMT, Including Jnk1 (Mapk8)

    doi: 10.1371/journal.pone.0057329

    Figure Lengend Snippet: Klf4 maintains epithelial morphology and prevents EMT. ( A ) NMuMG cells were transfected with either control siRNA (siControl) or Klf4-specific siRNA (siKlf4) in the absence as well as in the presence of TGFβ for 2 or 15 days, as indicated. The levels of Klf4 mRNA were determined by quantitative RT-PCR and the morphology of the cells evaluated by phase contrast microscopy. Filopodia and membrane protrusions are marked with closed arrowheads. Size bar, 100 µm. ( B ) Immunoblotting analysis of the expression of the epithelial marker ZO-1 and the mesenchymal markers N-cadherin and Vimentin during TGFβ-induced EMT in control NMuMG cells (siControl) and Klf4 depleted cells (siKlf4). ( C ) Immunofluorescence microscopy analysis of changes in the localization and expression levels of EMT markers during EMT. NMuMG cells transfected with either control siRNA (siControl) or with siRNA against Klf4 (siKlf4) were left untreated or treated with TGFβ for 2 days and stained with antibodies against the epithelial markers E-cadherin and ZO-1, against the mesenchymal markers N-cadherin and vimentin, against paxillin to detect focal adhesion plaques, and with phalloidin to visualize the actin cytoskeleton. Size bar, 50 µm. Statistical values were calculated by using a paired, two-tailed t-test and experiments were performed at least three times. *** = p≤0.001.

    Article Snippet: Immunoblotting Immun oblotting was performed as described previously using following antibodies: Klf4 (09–821, Millipore), E-Cadherin (610182, Transduction Laboratories), N-Cadherin (M142, Takara), ZO-1 (617300, Zymed), Fibronectin (F-3648, Sigma), ZO-1 (617300, Zymed), Vimentin (V2258, Sigma), Vinculin (SC-7649, Santa Cruz Biotechnology), Jnk1/3 (SC-474, Santa Cruz), GAPDH (G8795, Sigma or ab9485, Abcam for E9 and Py2T lysates, respectively) and Actin (SC-1616, Santa Cruz).

    Techniques: Transfection, Quantitative RT-PCR, Microscopy, Expressing, Marker, Immunofluorescence, Staining, Two Tailed Test

    High Klf4 expression correlates with good survival prognosis in patients and prevents metastasis in experimental models. ( A–C ) Kaplan-Meier survival analysis reveals a significant correlation between low Klf4 expression levels and poor overall disease-free survival (A) by correlating high or low Klf4 expression levels with all patient samples (Uppsala database). Correlation of Klf4 expression levels with disease-free survival was further determined after stratifying the patient samples into lymph node-positive (LN+; B) and estrogen receptor-positive (ER+; C) tumor groups. ( D ) Kaplan-Meier survival analysis reveals a significant correlation between poor relapse-free survival in ER+ breast cancer patients (TRANSBIG database) and low Klf4 expression. ( E ) Myc-Klf4-ER™ and firefly luciferase-expressing Py2T cells were orthotopically transplanted into the mammary fat pad of immune-compromised Balb/c nude mice. Mice were treated with normal food or with food containing Tamoxifen to induce Klf4 transcriptional activity. Shown are images of histological sections of primary tumors stained with hematoxylin and eosin. Note that Klf4 appears to repress the invasive morphology of the tumors. Size bar, 100 µm. ( F ) The percentages of apoptotic cells were quantified by cleaved caspase 3 staining of histological tumor sections described in (E). Activation of Klf4 by Tamoxifen repressed apoptosis in the primary tumors. ( G ) Primary tumor volumes were calculated after transplantation of Myc-Klf4-ER™ cells as described in (E) while tumor weights were calculated after sacrificing the tumor-bearing mice 27 days post-injection. Activation of Klf4 by Tamoxifen promoted tumor growth. ( H ) Metastatic spread was determined in the Myc-Klf4-ER™ cells -transplanted mice described in (E) by measuring luciferase activity in extracts of lungs and livers of the transplanted mice. Luciferase activity levels in the various organs were divided by the primary tumor weights within the same mice to establish the metastatic index, as shown. Activation of Klf4 by Tamoxifen represses metastatic spread of the transplanted tumor cells. Statistical values for panels F – H were calculated using an unpaired/paired, two-tailed t-test. * = p≤0.05; ** = p≤0.01; *** = p≤0.001.

    Journal: PLoS ONE

    Article Title: Klf4 Is a Transcriptional Regulator of Genes Critical for EMT, Including Jnk1 (Mapk8)

    doi: 10.1371/journal.pone.0057329

    Figure Lengend Snippet: High Klf4 expression correlates with good survival prognosis in patients and prevents metastasis in experimental models. ( A–C ) Kaplan-Meier survival analysis reveals a significant correlation between low Klf4 expression levels and poor overall disease-free survival (A) by correlating high or low Klf4 expression levels with all patient samples (Uppsala database). Correlation of Klf4 expression levels with disease-free survival was further determined after stratifying the patient samples into lymph node-positive (LN+; B) and estrogen receptor-positive (ER+; C) tumor groups. ( D ) Kaplan-Meier survival analysis reveals a significant correlation between poor relapse-free survival in ER+ breast cancer patients (TRANSBIG database) and low Klf4 expression. ( E ) Myc-Klf4-ER™ and firefly luciferase-expressing Py2T cells were orthotopically transplanted into the mammary fat pad of immune-compromised Balb/c nude mice. Mice were treated with normal food or with food containing Tamoxifen to induce Klf4 transcriptional activity. Shown are images of histological sections of primary tumors stained with hematoxylin and eosin. Note that Klf4 appears to repress the invasive morphology of the tumors. Size bar, 100 µm. ( F ) The percentages of apoptotic cells were quantified by cleaved caspase 3 staining of histological tumor sections described in (E). Activation of Klf4 by Tamoxifen repressed apoptosis in the primary tumors. ( G ) Primary tumor volumes were calculated after transplantation of Myc-Klf4-ER™ cells as described in (E) while tumor weights were calculated after sacrificing the tumor-bearing mice 27 days post-injection. Activation of Klf4 by Tamoxifen promoted tumor growth. ( H ) Metastatic spread was determined in the Myc-Klf4-ER™ cells -transplanted mice described in (E) by measuring luciferase activity in extracts of lungs and livers of the transplanted mice. Luciferase activity levels in the various organs were divided by the primary tumor weights within the same mice to establish the metastatic index, as shown. Activation of Klf4 by Tamoxifen represses metastatic spread of the transplanted tumor cells. Statistical values for panels F – H were calculated using an unpaired/paired, two-tailed t-test. * = p≤0.05; ** = p≤0.01; *** = p≤0.001.

    Article Snippet: Immunoblotting Immun oblotting was performed as described previously using following antibodies: Klf4 (09–821, Millipore), E-Cadherin (610182, Transduction Laboratories), N-Cadherin (M142, Takara), ZO-1 (617300, Zymed), Fibronectin (F-3648, Sigma), ZO-1 (617300, Zymed), Vimentin (V2258, Sigma), Vinculin (SC-7649, Santa Cruz Biotechnology), Jnk1/3 (SC-474, Santa Cruz), GAPDH (G8795, Sigma or ab9485, Abcam for E9 and Py2T lysates, respectively) and Actin (SC-1616, Santa Cruz).

    Techniques: Expressing, Luciferase, Mouse Assay, Activity Assay, Staining, Activation Assay, Transplantation Assay, Injection, Two Tailed Test

    Klf4 prevents EMT and cell migration and supports cell survival during EMT. ( A ) Immunofluorescence staining of NMuMG cells stably expressing a Myc-Klf4-ER™ fusion protein using an anti-Myc-tag antibody to visualize the nuclear localization of Klf4 with and without 4-hydroxy-Tamoxifen (± OHT) treatment in the absence as well as in the presence of TGFβ for 2 days. Size bar, 50 µm. ( B ) Induction of Klf4 transcriptional activity by treatment of Myc-Klf4-ER™-expressing NMuMG cells treated with 4-OHT (+OHT) results in a repression of the morphological changes occurring during TGFβ-induced EMT in control-treated cells (−OHT). Shown are phase contrast images of Myc-Klf4-ER™ expressing NMuMG cells treated with TGFβ for 0, 1, 4 and 7 days in the absence or presence of 4-OHT. Size bar, 100 µm. ( C ) Immunoblotting analysis of the expression of the epithelial markers E-cadherin and ZO-1 and the mesenchymal marker N-cadherin during TGFβ-induced EMT in control NMuMG cells and in Myc-Klf4-ER™-expressing NMuMG cells in which Klf4 transcriptional activity has been induced (+OHT) or not (−OHT). Activation of Klf4 in Klf4-ER (+OHT) cells results in the maintenance of the expression of epithelial markers and the failure to express mesenchymal markers. Immunoblotting for actin was used as loading control. ( D ) Activation of Klf4 by treatment of Myc-Klf4-ER™-expressing NMuMG cells with 4-OHT (+OHT) results in reduced cell migration in scratch wound closure assays as compared to control-treated Myc-Klf4-ER™-expressing NMuMG cells (−OHT). Cells have been treated with TGFβ for 15 days prior to the migration assays. ( E ) Activation of Klf4 (+OHT) represses cell migration in trans-well migration assays by using 20% FBS as a chemoattractant for 20 hours in 15 days TGFβ-treated Myc-Klf4-ER™-expressing NMuMG cells. ( F ) Induction of Klf4 transcriptional activity by treatment of Myc-Klf4-ER™-expressing NMuMG cells with 4-OHT (+OHT) results in reduced apoptosis as compared to Myc-Klf4-ER™-expressing NMuMG cells in the absence of 4-OHT (−OHT). Apoptosis was quantified by Annexin-V staining and flow cytometry. ( G ) Activation of Klf4 by treatment of Myc-Klf4-ER™-expressing NMuMG cells with 4-OHT (+OHT) results in a significant increase in cell numbers as compared to the same cells cultured in the absence of 4-OHT (−OHT). Statistical values were calculated using an unpaired/paired, two-tailed t-test and experiments were performed at least three times. ** = p≤0.01; *** = p≤0.001.

    Journal: PLoS ONE

    Article Title: Klf4 Is a Transcriptional Regulator of Genes Critical for EMT, Including Jnk1 (Mapk8)

    doi: 10.1371/journal.pone.0057329

    Figure Lengend Snippet: Klf4 prevents EMT and cell migration and supports cell survival during EMT. ( A ) Immunofluorescence staining of NMuMG cells stably expressing a Myc-Klf4-ER™ fusion protein using an anti-Myc-tag antibody to visualize the nuclear localization of Klf4 with and without 4-hydroxy-Tamoxifen (± OHT) treatment in the absence as well as in the presence of TGFβ for 2 days. Size bar, 50 µm. ( B ) Induction of Klf4 transcriptional activity by treatment of Myc-Klf4-ER™-expressing NMuMG cells treated with 4-OHT (+OHT) results in a repression of the morphological changes occurring during TGFβ-induced EMT in control-treated cells (−OHT). Shown are phase contrast images of Myc-Klf4-ER™ expressing NMuMG cells treated with TGFβ for 0, 1, 4 and 7 days in the absence or presence of 4-OHT. Size bar, 100 µm. ( C ) Immunoblotting analysis of the expression of the epithelial markers E-cadherin and ZO-1 and the mesenchymal marker N-cadherin during TGFβ-induced EMT in control NMuMG cells and in Myc-Klf4-ER™-expressing NMuMG cells in which Klf4 transcriptional activity has been induced (+OHT) or not (−OHT). Activation of Klf4 in Klf4-ER (+OHT) cells results in the maintenance of the expression of epithelial markers and the failure to express mesenchymal markers. Immunoblotting for actin was used as loading control. ( D ) Activation of Klf4 by treatment of Myc-Klf4-ER™-expressing NMuMG cells with 4-OHT (+OHT) results in reduced cell migration in scratch wound closure assays as compared to control-treated Myc-Klf4-ER™-expressing NMuMG cells (−OHT). Cells have been treated with TGFβ for 15 days prior to the migration assays. ( E ) Activation of Klf4 (+OHT) represses cell migration in trans-well migration assays by using 20% FBS as a chemoattractant for 20 hours in 15 days TGFβ-treated Myc-Klf4-ER™-expressing NMuMG cells. ( F ) Induction of Klf4 transcriptional activity by treatment of Myc-Klf4-ER™-expressing NMuMG cells with 4-OHT (+OHT) results in reduced apoptosis as compared to Myc-Klf4-ER™-expressing NMuMG cells in the absence of 4-OHT (−OHT). Apoptosis was quantified by Annexin-V staining and flow cytometry. ( G ) Activation of Klf4 by treatment of Myc-Klf4-ER™-expressing NMuMG cells with 4-OHT (+OHT) results in a significant increase in cell numbers as compared to the same cells cultured in the absence of 4-OHT (−OHT). Statistical values were calculated using an unpaired/paired, two-tailed t-test and experiments were performed at least three times. ** = p≤0.01; *** = p≤0.001.

    Article Snippet: Immunoblotting Immun oblotting was performed as described previously using following antibodies: Klf4 (09–821, Millipore), E-Cadherin (610182, Transduction Laboratories), N-Cadherin (M142, Takara), ZO-1 (617300, Zymed), Fibronectin (F-3648, Sigma), ZO-1 (617300, Zymed), Vimentin (V2258, Sigma), Vinculin (SC-7649, Santa Cruz Biotechnology), Jnk1/3 (SC-474, Santa Cruz), GAPDH (G8795, Sigma or ab9485, Abcam for E9 and Py2T lysates, respectively) and Actin (SC-1616, Santa Cruz).

    Techniques: Migration, Immunofluorescence, Staining, Stable Transfection, Expressing, Activity Assay, Marker, Activation Assay, Flow Cytometry, Cytometry, Cell Culture, Two Tailed Test