Journal: PLoS ONE

Article Title: Nuclear corepressor SMRT is a strong regulator of body weight independently of its ability to regulate thyroid hormone action

doi: 10.1371/journal.pone.0220717

Figure Lengend Snippet: (A) Hepatic triglyceride and cholesterol levels were measured in the 1 st cohort mice 12th weeks after tamoxifen treatment. Images of control and UBC-SKO mice the livers are shown. (B) The mRNA expression of lipogenic and gluconeogenic-related genes in the liver, PGWAT, and muscle (1 st cohort of mice) (C) mRNA expression of NCoR1 in the liver, skeletal muscle, and PGWAT were quantified by qPCR. Protein expression of NCoR1 in the liver and heart were assessed by Western blot, using a NCoR1 specific antibody (n = 3–4 animals per group). Control samples are indicated by gray lanes and UBC-SKO mice are indicated by black lanes. For panel A and all qPCR analyses in panels B, and C, the data were analyzed using an unpaired t-test. These results are shown as the mean±SEM, and the p-values as; ****, p< 0.001; ***, p< 0.001; **, p< 0.01; *, p< 0.05 (1 st cohort of mice, n = 6–7 mice/group).

Article Snippet: Briefly, blots of 50 μg protein lysate were probed for SMRT-specific antibody (rabbit polyclonal anti-SMRTe antibody, 06–891; Millipore) or NCoR1-specific (rabbit polyclonal anti-NCoR antibody, A301145A; Bethyl), and then appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized using ECL prime (GE Healthcare).

Techniques: Control, Expressing, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Nuclear hormone receptor corepressor promotes esophageal cancer cell invasion by transcriptional repression of interferon-γ–inducible protein 10 in a casein kinase 2–dependent manner

doi: 10.1091/mbc.E11-11-0947

Figure Lengend Snippet: CK2α phosphorylates Ser-2436 of NCoR. (A) Reciprocal immunoprecipitation analysis was performed using HeLa cell lysates with a CK2α or NCoR antibody, and immunoblotting was performed using their respective antibodies (right). HeLa cells were transfected with Myc-CK2α, and cell lysates were immunoprecipitated and immunoblotted with their respective antibodies (left). (B) A schematic of the deletion mutants of NCoR for in vitro translation (left). Bound proteins were eluted and analyzed by autoradiography (right, top). HeLa cells were transfected with FLAG-tagged NCoR-15/16 (1985–2440) and HA-tagged CK2α plasmids. Whole-cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies (right, bottom). (C and D) In vitro kinase assays were performed with CK2α and the indicated GST-fused NCoR-15/16 proteins. Bound proteins were eluted and analyzed by autoradiography (C) and scintillation counter (D). (E) In vitro kinase assays were performed with recombinant CK2α enzyme and the indicated GST-fused NCoR-15/16 proteins. Western blotting was performed with phospho-NCoR antibody. CBB , Coomassie blue staining. (F) Full-length GFP-NCoR plasmids were transfected into HeLa cells with or without Myc-CK2α and treated with TBB (50 μM) for 6 h. Cell lysates were analyzed by Western blotting with indicated antibodies. (G) HeLa cells were seeded on coverslips and transfected with the indicated expression plasmids in the presence or absence of TBB (50 μM). The permeabilized HeLa cells were incubated with indicated antibodies and/or 1 μg/ml of non-phosphopeptide (NPP) or phosphopeptide (PP) for 12 h, which was followed by PLA probes (PLUS and MINUS) treatment. The positive signal was analyzed using confocal microscopy. (H) In situ PLA analysis was performed under the same conditions as above without transfection of expression plasmids. The level of NCoR phosphorylation was assessed with αNCoR antibody and α-phospho-NCoR antibody.

Article Snippet: The following antibodies were used: anti-CK2 (Upstate, Charlottesville, VA), anti-hemagglutinin (anti-HA; Sigma-Aldrich, and Covance, New York, NY), anti-FLAG (Sigma-Aldrich), anti–β-actin (Sigma-Aldrich), anti-GAPDH (Millipore, Bedford, MA), anti-NCoR (ATGEN, Seongnam, Gyeonggido, Korea), anti–E-cadherin, anti–MMP-9 (Calbiochem), anti-vimentin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p300 (Upstate), anti–c-Jun (Epitomics, Burlingame, CA), anti–acetyl-histone H3, anti-IP-10 (Santa Cruz Biotechnology), and anti-Myc (Cell Signaling, Danvers, MA).

Techniques: Immunoprecipitation, Western Blot, Transfection, In Vitro, Autoradiography, Recombinant, Staining, Expressing, Incubation, Confocal Microscopy, In Situ

Journal: Molecular Biology of the Cell

Article Title: Nuclear hormone receptor corepressor promotes esophageal cancer cell invasion by transcriptional repression of interferon-γ–inducible protein 10 in a casein kinase 2–dependent manner

doi: 10.1091/mbc.E11-11-0947

Figure Lengend Snippet: CK2α-dependent NCoR phosphorylation increases NCoR stability via inhibition of the ubiquitin-dependent proteasomal pathway. (A) HeLa cells were transfected with full-length GFP-NCoR plasmids and treated with an increase amount of TBB (10, 50, 100 μM) and/or MG132 (10 μM) for 6 h. Cell lysates were analyzed by Western blotting. (B) HeLa cells were treated with an increase amount of TBB (10, 50 μM) and/or MG132, and cell lysates were analyzed by Western blotting with indicated antibodies. (C) HeLa cells were transfected with either FLAG-NCoR-15/16 WT or FLAG-NCoR-15/16 S2436A plasmid. After 2 d, cells were treated with cycloheximide (10 μg/ml), TBB (50 μM), and/or MG132 for various time periods, and cell lysates were analyzed by Western blotting. (D) HeLa cells were treated with TBB (50 μM) and/or MG132, and endogenous NCoR and phospho-NCoR levels were analyzed by confocal microscopy. (E and F) HeLa cells were treated with TBB (50 μM) or siRNAs against NCoR or CK2α, and then protein and mRNA levels were analyzed by Western blotting (E) and RT-PCR (F), respectively. (G) HeLa cells were treated with emodin (50 μM), TBB, LiCl 2 , and indicated siRNAs, and endogenous NCoR levels were then analyzed by confocal microscopy. (H) HeLa cells were treated with indicated inhibitors or siRNAs, and cell lysates were analyzed by Western blotting. (I) FLAG-NCoR-15/16 plasmids and HA-ubiquitin (Ub) were transfected into HeLa cells with TBB and/or MG132, and cell lysates were analyzed by Western blotting.

Article Snippet: The following antibodies were used: anti-CK2 (Upstate, Charlottesville, VA), anti-hemagglutinin (anti-HA; Sigma-Aldrich, and Covance, New York, NY), anti-FLAG (Sigma-Aldrich), anti–β-actin (Sigma-Aldrich), anti-GAPDH (Millipore, Bedford, MA), anti-NCoR (ATGEN, Seongnam, Gyeonggido, Korea), anti–E-cadherin, anti–MMP-9 (Calbiochem), anti-vimentin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p300 (Upstate), anti–c-Jun (Epitomics, Burlingame, CA), anti–acetyl-histone H3, anti-IP-10 (Santa Cruz Biotechnology), and anti-Myc (Cell Signaling, Danvers, MA).

Techniques: Inhibition, Transfection, Western Blot, Plasmid Preparation, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Biology of the Cell

Article Title: Nuclear hormone receptor corepressor promotes esophageal cancer cell invasion by transcriptional repression of interferon-γ–inducible protein 10 in a casein kinase 2–dependent manner

doi: 10.1091/mbc.E11-11-0947

Figure Lengend Snippet: NCoR selectively represses transcription of the anti-tumorigenic gene IP-10/CXCL10 in a CK2α-dependent manner. (A) HCE4 cells were transfected with siRNA against NCoR and CK2α, and the change in mRNA expression was analyzed by cDNA microarray analysis using the Illumina HumanRef-8 version 3 Expression BeadChip. Data outputs and the average intensity for each array were normalized against housekeeping genes located on each array. Differentially expressed genes were identified by comparison of the siCK2 sample set with the small-interfering control sample set, and the siNCoR-2 sample set with the small-interfering control sample set using p < 0.05 as the significance cutoff. Only fold changes greater than 2.0 were considered. (B) HCE4 cells were treated with siRNAs, and the levels of indicated genes were analyzed by real-time PCR (left). The relative levels of indicated genes between TE2 and HCE4 cells were analyzed by real-time PCR (right). All samples were normalized to human GAPDH. (C) HCE4 cells were treated with TBB (50 μM, 6 H) or indicated siRNAs, and the level of each gene was analyzed by real-time PCR. (D) HCE4 cells were treated with an increasing amount of indicated inhibitors, and then mRNAs were analyzed by real-time PCR. (E) HCE4 cells were treated with siRNAs against each HDAC, and the level of each gene was analyzed by real-time PCR. (F) HCE4 cells were treated with TBB (50 μM, 6 H) or indicated siRNAs, and the level of each gene was analyzed by real-time PCR. (G) TE2 cells were transfected with CK2α plasmid. After 24 h, cells were treated with indicated siRNAs or TBB (50 μm, 6 H), and the levels of the indicated genes were analyzed by real-time PCR. *, p < 0.01 vs. CK2α alone; **, p < 0.05 vs. CK2α alone; #, p < 0.05 vs. CK2α +.siCon; # #, p < 0.05 vs. CK2α + siCon.

Article Snippet: The following antibodies were used: anti-CK2 (Upstate, Charlottesville, VA), anti-hemagglutinin (anti-HA; Sigma-Aldrich, and Covance, New York, NY), anti-FLAG (Sigma-Aldrich), anti–β-actin (Sigma-Aldrich), anti-GAPDH (Millipore, Bedford, MA), anti-NCoR (ATGEN, Seongnam, Gyeonggido, Korea), anti–E-cadherin, anti–MMP-9 (Calbiochem), anti-vimentin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p300 (Upstate), anti–c-Jun (Epitomics, Burlingame, CA), anti–acetyl-histone H3, anti-IP-10 (Santa Cruz Biotechnology), and anti-Myc (Cell Signaling, Danvers, MA).

Techniques: Transfection, Expressing, Microarray, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Molecular Biology of the Cell

Article Title: Nuclear hormone receptor corepressor promotes esophageal cancer cell invasion by transcriptional repression of interferon-γ–inducible protein 10 in a casein kinase 2–dependent manner

doi: 10.1091/mbc.E11-11-0947

Figure Lengend Snippet: NCoR complexes repress IP-10 transcription at the epigenetic status via deacetylation of histone tails in a CK2α-dependent manner. (A and B) HCE4 cells were treated with TBB (50 μM, 6 H) or indicated siRNAs, and ChIP assays were performed with the indicated antibodies. The precipitated samples were analyzed by real-time PCR, and results are given as the percentage of input as means ± SD of three independent experiments. *, p < 0.005 vs. SiCon; #, P < 0.01 vs. SiCon; **, p < 0.05 vs. SiCon. (C) ChIP and reChIP assays were performed with the indicated antibodies. Error bars, SD ( n = 3). *, p < 0.05 vs. IgG; **, p < 0.01 vs. IgG. (D) TE2 cells were transfected with indicated siRNAs and/or plasmids, and treated with TBB (50 μM, 6 H). The invading cells were counted in BioCoat Matrigel invasion chambers. (E) Model of our findings. In tumor cells with high CK2α activity, active CK2α phosphorylates NCoR and HDAC3 to repress IP-10 transcription, enhancing tumorigenesis. Additionally, CK2α phosphorylates Snail1 to repress E-cadherin transcription via an NCoR-independent pathway. Thus selective CK2α inhibition may be promising for anticancer therapy.

Article Snippet: The following antibodies were used: anti-CK2 (Upstate, Charlottesville, VA), anti-hemagglutinin (anti-HA; Sigma-Aldrich, and Covance, New York, NY), anti-FLAG (Sigma-Aldrich), anti–β-actin (Sigma-Aldrich), anti-GAPDH (Millipore, Bedford, MA), anti-NCoR (ATGEN, Seongnam, Gyeonggido, Korea), anti–E-cadherin, anti–MMP-9 (Calbiochem), anti-vimentin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p300 (Upstate), anti–c-Jun (Epitomics, Burlingame, CA), anti–acetyl-histone H3, anti-IP-10 (Santa Cruz Biotechnology), and anti-Myc (Cell Signaling, Danvers, MA).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Activity Assay, Inhibition

Journal: Nature Communications

Article Title: Defining a conformational ensemble that directs activation of PPARγ

doi: 10.1038/s41467-018-04176-x

Figure Lengend Snippet: Nuclear receptor co-regulator interaction differentiates pharmacologically distinct PPARγ ligands. TR-FRET biochemical assay shows the effect of the compounds on the interaction between PPARγ LBD and peptides derived from the a MED1 coactivator and b NCoR corepressor, plotted as TR-FRET ratio (665 nm/620 nm) vs. ligand concentration ( n = 2, standard deviation). The data shown represents technical replicates from a single experiment and the experiment was repeated four times with similar results. The window of efficacy in these data is representative of ligand-induced changes in co-regulator affinity for PPARγ. An increase in TR-FRET ratio indicates a strengthening of affinity for MED1/NCoR compared to apo while a decrease indicates a ligand-induced weakening of affinity for the co-regulator. The effect of vehicle (DMSO) is negligible (Supplementary Fig. ); furthermore, the DMSO concentration is constant across the titration both in this figure and all other TR-FRET data presented

Article Snippet: Peptides were synthesized by Lifetein LLC (Somerset, NJ, USA) for the for MED1 peptide, sequence: NTKNHPMLMNLLKDNPAQD; and the NCoR peptide, sequence: GHSFADPASNLGLEDIIRKALMG (2251–2273).

Techniques: Derivative Assay, Concentration Assay, Standard Deviation, Titration

Journal: Nature Communications

Article Title: Defining a conformational ensemble that directs activation of PPARγ

doi: 10.1038/s41467-018-04176-x

Figure Lengend Snippet: Ligand-directed helix 12 ensemble dictates PPARγ–co-regulator interaction. a Plot of mean 19 F NMR chemical shift values (PPARγ K502C -BTFA) vs. TR-FRET endpoint data for the recruitment of MED1, NCoR, and CBP peptides to PPARγ K502C -BTFA (top panels) and wt PPARγ LBD (bottom panels) for the set of 16 pharmacologically distinct synthetic PPARγ ligands and apoprotein (select ligands for CBP). Error bars are relatively small for endpoint TR-FRET values (Fig. and Supplementary Figure ) and are excluded for clarity. b Plot of mean 19 F NMR chemical shift values (PPARγ K502C -BTFA) vs. MED1, NCoR, and SMRT peptide dissociation constant ( K d ) for PPARγ K502C -BTFA (top panels) and wt PPARγ LBD (bottom panels) as measured by fluorescence polarization for a subset of the ligands in a . Linear regression fit is shown as a solid line and the correlation coefficient ( R 2 ) for the fitted line is indicated. The ligands with the highest efficacy for MED1 and NCoR recruitment for PPARγ K502C -BTFA are highlighted. Error bars represent standard deviation of two (K502C-BTFA K d and wt SMRT K d ) or three (wt MED1 and NCoR K d ) independent experiments

Article Snippet: Peptides were synthesized by Lifetein LLC (Somerset, NJ, USA) for the for MED1 peptide, sequence: NTKNHPMLMNLLKDNPAQD; and the NCoR peptide, sequence: GHSFADPASNLGLEDIIRKALMG (2251–2273).

Techniques: Fluorescence, Standard Deviation

Journal: Nature Communications

Article Title: Defining a conformational ensemble that directs activation of PPARγ

doi: 10.1038/s41467-018-04176-x

Figure Lengend Snippet: Co-regulator-binding shifts the helix 12 conformational ensemble. a Deconvoluted 19 F NMR spectra of apo PPARγ C313A,K502C -BTFA and PPARγ K502C -BTFA bound to GW1929 or T0070907 in the absence and presence of MED1 coactivator or NCoR corepressor peptides. The percent of total signal area found in the left sharp peak is shown for T0070907 and GW9662 spectra. The small sharp peak at ~−83.3 ppm is free BTFA. b Mean-weighted chemical shift values from the plots in a . c Fraction of the total peak areas in the four colored boxed regions in a , which roughly correspond to the clustered spectral regions from Fig. ; with the two middle regions corresponding to the two peaks observed for apoprotein. Agonist-bound PPARγ is changed little by MED1 binding, whereas NCoR binding changes the spectrum drastically and vice versa for inverse-agonist (T0070907)-bound PPARγ. *SMRT-induced mean chemical shift in GW1929-bound PPARγ K502C -BTFA is the same as NCoR. These experiments were performed once

Article Snippet: Peptides were synthesized by Lifetein LLC (Somerset, NJ, USA) for the for MED1 peptide, sequence: NTKNHPMLMNLLKDNPAQD; and the NCoR peptide, sequence: GHSFADPASNLGLEDIIRKALMG (2251–2273).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Defining a conformational ensemble that directs activation of PPARγ

doi: 10.1038/s41467-018-04176-x

Figure Lengend Snippet: Heterodimerization of PPARγ LBD with RXRα LBD favors coactivator binding. a 19 F NMR of PPARγ K502C -BTFA bound to T0070907 and PPARγ C313A,502C -BTFA bound to MRL24 or GW1929 or with no ligand bound was performed in the presence (orange) or absence (black) of RXRα LBD. These experiments were performed once. d The 19 F NMR signal from the MRL24 ligand. Broadening and consequent reduction in signal intensity is expected as a consequence of the increased rotational correlation time of the heterodimer complex. b , c TR-FRET was used to measure interaction between wt PPARγ LBD and MED1 or NCoR in the presence or absence of equimolar concentrations of RXRα. Error bars represent standard deviation of two technical replicates within a single experiment. The experiment was repeated twice and gave similar results each time. e Heterodimerization favors MED1 binding ( p = 0.0017) and disfavors NCoR binding ( p = 0.0076) to apo PPARγ. In addition, visual and statistical comparison of NCoR and MED1 recruitment to PPARγ LBD saturated with ligand (four highest concentrations of ligands) indicates that RXRα affects co-regulator recruitment to T0070907-bound PPARγ (NCoR, p = 0.0042; MED1, p = 0.0027) more than GW1929 (NCoR, p = 0.44; MED1, p = 0.34) or MRL24 (NCoR, p = 0.0141; MED1, p = 0.061). All p values are derived from a two-tailed t test

Article Snippet: Peptides were synthesized by Lifetein LLC (Somerset, NJ, USA) for the for MED1 peptide, sequence: NTKNHPMLMNLLKDNPAQD; and the NCoR peptide, sequence: GHSFADPASNLGLEDIIRKALMG (2251–2273).

Techniques: Binding Assay, Standard Deviation, Comparison, Derivative Assay, Two Tailed Test