Journal: The Journal of Clinical Investigation

Article Title: Epigenetic regulator UHRF1 orchestrates proinflammatory gene expression in rheumatoid arthritis in a suppressive manner

doi: 10.1172/JCI150533

Figure Lengend Snippet: ( A ) Protocol for analysis of collagen antibody-induced arthritis (CAIA) model. PBS (Ctrl P ) or LPS (Ctrl L ) was administered as a control. ( B ) PCA using microarray data obtained from ankle tissue. ( C ) Heatmap of differentially expressed gene probes in ankle tissue (log 2 FC > |1|, P < 0.01). Log 10 transformed read counts are scaled from 1.0 to 3.0. ( D ) Expression of genes related to epigenetic regulation classified by GSEA. Log 10 transformed read counts are scaled to minimum to maximum values. ( E ) Relative probe counts detected in CAIA ankle compared with Ctrl P or Ctrl L ankles. ( F ) RT-qPCR of Uhrf1 mRNA expression in CAIA ( n = 4) and STA ( n = 3) ankles. ( G ) UHRF1 mRNA expression in synovium biopsies from healthy individuals, patients with OA, and patients with RA by RNA-Seq. Data are registered in the Gene Expression Omnibus (GEO GSE89408). ( H ) Left, representative images of immunofluorescence staining for Uhrf1 (red); Pdpn, Fap, Thy-1, CD45, F4/80, and CD3 (green); and DAPI (blue) in WT STA ankle tissue. Scale bar: 50 μm. Right, quantification of Uhrf1 + marker cells in hyperplastic synovium. Cell number in 1 field per similar region of independent mice was calculated. ( I ) Uhrf1 mRNA expression in SFs treated with 20 ng/mL Tnf-α for 24 hours. Mean ± SD is shown. * P < 0.05 and ** P < 0.01 by ANOVA followed by Tukey’s test in F (left), G , and H, and unpaired t test in F (right) and I . Data in A – F and H – I were obtained from 3 to 5 independent experiments.

Article Snippet: Flow cytometry antibodies used included FITC-conjugated rat antibody against mouse CD45 (BioLegend, 30-F11); PE-conjugated rat antibody against mouse CD4 (BioLegend, GK1.5); Alexa Fluor 647–conjugated rat antibody against mouse Ccr6 (BD Biosciences, 140706); APC-conjugated mouse antibody against human CD45 (Miltenyi Biotec, 5B1); FITC-conjugated mouse antibody against human CD4 (BioLegend, OKT4); and PE-conjugated mouse antibody against human CCR6 (BioLegend, G034E3).

Techniques: Control, Microarray, Transformation Assay, Expressing, Quantitative RT-PCR, RNA Sequencing, Gene Expression, Immunofluorescence, Staining, Marker

Journal: The Journal of Clinical Investigation

Article Title: Epigenetic regulator UHRF1 orchestrates proinflammatory gene expression in rheumatoid arthritis in a suppressive manner

doi: 10.1172/JCI150533

Figure Lengend Snippet: ( A ) Quantification of methylated DNA after enrichment from genome DNA using MBD beads. ( B ) Distribution of Uhrf1-mediated methylated DNA annotated using given intervals and scores with genome features by CEAS. ( C ) Venn diagram to compare Uhrf1-mediated methylated DNA loci between SFs and chondrocytes using MBD-Seq data from this study and GEO GSE99335. ( D ) Venn diagram for 171 genes having upregulated expression in Uhrf1 ΔCol6a1 SFs and 105 genes having Uhrf1-mediated methylated DNA peaks within the transcriptional start site region (± 50 kb). ( E ) Representative Uhrf1-mediated methylated DNA peaks visualized by Integrative Genomics Viewer. ( F ) KEGG pathway analysis of 105 upregulated with peaks assigned using DAVID Bioinformatics Resources. Significantly enriched pathways illustrated by gene counts and P values. Representative mRNA expression of genes included in the ( G ) KEGG pathways “Rheumatoid arthritis” and “Cytokine-cytokine receptor interaction” and ( H ) GO biological process “Negative regulation of apoptotic process” in SFs from Uhrf1 fl/fl and Uhrf1 ΔCol6a1 mice ( n = 3) as measured by RT-qPCR. ( I ) ChIP-qPCR assay of Uhrf1 for Ccl20 locus in Uhrf1 ΔCol6a1 and Uhrf1 fl/fl SFs. ( J ) Quantification of Ccl20 serum levels in Uhrf1 fl/fl and Uhrf1 ΔCol6a1 on day 0 ( n = 10) and day 10 ( n = 16) after STA induction. ( K ) Left, flow cytometry analysis of the population of Th17 cells (CD45 + , CD4 + , Ccr6 + ) in Uhrf1 fl/fl and Uhrf1 ΔCol6a1 derived from STA mice on day 4 ( n = 6–8) and day 10 ( n = 9–10). Right, quantification of CD45 + CD4 + Ccr6 + cells among CD45 + cells. Mean ± SD shown. NS, not significant versus Uhrf1 fl/fl . * P < 0.05 and ** P < 0.01 by unpaired t test in G – J and ANOVA followed by Tukey’s test in K . Data in A , G , and H obtained from 3 independent experiments. Data in B – F obtained from combined read data from 3 independent experiments. Data in I were technically replicated 3 times. Data in J and K obtained from 6–10 independent experiments.

Article Snippet: Flow cytometry antibodies used included FITC-conjugated rat antibody against mouse CD45 (BioLegend, 30-F11); PE-conjugated rat antibody against mouse CD4 (BioLegend, GK1.5); Alexa Fluor 647–conjugated rat antibody against mouse Ccr6 (BD Biosciences, 140706); APC-conjugated mouse antibody against human CD45 (Miltenyi Biotec, 5B1); FITC-conjugated mouse antibody against human CD4 (BioLegend, OKT4); and PE-conjugated mouse antibody against human CCR6 (BioLegend, G034E3).

Techniques: Methylation, Expressing, Quantitative RT-PCR, ChIP-qPCR, Flow Cytometry, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Epigenetic regulator UHRF1 orchestrates proinflammatory gene expression in rheumatoid arthritis in a suppressive manner

doi: 10.1172/JCI150533

Figure Lengend Snippet: ( A ) Expression levels of UHRF1 and DNMTs ( DNMT1 , DNMT3A , and DNMT3B ) mRNA in synovium obtained from patients with OA ( n = 32) and RA ( n = 30). ( B ) Spearman’s correlation between UHRF1 mRNA expression in RA synovium ( n = 30) and disease activity score 28-CRP (DAS28) as well as levels of C-reactive protein (CRP) and age. ( C ) Correlation of UHRF1 expression in RA synovium with 6-month response to DMARD treatment measured by ΔDAS28-CRP ( https://peac.hpc.qmul.ac.uk/ ). ( D ) Top, Western blot analysis of OA synovium ( n = 5) and RA synovium ( n = 5). Bottom, quantification of relative UHRF1 protein levels. ( E ) H&E staining and immunofluorescence staining for UHRF1 (red); PDPN, FAP, CD45 (green); and DAPI (blue) in specimens from multiple patients with RA (P1–P4). Scale bar: 100 μm. Arrow and arrowhead indicate UHRF1 + cells in cells positive for SF markers PDPN and FAP and leukocyte marker CD45, respectively. ( F ) Quantification of UHRF1 + cell number in CD45 + cells and CD45 – cells among total UHRF1 + cells ( n = 19). ( G ) Spearman’s correlation between DAS28 and number of UHRF1 + cells per tissue area ( n = 19). Mean ± SD is shown. * P < 0.05 and ** P < 0.01 by Mann-Whitney U test in A and D , and by Wilcoxon signed-rank test in F . All data were obtained from 5–32 independent experiments.

Article Snippet: Flow cytometry antibodies used included FITC-conjugated rat antibody against mouse CD45 (BioLegend, 30-F11); PE-conjugated rat antibody against mouse CD4 (BioLegend, GK1.5); Alexa Fluor 647–conjugated rat antibody against mouse Ccr6 (BD Biosciences, 140706); APC-conjugated mouse antibody against human CD45 (Miltenyi Biotec, 5B1); FITC-conjugated mouse antibody against human CD4 (BioLegend, OKT4); and PE-conjugated mouse antibody against human CCR6 (BioLegend, G034E3).

Techniques: Expressing, Activity Assay, Western Blot, Staining, Immunofluorescence, Marker, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Epigenetic regulator UHRF1 orchestrates proinflammatory gene expression in rheumatoid arthritis in a suppressive manner

doi: 10.1172/JCI150533

Figure Lengend Snippet: ( A ) mRNA expression levels of UHRF1 and CCL20 in RASFs transfected with UHRF1 siRNA ( n = 4–5). ( B ) Left, flow cytometry to measure proportion of Th17 cells (CD45 + CD4 + CCR6 + ) in OA ( n = 14) and RA ( n = 21) synovium tissue. Right, quantification of total CD45 + cells, CD45 + CD4 – CCR6 – cells, CD45 + CD4 – CCR6 + cells, CD45 + CD4 + CCR6 – cells, and CD45 + CD4 + CCR6 + cells among 7-AAD – cells. ( C ) Spearman’s correlation between proportion of Th17 cells and UHRF1 mRNA expression level in OASFs ( n = 10) and RASFs ( n = 12) obtained from synovium of the same patients. ( D ) Schematic protocol of consecutive UHRF1 knockdown and experimental apoptosis induction in RASFs. ( E ) Left, phalloidin (green) and DAPI (blue) staining of RASFs transfected twice with UHRF1 siRNA ( n = 9) after treatment with 0.5 μg/mL anti-FAS antibody for 16 hours. Scale bar: 200 μm. Right, quantification of cell numbers after apoptosis induction relative to that for vehicle treatment. Mean ± SD is shown. * P < 0.05 and ** P < 0.01 by ANOVA followed by Tukey’s test in A and E , and Mann-Whitney U test in B . All data were obtained from 4 to 21 independent experiments.

Article Snippet: Flow cytometry antibodies used included FITC-conjugated rat antibody against mouse CD45 (BioLegend, 30-F11); PE-conjugated rat antibody against mouse CD4 (BioLegend, GK1.5); Alexa Fluor 647–conjugated rat antibody against mouse Ccr6 (BD Biosciences, 140706); APC-conjugated mouse antibody against human CD45 (Miltenyi Biotec, 5B1); FITC-conjugated mouse antibody against human CD4 (BioLegend, OKT4); and PE-conjugated mouse antibody against human CCR6 (BioLegend, G034E3).

Techniques: Expressing, Transfection, Flow Cytometry, Knockdown, Staining, MANN-WHITNEY

Journal: mSphere

Article Title: Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines

doi: 10.1128/mSphere.00305-17

Figure Lengend Snippet: EBV upregulates CD226 during primary B-cell outgrowth into LCLs. (A) Representative flow cytometry plot of peripheral blood mononuclear cells (PBMCs). B cells are CD19 + . (B) Overlaid CD226/CFSE flow cytometry plots of uninfected primary B cells (red), proliferating EBV-infected B cells (blue), and LCLs (green) from a matched biological donor. (C) CD226 flow cytometry of primary B cells at select days following EBV infection. The mean and standard deviation (SD) from two donors is shown.

Article Snippet: For labeling, cells were incubated with allophycocyanin (APC)-conjugated mouse anti-human CD226 antibody (Miltenyi Biotech, clone DX11), phycoerythrin (PE)-conjugated mouse anti-human CD226 antibody (BioLegend, clone 11A8), or PE-conjugated mouse anti-human CD19 antibody (BD Pharmingen) according to the manufacturer’s protocol.

Techniques: Flow Cytometry, Infection, Standard Deviation

Journal: mSphere

Article Title: Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines

doi: 10.1128/mSphere.00305-17

Figure Lengend Snippet: EBV, but not CpG DNA, upregulates CD226 upon cell proliferation. Representative flow cytometry plots of EBV-infected (A), CpG-activated (B), or CD40L/IL-4-activated B cells (C) at day 6 posttreatment/infection. Lower CFSE or CellTrace violet staining indicates proliferating cells. (D) CD226 mean fluorescence intensity (MFI) of proliferating population normalized to that of the nonproliferating population in EBV-infected (10 donors), CpG-activated (4 donors), and CD40L/IL-4-activated (4 donors) B cells. ***, P < 0.001 by one-way analysis of variance (ANOVA).

Article Snippet: For labeling, cells were incubated with allophycocyanin (APC)-conjugated mouse anti-human CD226 antibody (Miltenyi Biotech, clone DX11), phycoerythrin (PE)-conjugated mouse anti-human CD226 antibody (BioLegend, clone 11A8), or PE-conjugated mouse anti-human CD19 antibody (BD Pharmingen) according to the manufacturer’s protocol.

Techniques: Flow Cytometry, Infection, Staining, Fluorescence

Journal: mSphere

Article Title: Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines

doi: 10.1128/mSphere.00305-17

Figure Lengend Snippet: EBV-positive lymphoblasts express higher levels of CD226 than EBV-negative lymphoblasts. (A) qRT-PCR and (B) flow cytometry assessed CD226 mRNA and surface expression across a range of EBV-negative and EBV-positive (latency III, latency I, and Wp-restricted) B-lymphoblast (BL) cell lines.

Article Snippet: For labeling, cells were incubated with allophycocyanin (APC)-conjugated mouse anti-human CD226 antibody (Miltenyi Biotech, clone DX11), phycoerythrin (PE)-conjugated mouse anti-human CD226 antibody (BioLegend, clone 11A8), or PE-conjugated mouse anti-human CD19 antibody (BD Pharmingen) according to the manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Flow Cytometry, Expressing

Journal: mSphere

Article Title: Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines

doi: 10.1128/mSphere.00305-17

Figure Lengend Snippet: LMP1 and NF-κB regulate CD226 expression. (A) ChIP-seq of NF-κB transcription factors (RelA, RelB, cRel, and p52) and EBV nuclear antigens (EBNA2 and EBNA3C) at the CD226 locus. RNA-seq coverage at the CD226 locus is shown below from early-infected B cells and LCLs. (B) BL41 cells transduced with an LMP1-expressing retrovirus or mock transduced with an empty vector control and then assayed by flow cytometry for CD226 surface expression. (C) BL41 cells were transduced with tetracycline-responsive LMP1 and assayed for surface levels of CD226 and LMP1 expression by flow cytometry at 18-h intervals following treatment with tetracycline. (D) LCLs were sorted into low-, middle-, and high-ICAM-1-expressing populations using FACS. mRNA levels of CD226, LMP1, and NF-κB targets (TRAF1, ICAM-1, and c-FLIP) were determined by qRT-PCR from two independent experiments. Data are reported as means ± SD. (E) LCLs were treated with 5 μM IKKβ inhibitor IV and DMSO and then analyzed for CD226 RNA expression with qRT-PCR. Data are reported as means ± SD. (F) CD226 mRNA expression on GCB DLBCLs, ABC DLBCLs, and LCLs was determined using microarray analysis. *, P < 0.05; ***, P < 0.001.

Article Snippet: For labeling, cells were incubated with allophycocyanin (APC)-conjugated mouse anti-human CD226 antibody (Miltenyi Biotech, clone DX11), phycoerythrin (PE)-conjugated mouse anti-human CD226 antibody (BioLegend, clone 11A8), or PE-conjugated mouse anti-human CD19 antibody (BD Pharmingen) according to the manufacturer’s protocol.

Techniques: Expressing, ChIP-sequencing, RNA Sequencing, Infection, Transduction, Plasmid Preparation, Control, Flow Cytometry, Quantitative RT-PCR, RNA Expression, Microarray

Journal: mSphere

Article Title: Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines

doi: 10.1128/mSphere.00305-17

Figure Lengend Snippet: Generation of sgCD226 mutant LCLs using the CRISPR/Cas9 system. (A) Short guide RNAs (sgRNAs) were designed to target coding sequences of the CD226 gene. (B) Genetic lesions in the CD226 gene resulting from Cas9 cleavage and subsequent error-prone DNA repair were sequenced. The guide target is in blue, PAM is in red, and insertions are in green. (C) CD226 mRNA levels from the wild-type (WT) LCL, Cas9-only expressing LCL (Cas9), sgAAVS1-transduced Cas9-expressing LCL (sgAAVS1), and ΔCD226 LCLs (sgCD226-1 and sgCD226-2) were assayed by qRT-PCR from three independent experiments. Data are shown as means ± SD. *, P < 0.05 by Student’s t test. (D) The same cell lines in panel C were assayed for CD226 surface expression by flow cytometry in three independent experiments. Data are shown as means ± SD. ****, P < 0.0001 by Student’s t test.

Article Snippet: For labeling, cells were incubated with allophycocyanin (APC)-conjugated mouse anti-human CD226 antibody (Miltenyi Biotech, clone DX11), phycoerythrin (PE)-conjugated mouse anti-human CD226 antibody (BioLegend, clone 11A8), or PE-conjugated mouse anti-human CD19 antibody (BD Pharmingen) according to the manufacturer’s protocol.

Techniques: Mutagenesis, CRISPR, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: mSphere

Article Title: Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines

doi: 10.1128/mSphere.00305-17

Figure Lengend Snippet: CD226 is not required for LCL proliferation or sensitivity to apoptosis relative to controls. (A) Growth of the WT LCL, sgAAVS1, and sgCD226-1 and -2 LCLs seeded at 200,000 cells/ml from three independent experiments. Data are shown as means ± SD. (B) The WT LCL, sgAAVS1, and sgCD226-1 and -2 LCLs were treated with Nutlin-3 (10 μM [two independent experiments]) or camptothecin (CPT; 15 μM [three independent experiments]) for 24 h. Apoptosis was quantified by flow cytometry for annexin V positivity. Data are reported as means ± SD. (C) Relative binding of WT, sgAAVS1, and sgCD226-1 and -2 LCLs to activated HUVECs from two independent experiments performed in triplicate. Data are reported as means ± SD. (D) Bright-field images of WT, sgAAVS1, and sgCD226-1 and -2 LCLs during culture (magnification, 40×).

Article Snippet: For labeling, cells were incubated with allophycocyanin (APC)-conjugated mouse anti-human CD226 antibody (Miltenyi Biotech, clone DX11), phycoerythrin (PE)-conjugated mouse anti-human CD226 antibody (BioLegend, clone 11A8), or PE-conjugated mouse anti-human CD19 antibody (BD Pharmingen) according to the manufacturer’s protocol.

Techniques: Flow Cytometry, Binding Assay