Journal: Molecular Pharmacology

Article Title: TCL1A Single-Nucleotide Polymorphisms and Estrogen-Mediated Toll-Like Receptor-MYD88–Dependent Nuclear Factor- κ B Activation: Single-Nucleotide Polymorphism– and Selective Estrogen Receptor Modulator–Dependent Modification of Inflammation and Immune Response

doi: 10.1124/mol.117.108340

Figure Lengend Snippet: The SNP- and estrogen-dependent effects on mRNA expression of TCL1A, cytokines, and chemokines in LCLs. (A) Schematic diagrams of the two TCL1A SNPs, rs7359033 and rs7160302, in tight LD with rs11849538, the top hit signal from the MA.27 GWAS. Locations of EREs are shown as boxes for these three SNPs that map between the 3′-termini of TCL1A and TCL1B. ER blockade by 4-hydroxytamoxifen (4OH) or fulvestrant (ICI) treatment resulted in the reversal of TCL1A SNP and estrogen-dependent TCL1A expression patterns (B and C) and downstream effects on the expression of CCR6, CCL20, IL17RA, and IL17A (D–G) Values are mean ± S.E.M. of three assays. *P < 0.0001. Adapted from Fig. 2 and Fig. 3 in Ho et al. (2016).

Article Snippet: The membranes were incubated overnight with primary antibodies: TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, UNC93B1 (Novus Biologicals, Littleton, CO), and ACTB at a 1:500 dilution at 4°C.

Techniques: Expressing

Journal: Molecular Pharmacology

Article Title: TCL1A Single-Nucleotide Polymorphisms and Estrogen-Mediated Toll-Like Receptor-MYD88–Dependent Nuclear Factor- κ B Activation: Single-Nucleotide Polymorphism– and Selective Estrogen Receptor Modulator–Dependent Modification of Inflammation and Immune Response

doi: 10.1124/mol.117.108340

Figure Lengend Snippet: SNP- and estrogen-dependent mRNA expression of TCL1A (A), TLR2 (B), TLR7 (C), TLR9 (D), TLR10 (E), MYD88 (F), and UNC93B1 (G) in LCLs (H). Western blot analysis was performed for TCL1A, TLR2, TLR7, TLR9 TLR10, MYD88, UNC93B1, and ACTB in LCLs with known TCL1A SNP genotypes. The cells were treated with 0.1 nM E2 or with 0.1 nM E2 plus 10−7µM 4-hydroxytamoxifen (4OH-TAM) for an additional 24 hours. Eight cell lines homozygous for the variant (V) genotypes for all three of the TCL1A SNPs and eight cell lines homozygous for WT genotypes were used in these experiments. ***P < 0.0001.

Article Snippet: The membranes were incubated overnight with primary antibodies: TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, UNC93B1 (Novus Biologicals, Littleton, CO), and ACTB at a 1:500 dilution at 4°C.

Techniques: Expressing, Western Blot, Variant Assay

Journal: Molecular Pharmacology

Article Title: TCL1A Single-Nucleotide Polymorphisms and Estrogen-Mediated Toll-Like Receptor-MYD88–Dependent Nuclear Factor- κ B Activation: Single-Nucleotide Polymorphism– and Selective Estrogen Receptor Modulator–Dependent Modification of Inflammation and Immune Response

doi: 10.1124/mol.117.108340

Figure Lengend Snippet: Correlations of TCL1A mRNA expression and those of toll-like receptors and MYDBB in the Human Variation Panel of 300 LCLs

Article Snippet: The membranes were incubated overnight with primary antibodies: TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, UNC93B1 (Novus Biologicals, Littleton, CO), and ACTB at a 1:500 dilution at 4°C.

Techniques: Expressing

Journal: Molecular Pharmacology

Article Title: TCL1A Single-Nucleotide Polymorphisms and Estrogen-Mediated Toll-Like Receptor-MYD88–Dependent Nuclear Factor- κ B Activation: Single-Nucleotide Polymorphism– and Selective Estrogen Receptor Modulator–Dependent Modification of Inflammation and Immune Response

doi: 10.1124/mol.117.108340

Figure Lengend Snippet: TCL1A could modulate TLR2, TLR7, TLR9, TLR10, MYD88, and UNC93B1 expression in LCLs. Relative mRNA expression (A and B) of TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, and UNC93B1 after knockdown of TCL1A in LCLs with known TCL1A SNP genotypes using pooled siRNA. Eight cell lines of each genotype were used in these experiments. *P < 0.05. Student’s t test was performed to compare gene expression in LCLs with differing TCL1A SNP genotypes before and after gene knockdown, *P value ≤ 0.05 was considered statistically significant. All values are mean ±S.E.M for three separate independent assays. Protein expression was determined by Western blot analysis (C). Relative mRNA expression (D and E) of TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, and UNC93B1 after knockdown of MYD88 in LCLs with known TCL1A SNP genotypes using pooled siRNA. (F) Western blot analysis was performed for TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, and UNC93B1 after knockdown of MYD88.

Article Snippet: The membranes were incubated overnight with primary antibodies: TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, UNC93B1 (Novus Biologicals, Littleton, CO), and ACTB at a 1:500 dilution at 4°C.

Techniques: Expressing, Knockdown, Gene Expression, Western Blot

Journal: Molecular Pharmacology

Article Title: TCL1A Single-Nucleotide Polymorphisms and Estrogen-Mediated Toll-Like Receptor-MYD88–Dependent Nuclear Factor- κ B Activation: Single-Nucleotide Polymorphism– and Selective Estrogen Receptor Modulator–Dependent Modification of Inflammation and Immune Response

doi: 10.1124/mol.117.108340

Figure Lengend Snippet: TCL1A SNP- and estrogen-dependent NF-κB activation as determined by NF-κB reporter assays could be altered by the knockdown or inhibition of MYD88. ER blockade by fulvestrant (ICI) or 4-hydroxytamoxifen (4OH) treatment resulted in TCL1A SNP -dependent NF-κB activation (A and B). TCL1A SNP and estrogen-dependent NF-κB activation could be blocked by MYD88 siRNA knockdown (C) or by exposure to MYD88 inhibitory peptide (100 µM) (D). Specifically, LCLs were cotransfected with an NF-κB reporter construct and siRNA (MYD88 or control siRNA); 24 hours after transfection, cells were treated with either vehicle or 0.1 nM E2 for 24 hours, followed by 10−7µM 4OH or ICI for an additional 24 hours. In some experiments, cells were exposed to MYD88 inhibitory peptide for 24 hours before E2 treatment. Luciferase activity was measured 72 hours after transfection. The firefly luciferase activity derived from the NF-κB responsive reporter was normalized by the use of Renilla luciferase activity as a control to correct for possible variation in transfection efficiency. All experiments were repeated three times in triplicate. **P < 0.001. (E) Coimmunoprecipitation was used to determine whether TCL1A protein could interact with MYD88 in LCLs. Whole-cell lysates from 1 × 107 LCLs were immunoprecipitated with anti-TCL1A (1:50) antibodies or anti-IgG antibodies. Whole-cell lysate (input, left panel) and immunoprecipitated samples (middle and right panels) were immunoblotted and probed with antibodies against TCL1A and MYD88.

Article Snippet: The membranes were incubated overnight with primary antibodies: TCL1A, TLR2, TLR7, TLR9, TLR10, MYD88, UNC93B1 (Novus Biologicals, Littleton, CO), and ACTB at a 1:500 dilution at 4°C.

Techniques: Activation Assay, Knockdown, Inhibition, Construct, Control, Transfection, Luciferase, Activity Assay, Derivative Assay, Immunoprecipitation

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: TCL1A , a Novel Transcription Factor and a Coregulator of Nuclear Factor κ B p65: Single Nucleotide Polymorphism and Estrogen Dependence

doi: 10.1124/jpet.118.247718

Figure Lengend Snippet: (A) Coimmunoprecipitation was used to determine whether TCL1A protein could interact with p65 in LCLs. Whole cell lysates from 1 × 107 LCLs were immunoprecipitated with (left panel) anti-TCL1A (1:50) antibodies or anti-IgG antibodies and protein samples were immunoblotted and probed with antibodies against TCL1A. Reversed IP was preformed to confirm that p65 and TCL1A interacted (right panel). (B) Heat-map plots showing the association between TCL1A binding and p65 binding in LCLs (Zhao et al., 2014). The signals for TCL1A ChIP-seq peaks and p65 ChIP-seq peaks are shown as heat-maps using red (the strongest signal) and white (the weakest signal) color schemes. Each row shows ±500 bp centered on the TCL1A ChIP-seq peak summits. (C) TCL1A occupancy versus p65 occupancy in LCLs. Scatter plots depicting the Pearson correlations (r) between TCL1A ChIP tag density (y axis) and p65 ChIP tag density (x axis) for the 357 TCL1A target genes are shown in Fig. 1C.

Article Snippet: TCL1A Double Nickase Plasmid sc-402028-NIC (Santa Cruz Biotechnology, Dallas, TX) was transfected into LCLs using the program X-001 of Amaxa Nucleofector (Lonza, Köln, Germany), and re-covered for 24 hours in RPMI1640 medium (Cellgro, Manassas, VA) supplemented with 15% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA).

Techniques: Immunoprecipitation, Binding Assay, ChIP-sequencing

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: TCL1A , a Novel Transcription Factor and a Coregulator of Nuclear Factor κ B p65: Single Nucleotide Polymorphism and Estrogen Dependence

doi: 10.1124/jpet.118.247718

Figure Lengend Snippet: (A) Schematic diagram of three TCL1A SNPs: rs11849538, the “top hit” signal from the MA.27 musculoskeletal adverse event GWAS, rs7359033, and rs7160302. All three of these SNPs map near the 3′-terminus of TCL1A and are in tight linkage disequilibrium. Locations of estrogen response elements (EREs) are shown as boxes. (B) SNP- and estrogen-related variation of TCL1A mRNA expression in lymphoblastoid cell lines with known TCL1A SNP genotypes after exposure to E2 with or without 4OH-TAM. Student’s t test was performed to compare gene expression in LCLs with differing TCL1A SNP genotypes for each treatment condition, ***P < 0.001. (C) Heat map showing expression profiles for 357 genes regulated by TCL1A SNPs in an estrogen-dependent fashion, all of which could be reversed by 4OH-TAM treatment, as determined by RNA-seq using LCLs with either homozygous wild-type (W/W) (n = 2) or homozygous variant (V/V) (n = 3) genotypes for the TCL1A SNPs.

Article Snippet: TCL1A Double Nickase Plasmid sc-402028-NIC (Santa Cruz Biotechnology, Dallas, TX) was transfected into LCLs using the program X-001 of Amaxa Nucleofector (Lonza, Köln, Germany), and re-covered for 24 hours in RPMI1640 medium (Cellgro, Manassas, VA) supplemented with 15% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA).

Techniques: Expressing, RNA Sequencing Assay, Variant Assay

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: TCL1A , a Novel Transcription Factor and a Coregulator of Nuclear Factor κ B p65: Single Nucleotide Polymorphism and Estrogen Dependence

doi: 10.1124/jpet.118.247718

Figure Lengend Snippet: (A) Genome-wide TCL1A occupancy profiles. Regions (Tss ± 2 kb) were clustered based on their profiles for TCL1A ChIP enrichment over input for all four samples using k-means clustering. Clustering was used to identify regions with distinct binding intensities. The gradient blue-to-red color indicates high-to-low counts in the corresponding genome region. (B) Percentage distributions of TCL1A ChIP-seq peaks for all four samples. (C) Identification of TCL1A consensus binding sequence motifs using HOMER and MEME.

Article Snippet: TCL1A Double Nickase Plasmid sc-402028-NIC (Santa Cruz Biotechnology, Dallas, TX) was transfected into LCLs using the program X-001 of Amaxa Nucleofector (Lonza, Köln, Germany), and re-covered for 24 hours in RPMI1640 medium (Cellgro, Manassas, VA) supplemented with 15% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA).

Techniques: Genome Wide, Binding Assay, ChIP-sequencing, Sequencing

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: TCL1A , a Novel Transcription Factor and a Coregulator of Nuclear Factor κ B p65: Single Nucleotide Polymorphism and Estrogen Dependence

doi: 10.1124/jpet.118.247718

Figure Lengend Snippet: (A) EMSA results showing that one TCL1A DNA binding motif sequence, CCATATAGG, is sufficient for DNA–TCL1A protein interaction. (B) Immunofluorescence staining showing TCL1A nuclear translocation in response to E2 (0.1 nM) treatment in LCLs. (C) Representative examples of TCL1A occupancy peaks depicted for the CCR6 and IL17RA loci in LCLs with known TCL1A SNP genotypes in the absence of E2 or in the presence of E2 (0.1 nM). Red boxes indicate that TCL1A binding in both CCR6 and IL17RA is significantly increased in response to E2 treatment, but that only occurs in cells with homozygous variant genotypes for TCL1A SNPs. (D) Changes in TCL1A occupancy are highly correlated with changes in mRNA expression levels for CCR6 and IL17RA. Student’s t test was performed to compare gene expression in LCLs with differing TCL1A SNPs (homozygous wild-type versus homozygous variant) for each treatment condition, ***P < 0.001.

Article Snippet: TCL1A Double Nickase Plasmid sc-402028-NIC (Santa Cruz Biotechnology, Dallas, TX) was transfected into LCLs using the program X-001 of Amaxa Nucleofector (Lonza, Köln, Germany), and re-covered for 24 hours in RPMI1640 medium (Cellgro, Manassas, VA) supplemented with 15% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA).

Techniques: Binding Assay, Sequencing, Immunofluorescence, Staining, Translocation Assay, Variant Assay, Expressing

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: TCL1A , a Novel Transcription Factor and a Coregulator of Nuclear Factor κ B p65: Single Nucleotide Polymorphism and Estrogen Dependence

doi: 10.1124/jpet.118.247718

Figure Lengend Snippet: (A) Venn diagram showing 357 gene that displayed TCL1A SNP- and estrogen-dependent gene expression patterns as determined by RNA-seq, and 57 of those 357 genes that displayed TCL1A SNP- and estrogen-dependent TCL1A occupancy. (B and C) TCL1A ChIP assays were performed to confirm results obtained from TCL1A ChIP-seq for all 57 genes, all of which showed greater TCL1A binding in the presence of E2, but only in cells homozygous variant for the TCL1A SNP genotypes. In contrast, in the presence of 4OH-TAM, this binding pattern was reversed for all 57 genes (n = 4 for each genotype group). (D and E) Changes in TCL1A binding were correlated with changes in mRNA expression for all 57 genes using the same cell lines from which the data shown in B and C were obtained. Specifically, in the presence of E2, all those genes showed significant induction only for the variant genotype. However, the expression pattern could be reversed by 4OH-TAM, thus confirming that changes in TCL1A occupancy were highly correlated with transcription.

Article Snippet: TCL1A Double Nickase Plasmid sc-402028-NIC (Santa Cruz Biotechnology, Dallas, TX) was transfected into LCLs using the program X-001 of Amaxa Nucleofector (Lonza, Köln, Germany), and re-covered for 24 hours in RPMI1640 medium (Cellgro, Manassas, VA) supplemented with 15% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA).

Techniques: Expressing, RNA Sequencing Assay, ChIP-sequencing, Binding Assay, Variant Assay

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: TCL1A , a Novel Transcription Factor and a Coregulator of Nuclear Factor κ B p65: Single Nucleotide Polymorphism and Estrogen Dependence

doi: 10.1124/jpet.118.247718

Figure Lengend Snippet: (A and B) p65 ChIP-re-ChIP assays were performed to confirm the co-occupancy of TCL1A and p65 on 57 selected binding regions as shown in Fig. 4, B and C (n = 4 for each genotype group).

Article Snippet: TCL1A Double Nickase Plasmid sc-402028-NIC (Santa Cruz Biotechnology, Dallas, TX) was transfected into LCLs using the program X-001 of Amaxa Nucleofector (Lonza, Köln, Germany), and re-covered for 24 hours in RPMI1640 medium (Cellgro, Manassas, VA) supplemented with 15% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA).

Techniques: Binding Assay

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: TCL1A , a Novel Transcription Factor and a Coregulator of Nuclear Factor κ B p65: Single Nucleotide Polymorphism and Estrogen Dependence

doi: 10.1124/jpet.118.247718

Figure Lengend Snippet: (A) Western blot analysis after TCL1A knockout or p65 knockdown in LCLs. (B) Knockdown of p65 resulted in the abolition of SNP- and estrogen-dependent TCL1A occupancy. (C) Knockdown of TCL1A abolished the TCL1A SNP- and estrogen-dependent p65 binding. (D and E) TCL1A SNP- and estrogen-dependent gene expression was lost after the knockdown of p65 or the knockout of TCL1A.

Article Snippet: TCL1A Double Nickase Plasmid sc-402028-NIC (Santa Cruz Biotechnology, Dallas, TX) was transfected into LCLs using the program X-001 of Amaxa Nucleofector (Lonza, Köln, Germany), and re-covered for 24 hours in RPMI1640 medium (Cellgro, Manassas, VA) supplemented with 15% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA).

Techniques: Western Blot, Knock-Out, Binding Assay, Expressing

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: TCL1A , a Novel Transcription Factor and a Coregulator of Nuclear Factor κ B p65: Single Nucleotide Polymorphism and Estrogen Dependence

doi: 10.1124/jpet.118.247718

Figure Lengend Snippet: Coimmunoprecipitation was used to determine whether TCL1A protein could interact with NF-kB subunits in LCLs.

Article Snippet: TCL1A Double Nickase Plasmid sc-402028-NIC (Santa Cruz Biotechnology, Dallas, TX) was transfected into LCLs using the program X-001 of Amaxa Nucleofector (Lonza, Köln, Germany), and re-covered for 24 hours in RPMI1640 medium (Cellgro, Manassas, VA) supplemented with 15% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA).

Techniques:

Journal: Leukemia

Article Title: Tumor antigen ROR1 targeted drug delivery mediated selective leukemic but not normal B cell cytotoxicity in chronic lymphocytic leukemia

doi: 10.1038/leu.2014.199

Figure Lengend Snippet: Gene expression analysis of CLL cells treated with OSU-2S. ( a ) Heat map of 266 differently expressed genes in OSU-2S treated CLL. RNA was isolated from CLL cells (N=3) treated with vehicle or OSU-2S(8µM) at 16 hours and subjected to microarray analyses using GeneChip Human Genome U133 plus 2.0 (Affymetrix, GPL570). After statistical analyses, genes that changed by ≥ 2 fold and p<0.0005 in response to OSU-2S treatment were used for constructing a heat map. ( b ) Top five molecular and cellular functions revealed through Ingenuity Pathway analysis (IPA) of genes modulated by OSU-2S in CLL. ( c ) OSU-2S down modulates TCL1A. RNA was extracted from vehicle or OSU-2S treated (16hr) CLL patients cells (N=7) and TCL1A expression relative to 18S was determined by real-time RT-PCR analysis and values are normalized to vehicle control; bars represent SD. Immunoblot of TCL1A in CLL cells treated with vehicle or OSU-2S for 24 hours is also shown.

Article Snippet: Antibodies against phospho S591 SHP1 (ECM Biosciences); GAPDH, SHP1, PP2Ac (Millipore); Brg1, (Santa Cruz Biotechnology, Dallas, TX); TCL1A (MBL International).

Techniques: Expressing, Isolation, Microarray, Quantitative RT-PCR, Western Blot