Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 7) or HD-PBMCs ( n = 7). ( B ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 9) and with mogamulizumab (antiCCR4) in a dose-dependent manner for 72 hours. ( C ) The comparison of RGMA mRNA gene expression levels using DNA microarray among normal CD4 + T cells (HD CD4 + : n = 4), HAM patient–derived CD4 + T cells (HAM CD4 + : n = 4), ACs ( n = 2), and smoldering/chronic-type-ATL patient–derived ( n = 3) HTLV-1–infected CD4 + T cells (Non-HAM infected CD4 + T cells: n = 5), and acute-type-ATL patient–derived HTLV-1–infected CD4 + T cells (Acute ATL infected cells: n = 3). ( D ) The comparison of the expression levels of the genes associated with the inhibition of neuroregeneration ( OMG , MAG , RTN4 , and WNT5A ) between HD CD4 + ( n = 4) and HAM CD4 + T cells ( n = 4). ( E ) The enrichment levels of H3K27me3 –2916 bp upstream from the TSS of the RGMA gene locus in HD CD4 + ( n = 3), HAM CD4 + ( n = 4), and acute-ATL infected cells ( n = 4). Data are shown as mean ± SD. ** P < 0.01; *** P < 0.001 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test ( B , C , and E ). NF-L, neurofilament light chain.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Concentration Assay, Comparison, Gene Expression, Microarray, Derivative Assay, Infection, Expressing, Inhibition

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The validation of RGMA mRNA gene expression levels using qRT-PCR in HD CD4 + ( n = 6) and HAM CD4 + T cells ( n = 6). ( B ) Expression of RGMa protein in CD3 + CD4 + CCR4 + T cells from HAM-PBMCs. Representative dot plots of CCR4 and normal goat IgG (upper) or RGMa expression (bottom) in CD3 + CD4 + gated cells from HD-PBMCs (left) or HAM-PBMCs (right) cultured for 2 days. ( C ) Graph shows the percentage of RGMa protein–expressing cells in CCR4 – cells or CCR4 + cells in CD3 + CD4 + gated cells from HAM-PBMCs ( n = 8) cultured for 2 days, compared with the isotype control, normal goat IgG. ( D ) Graph shows the percentage of RGMa protein–expressing cells among CD3 + CD4 + CCR4 + gated cells from HD-PBMCs ( n = 5) or HAM-PBMCs ( n = 8) cultured for 2 days. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Expressing, Cell Culture, Control, Comparison

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) Tax (left), HBZ (middle), and RGMA (right) gene expression levels in cultured HAM-PBMCs ( n = 7) in a time-dependent manner. RPL19 was used as an internal control. ( B ) Tax-dependent RGMA mRNA gene induction in Jurkat cells, which were infected with lentivirus carrying the Tax gene. Top: Tax expression in the Jurkat cells was confirmed by Western blotting. β-Actin was measured as an internal control. Bottom: The induction levels of the RGMA gene were evaluated by qRT-PCR in a time-dependent manner ( n = 3). ( C ) Tax -dependent RGMA mRNA gene induction in JPX9 cells treated with 20 μM CdCl 2 in a time-dependent manner. Tax mRNA (upper) and RGMA mRNA (bottom) were measured by qRT-PCR ( n = 3). GAPDH was measured as an internal control. ( D ) Tax-dependent RGMa protein induction in JPX9 cells treated with 20 μM CdCl 2 for 3 days. Dot plots of Tax and normal goat IgG (upper) or RGMa expression (bottom) in JPX9 cells. JPX9(-), untreated JPX9 cells; 20 μM CdCl 2 JPX9, CdCl 2 -supplemented JPX9 cells. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 by 1-way ANOVA with Dunnett’s multiple-comparison test ( A ), 2-sided Student’s t test ( B ), or an unpaired t test ( C ). Experiments were performed in triplicate ( B and C ).

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Gene Expression, Cell Culture, Control, Infection, Expressing, Western Blot, Quantitative RT-PCR, Comparison

Journal: Nature medicine

Article Title: Enhancer mapping uncovers phenotypic heterogeneity and evolution in patients with luminal breast cancer

doi: 10.1038/s41591-018-0091-x

Figure Lengend Snippet: A) Global Kaplan-Meier analysis summarize univariate analysis for 22278 genes included in the Affymetrix microarray platform. Hazard Ratios are plotted in the X axis B) SLC9A3R1 RNA levels pre- and post- short-term aromatase inhibitor treatment in responder and non-responder patients 61 . Oestrogen-dependent expression of progesterone receptor mRNA is shown as comparison C) Silencing SLC9A3R1 leads to proliferation arrest in response to estrogen stimulation in MCF7 and estrogen independent growth in LTED cells. Proliferation assays were conducted in biological triplicate. Symbol and error bars indicate average and 95% confidence intervals. Asterisks represent significance at P<0.05, 0.01, 0.001 and 0.0001 after two-way ANOVA with Bonferroni’s correction D) RIs for the SLC9A3R1 enhancer within all the individual patients included in the current study. SLC9A3R1 enhancer location and its 3D interactions are shown in the top right inset E) SLC9A3R1 enhancer ranking analysis of available Epigenome Roadmap H3K27ac datasets. Tissues are displayed from the strongest to the weakest SLC9A3R1 enhancer activity (based on RI). Representative IHC analysis of normal tissues stained with a SLC9A3R1 antibody are shown (Scale bars, 50 μm). F-G) YY1 and SLC9A3R1 IHC analysis of BC patients profiled using H3K27ac ChIP-seq. Predicted activity (RI) of YY and SLC9A3R1 enhancers is shown on the X axis. The number of cells positively stained for YY1 and SLC9A3R1 protein is indicated on the Y axis. Representative images are shown in the inbox. We stained one slide for each patient. Linear regression R square, confidence intervals and representative staining are also shown.

Article Snippet: For SLC9A3R1 (HPA9672 and HPA27247, Atlas Antibodies Cat#HPA009672, RRID:AB_1857215 and Atlas Antibodies Cat#HPA027247, RRID:AB_10601162 respectively) the following conditions were used.

Techniques: Microarray, Expressing, Comparison, Activity Assay, Staining, ChIP-sequencing

Journal: Nature medicine

Article Title: Enhancer mapping uncovers phenotypic heterogeneity and evolution in patients with luminal breast cancer

doi: 10.1038/s41591-018-0091-x

Figure Lengend Snippet: A) Theoretical framework of the analysis. The relative size of phenotypic clones can be tracked using enhancer ranking (RIs). Phenotypic clones can be positively or negatively selected during BC progression in response to endocrine therapies. B) Expanding or contracting phenotypic clones were defined based on the RI-ratio in primary and metastatic samples (RI P /RI M ). Distribution of RI-ratio shows that YY1 enhancers RI does not change significantly during progression compared to other enhancers, while SLC9A3R1 RI ranks among the enhancers with stronger increase in activity during progression. Vertical bars represent 1σ (Standard Deviation) increments from the population median C) Scatterplot of YY1 and SLC9A3R1 enhancer ranking according to patient stage. Bars indicate mean and 95% confidence intervals. Asterisks represent significance at P<0.05 after students two-tail T-Test D) IHC staining for YY1 and SLC9A3R1 positive cells in an independent matched longitudinal cohort of 22 ERα breast cancer patients (Scale bars, 100 μm). All normal and primaries are treatment naïve. All metastatic have received endocrine therapies (Tamoxifen or Aromatase inhibitors). Statistical significance was calculated using a pair-wise, two-tail T-test. Representative images are also shown E) Enhancer and promoter stratification based on frequency of usage in primary and metastatic patients. Percentages were calculated for each regulatory region for each stage (primary and metastatic) and differential was then derived and plotted on the X-axis. All enhancers and promoters called in were used. PE and ME were called by taking the top 1/1000 in the distribution that also satisfied a Fisher-exact test p<0.05. F) Dot-plot represent RI indexes for all PE (324) and ME (301) are plotted. As a control, RI for common enhancers (CE=320) were also plotted. Bottom plot: permutation was used to assess changes in RI in 50 randomly selected sets of 320 CE. Bow and whiskers represent median and 1-99 percentile for P-Value distribution. A Wilcoxon matched-pairs signed rank test was used to test for statistical significance G) Kaplan-Meier analysis using 1427 ERα-positive patients and averaged RNA expression of genes associated with PE or ME regulatory regions. Confidence interval for PE (0.39-0.61). Confidence interval for ME (1.1-1.67). Comparison of survival curves was performed using a Log-rank (Mantel-Cox) test. Genes were assigned considering CTCF insulated perimeters. Multivariate correction for the comparisons is also shown H) Pathway analysis for genes associated with PE or ME regulatory regions. Pathways were identified using GREAT and are listed in order of significance (symbols indicate qValue).

Article Snippet: For SLC9A3R1 (HPA9672 and HPA27247, Atlas Antibodies Cat#HPA009672, RRID:AB_1857215 and Atlas Antibodies Cat#HPA027247, RRID:AB_10601162 respectively) the following conditions were used.

Techniques: Clone Assay, Activity Assay, Standard Deviation, Immunohistochemistry, Derivative Assay, Control, RNA Expression, Comparison