Journal: JCI Insight

Article Title: TET2 regulates immune tolerance in chronically activated mast cells

doi: 10.1172/jci.insight.154191

Figure Lengend Snippet: ( A ) Tet2 expression relative to Hprt1 in primary unstimulated Tet2 +/+ (control) and Tet2 +/+ KITD816V mast cells or Tet2 +/+ cells stimulated with 10 ng/mL IL4, IL6, or IL10 or 100 ng/mL IFNγ for 4 hours ( n = 4; mean ± SD; the means of all groups were significantly different by 1-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001 for Dunnett’s multiple-comparison test, each treatment versus control). ( B ) Heatmap of differentially methylated regions (DMRs) determined by eRRBS analysis of biological replicates of each cell population. The first lane of each sample is the sum of the 2 independent replicates shown in other 2 lanes, and it was used for clustering. ( C ) Venn diagram showing overlap (1 bp) of hypermethylated regions (HMR) in Tet2 +/+ versus Tet2 +/+ KITD816V , Tet2 –/– , or Tet2 –/– KITD816V mast cells and consistent between 2 independent biological replicates. ( D ) Histogram (left) shows DMR ranking according to H3K27ac ChIP-Seq signal. Dotted lines crossed by red slope indicate the threshold for high versus low signal subgroups. Histogram (right) shows the enrichment of DMRs associated to each genotype in either the high or low H3K27ac subgroup. Dotted line shows threshold, above which enrichment is considered statistically significant. ( E ) Heatmap showing results of gene ontology analysis for genes associated to HMR in Tet2 +/+ versus Tet2 +/+ KITD816V (lane 1), Tet2 –/– (lane 2), or Tet2 –/– KITD816V (lane 3) mast cells. Immune-related pathways are highlighted in red.

Article Snippet: We have used a model of somatic mutation of Tet2 for our studies, since in mastocytosis, clonal mutation of TET2 occurs during early hematopoiesis and KITD816V mutation is secondary ( , ).

Techniques: Expressing, Control, Comparison, Methylation, ChIP-sequencing

Journal: JCI Insight

Article Title: TET2 regulates immune tolerance in chronically activated mast cells

doi: 10.1172/jci.insight.154191

Figure Lengend Snippet: ( A ) Binary comparison of ChIP-Seq data by density scatterplot of log 2 mean H3K27ac counts reads. Samples being compared are labeled on the x and y axes. Data are based on mean ChIP-Seq peaks of n = 2 replicates per sample. ( B ) Aggregation plots showing H3K27ac normalized mean tag density in each sample at non-HMR and HMR identified in Tet2 –/– KITD816V cells. ( C ) Histogram showing results of gene ontology analysis for genes associated to differentially regulated H3K27ac signals in Tet2 +/+ KITD816V versus Tet2 –/– KITD816V mast cells. ( D ) Fold change in signal at immune gene loci from Cut&Run using H3K27ac-specific antibodies in Tet2 –/– KITD816V compared with Tet2 +/+ KITD816V mast cells. Each point is derived from 2 independent mast cell cultures for each genotype that were pooled at the time of assay. Graph show the combined results from 2 independent experiments (mean ± SEM). Immune genes also associated to HMR in Tet2 –/– KITD816V are highlighted in red. ( E ) Fold enrichment of quantitative PCR signal in STAT5 ChIP compared with ChIP using a nonspecific IgG antibody with primers directed against STAT5 binding sites at specific gene loci. Each point is derived from 2 independent mast cell cultures for each genotype that were pooled at the time of assay. Graph shows the combined results from 2 independent experiments (mean ± SEM).

Article Snippet: We have used a model of somatic mutation of Tet2 for our studies, since in mastocytosis, clonal mutation of TET2 occurs during early hematopoiesis and KITD816V mutation is secondary ( , ).

Techniques: Comparison, ChIP-sequencing, Labeling, Derivative Assay, Real-time Polymerase Chain Reaction, Binding Assay

Journal: JCI Insight

Article Title: TET2 regulates immune tolerance in chronically activated mast cells

doi: 10.1172/jci.insight.154191

Figure Lengend Snippet: ( A ) Heatmap of normalized RNA-Seq read counts used as signals for gene expression. The top 500 genes with highest variances among conditions were filtered in, and normalized counts were finally standardized as Z score. Samples and genes were clustered using Spearman and Pearson correlation, respectively. ( B ) Venn diagram showing the number of genes differentially expressed between Tet2 +/+ mast cells versus Tet2 +/+ KITD816V , Tet2 –/– , or Tet2 –/– KITD816V cells as determined by RNA-Seq and consistent between 2 independent biological replicates for each cell population. ( C ) Heatmap showing results of gene ontology analysis for genes differentially expressed in Tet2 +/+ versus Tet2 +/+ KITD816V (lane 1), Tet2 –/– (lane 2), or Tet2 –/– KITD816V (lane 3) mast cells. Immune-associated pathways are highlighted in red. ( D ) Radar plots of normalized enrichment scores (NES) from gene set enrichment analysis comparing genes downregulated (lower plot) or upregulated (upper plot) in Tet2 +/+ versus Tet2 +/+ KITD816V (gray line) or Tet2 –/– KITD816V (blue line) to genes enriched in indicated cell types compared with WT mast cells.

Article Snippet: We have used a model of somatic mutation of Tet2 for our studies, since in mastocytosis, clonal mutation of TET2 occurs during early hematopoiesis and KITD816V mutation is secondary ( , ).

Techniques: RNA Sequencing, Gene Expression

Journal: JCI Insight

Article Title: TET2 regulates immune tolerance in chronically activated mast cells

doi: 10.1172/jci.insight.154191

Figure Lengend Snippet: ( A ) Differentially expressed genes (FDR 5%, absolute [log 2 FC] > 1) between Tet2 +/+ KITD816V and Tet2 –/– KITD816V conditions were crossed with methylomics data from the same conditions. Methylation bin signals located at genes TSS (up to 1 kb upstream and downstream TSS) were filtered in and averaged per gene promoter. Methylation values were then categorized as 4 quartiles (Low [0,0.25], MidLow [0.25,0.5], MidHigh [0.5,0.75], High [0.75,1]). Bracketed numbers refer to quartile range. ( B ) As in A , but methylation values were categorized as 20% up (hypermethylated) or down (hypomethlated) between Tet2 +/+ KITD816V and Tet2 –/– KITD816V conditions. Genes absent from either of the data sets were discarded. ( C ) Heatmap of scaled gene expression measured by RNA-Seq for 2 groups of mast cell activation genes described in main text. Genes associated to HMR are highlighted in red.

Article Snippet: We have used a model of somatic mutation of Tet2 for our studies, since in mastocytosis, clonal mutation of TET2 occurs during early hematopoiesis and KITD816V mutation is secondary ( , ).

Techniques: Methylation, Gene Expression, RNA Sequencing, Activation Assay

Journal: JCI Insight

Article Title: TET2 regulates immune tolerance in chronically activated mast cells

doi: 10.1172/jci.insight.154191

Figure Lengend Snippet: ( A ) Schematic showing chronic mast cell activation assay. ( B ) Quantification of mean fluorescent intensity (MFI) of Cell Trace reagent (CFSE), gated on live BM cells after 6 days of culture in conditioned media derived from either Tet2 +/+ KITD816V or Tet2 –/– KITD816V mast cell cultures. P value for paired, 2-tailed t test is shown. ( C ) FACS analysis of BM at 3 days culture in conditioned media derived from either Tet2 +/+ KITD816V or Tet2 –/– KITD816V mast cell cultures. Panels show representative profiles gated on live, single-cell, CD11b + lymphocyte populations. ( D ) Graphs show quantification of C for a total of 8 paired analyses: n = 4 different starting BM preparations cultured with either Tet2 +/+ KITD816V or Tet2 –/– KITD816V mast cell conditioned media, n = 2 each. Results shown were reproduced 3 times in independent experiments. P values for paired, 2-tailed t test are associated to each plot. ( E ) Quantification of immune genes by qPCR, relative to Hprt1 , in total BM cultured with mast cell conditioned media for 24 hours. Each square shows mean expression for n = 4. Linear regression with 95% CI is shown. Slope is 0.81 ± 0.045 and shows a significant deviation from zero; P < 0.0001.

Article Snippet: We have used a model of somatic mutation of Tet2 for our studies, since in mastocytosis, clonal mutation of TET2 occurs during early hematopoiesis and KITD816V mutation is secondary ( , ).

Techniques: Activation Assay, Derivative Assay, Cell Culture, Expressing

Journal: JCI Insight

Article Title: TET2 regulates immune tolerance in chronically activated mast cells

doi: 10.1172/jci.insight.154191

Figure Lengend Snippet: ( A ) Schematic of acute mast cell activation assay. ( B ) FACS profiles for acute mast cell activation assay measuring TNF and IL6 levels by intracellular staining in response to IgE-OVA. Panels show representative profiles gated on live, single cells. ( C ) Quantification of B . Pooled results from 2 independent experiments are shown ( n = 4 each; mean ± SD; * P < 0.05, unpaired, 2-tailed t test). ( D ) Quantification of acute mast cell activation in control or in response to 10 ng/mL IL10 or 0.5 μg/mL LPS ( n = 4; mean ± SD; * P < 0.05, unpaired, 2-tailed t test). ( E ) Heatmap showing the mean fold change in expression ( n = 4, qPCR) of mast cell activation genes in response to 10 ng/mL IL10 or 0.5 μg/mL LPS compared with control, as in D . Genes associated to HMR in Tet2 –/– KITD816V cells are highlighted in red.

Article Snippet: We have used a model of somatic mutation of Tet2 for our studies, since in mastocytosis, clonal mutation of TET2 occurs during early hematopoiesis and KITD816V mutation is secondary ( , ).

Techniques: Activation Assay, Staining, Control, Expressing

Journal: JCI Insight

Article Title: TET2 regulates immune tolerance in chronically activated mast cells

doi: 10.1172/jci.insight.154191

Figure Lengend Snippet: ( A ) ChIP-qPCR analyses showing enrichment in signal in TET2 ChIP over ChIP using a nonspecific IgG antibody at HMR and at regions 4 kb upstream the HMR in Tet2 +/+ KITD816V (gray) and Tet2 –/– KITD816V cells (blue; negative control for TET2 antibody). Each point is derived from 3 independent mast cell cultures for each genotype that were pooled at the time of assay. Graphs show the combined results from 2 independent experiments (mean ± SEM). ( B ) Expression levels of genes relative to Hprt1 in untreated Tet2 +/+ KITD816V and Tet2 +/– KITD816V mast cells or after 24 hours of treatment with 250 μM vitamin C ( n = 3; mean ± SD; the means of all groups were significantly different by 1-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001 for Dunnett’s multiple-comparison test, each treatment versus control, or unpaired, 2-tailed t test to compare Tet2 +/– KITD816V with or without vitamin C. Only significant differences are shown in the figure). ( C ) Representative methylation profile of HMR associated regions in Tet2 +/+ KITD816V and Tet2 +/– KITD816V mast cells obtained by performing bisulfite sequencing. Samples were untreated or treated for 24 hours with 250 μM vitamin C. Black circles represent methylated CpG dinucleotide, while gray and white circles indicate partially methylated or unmethylated CpGs, respectively (mean methylation of n = 3). ( D ) Enrichment of 5hmC at HMR associated loci in Tet2 +/– KITD816V mast cells with or without vitamin C treatment (250 μM for 24 hours) evaluated by using hydroxy-methyl DNA immunoprecipitation (hMeDIP) ( n = 3; mean ± SD; * P < 0.05, ** P < 0.01 unpaired, 2-tailed t test). ( E ) Quantification analysis for acute mast cell activation assay measuring TNF and IL6 levels by intracellular staining. Cells were pretreated for 24 hours with 250 μM vitamin C, prior to acute mast cell activation assay using 10 ng/mL IL10 or 0.5 μg/mL LPS as in A. Fold change is relative to control for each cell population. Each point is derived from 3 independent mast cell cultures for each genotype that were pooled at the time of assay. Graph show the combined results from 2 independent experiments (mean ± SD; ** P < 0.01 unpaired, 2-tailed t test).

Article Snippet: We have used a model of somatic mutation of Tet2 for our studies, since in mastocytosis, clonal mutation of TET2 occurs during early hematopoiesis and KITD816V mutation is secondary ( , ).

Techniques: ChIP-qPCR, Negative Control, Derivative Assay, Expressing, Comparison, Control, Methylation, Methylation Sequencing, Immunoprecipitation, Activation Assay, Staining

Journal: JCI Insight

Article Title: TET2 regulates immune tolerance in chronically activated mast cells

doi: 10.1172/jci.insight.154191

Figure Lengend Snippet: ( A ) Tet2 expression relative to Hprt1 in primary unstimulated Tet2 +/+ (control) and Tet2 +/+ KITD816V mast cells or Tet2 +/+ cells stimulated with 10 ng/mL IL4, IL6, or IL10 or 100 ng/mL IFNγ for 4 hours ( n = 4; mean ± SD; the means of all groups were significantly different by 1-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001 for Dunnett’s multiple-comparison test, each treatment versus control). ( B ) Heatmap of differentially methylated regions (DMRs) determined by eRRBS analysis of biological replicates of each cell population. The first lane of each sample is the sum of the 2 independent replicates shown in other 2 lanes, and it was used for clustering. ( C ) Venn diagram showing overlap (1 bp) of hypermethylated regions (HMR) in Tet2 +/+ versus Tet2 +/+ KITD816V , Tet2 –/– , or Tet2 –/– KITD816V mast cells and consistent between 2 independent biological replicates. ( D ) Histogram (left) shows DMR ranking according to H3K27ac ChIP-Seq signal. Dotted lines crossed by red slope indicate the threshold for high versus low signal subgroups. Histogram (right) shows the enrichment of DMRs associated to each genotype in either the high or low H3K27ac subgroup. Dotted line shows threshold, above which enrichment is considered statistically significant. ( E ) Heatmap showing results of gene ontology analysis for genes associated to HMR in Tet2 +/+ versus Tet2 +/+ KITD816V (lane 1), Tet2 –/– (lane 2), or Tet2 –/– KITD816V (lane 3) mast cells. Immune-related pathways are highlighted in red.

Article Snippet: High variance and the rare number of fresh biopsies from patients with mastocytosis, TET2 mutation, and sufficient mast cell burden in the BM biopsies precluded statistical analysis of RNA-Seq on mast cells sorted from mastocytosis patients with KITD816V and TET2 mutation.

Techniques: Expressing, Control, Comparison, Methylation, ChIP-sequencing

Journal: JCI Insight

Article Title: TET2 regulates immune tolerance in chronically activated mast cells

doi: 10.1172/jci.insight.154191

Figure Lengend Snippet: ( A ) Binary comparison of ChIP-Seq data by density scatterplot of log 2 mean H3K27ac counts reads. Samples being compared are labeled on the x and y axes. Data are based on mean ChIP-Seq peaks of n = 2 replicates per sample. ( B ) Aggregation plots showing H3K27ac normalized mean tag density in each sample at non-HMR and HMR identified in Tet2 –/– KITD816V cells. ( C ) Histogram showing results of gene ontology analysis for genes associated to differentially regulated H3K27ac signals in Tet2 +/+ KITD816V versus Tet2 –/– KITD816V mast cells. ( D ) Fold change in signal at immune gene loci from Cut&Run using H3K27ac-specific antibodies in Tet2 –/– KITD816V compared with Tet2 +/+ KITD816V mast cells. Each point is derived from 2 independent mast cell cultures for each genotype that were pooled at the time of assay. Graph show the combined results from 2 independent experiments (mean ± SEM). Immune genes also associated to HMR in Tet2 –/– KITD816V are highlighted in red. ( E ) Fold enrichment of quantitative PCR signal in STAT5 ChIP compared with ChIP using a nonspecific IgG antibody with primers directed against STAT5 binding sites at specific gene loci. Each point is derived from 2 independent mast cell cultures for each genotype that were pooled at the time of assay. Graph shows the combined results from 2 independent experiments (mean ± SEM).

Article Snippet: High variance and the rare number of fresh biopsies from patients with mastocytosis, TET2 mutation, and sufficient mast cell burden in the BM biopsies precluded statistical analysis of RNA-Seq on mast cells sorted from mastocytosis patients with KITD816V and TET2 mutation.

Techniques: Comparison, ChIP-sequencing, Labeling, Derivative Assay, Real-time Polymerase Chain Reaction, Binding Assay

Journal: JCI Insight

Article Title: TET2 regulates immune tolerance in chronically activated mast cells

doi: 10.1172/jci.insight.154191

Figure Lengend Snippet: ( A ) Heatmap of normalized RNA-Seq read counts used as signals for gene expression. The top 500 genes with highest variances among conditions were filtered in, and normalized counts were finally standardized as Z score. Samples and genes were clustered using Spearman and Pearson correlation, respectively. ( B ) Venn diagram showing the number of genes differentially expressed between Tet2 +/+ mast cells versus Tet2 +/+ KITD816V , Tet2 –/– , or Tet2 –/– KITD816V cells as determined by RNA-Seq and consistent between 2 independent biological replicates for each cell population. ( C ) Heatmap showing results of gene ontology analysis for genes differentially expressed in Tet2 +/+ versus Tet2 +/+ KITD816V (lane 1), Tet2 –/– (lane 2), or Tet2 –/– KITD816V (lane 3) mast cells. Immune-associated pathways are highlighted in red. ( D ) Radar plots of normalized enrichment scores (NES) from gene set enrichment analysis comparing genes downregulated (lower plot) or upregulated (upper plot) in Tet2 +/+ versus Tet2 +/+ KITD816V (gray line) or Tet2 –/– KITD816V (blue line) to genes enriched in indicated cell types compared with WT mast cells.

Article Snippet: High variance and the rare number of fresh biopsies from patients with mastocytosis, TET2 mutation, and sufficient mast cell burden in the BM biopsies precluded statistical analysis of RNA-Seq on mast cells sorted from mastocytosis patients with KITD816V and TET2 mutation.

Techniques: RNA Sequencing, Gene Expression

Journal: JCI Insight

Article Title: TET2 regulates immune tolerance in chronically activated mast cells

doi: 10.1172/jci.insight.154191

Figure Lengend Snippet: ( A ) Differentially expressed genes (FDR 5%, absolute [log 2 FC] > 1) between Tet2 +/+ KITD816V and Tet2 –/– KITD816V conditions were crossed with methylomics data from the same conditions. Methylation bin signals located at genes TSS (up to 1 kb upstream and downstream TSS) were filtered in and averaged per gene promoter. Methylation values were then categorized as 4 quartiles (Low [0,0.25], MidLow [0.25,0.5], MidHigh [0.5,0.75], High [0.75,1]). Bracketed numbers refer to quartile range. ( B ) As in A , but methylation values were categorized as 20% up (hypermethylated) or down (hypomethlated) between Tet2 +/+ KITD816V and Tet2 –/– KITD816V conditions. Genes absent from either of the data sets were discarded. ( C ) Heatmap of scaled gene expression measured by RNA-Seq for 2 groups of mast cell activation genes described in main text. Genes associated to HMR are highlighted in red.

Article Snippet: High variance and the rare number of fresh biopsies from patients with mastocytosis, TET2 mutation, and sufficient mast cell burden in the BM biopsies precluded statistical analysis of RNA-Seq on mast cells sorted from mastocytosis patients with KITD816V and TET2 mutation.

Techniques: Methylation, Gene Expression, RNA Sequencing, Activation Assay

Journal: JCI Insight

Article Title: TET2 regulates immune tolerance in chronically activated mast cells

doi: 10.1172/jci.insight.154191

Figure Lengend Snippet: ( A ) Schematic showing chronic mast cell activation assay. ( B ) Quantification of mean fluorescent intensity (MFI) of Cell Trace reagent (CFSE), gated on live BM cells after 6 days of culture in conditioned media derived from either Tet2 +/+ KITD816V or Tet2 –/– KITD816V mast cell cultures. P value for paired, 2-tailed t test is shown. ( C ) FACS analysis of BM at 3 days culture in conditioned media derived from either Tet2 +/+ KITD816V or Tet2 –/– KITD816V mast cell cultures. Panels show representative profiles gated on live, single-cell, CD11b + lymphocyte populations. ( D ) Graphs show quantification of C for a total of 8 paired analyses: n = 4 different starting BM preparations cultured with either Tet2 +/+ KITD816V or Tet2 –/– KITD816V mast cell conditioned media, n = 2 each. Results shown were reproduced 3 times in independent experiments. P values for paired, 2-tailed t test are associated to each plot. ( E ) Quantification of immune genes by qPCR, relative to Hprt1 , in total BM cultured with mast cell conditioned media for 24 hours. Each square shows mean expression for n = 4. Linear regression with 95% CI is shown. Slope is 0.81 ± 0.045 and shows a significant deviation from zero; P < 0.0001.

Article Snippet: High variance and the rare number of fresh biopsies from patients with mastocytosis, TET2 mutation, and sufficient mast cell burden in the BM biopsies precluded statistical analysis of RNA-Seq on mast cells sorted from mastocytosis patients with KITD816V and TET2 mutation.

Techniques: Activation Assay, Derivative Assay, Cell Culture, Expressing

Journal: JCI Insight

Article Title: TET2 regulates immune tolerance in chronically activated mast cells

doi: 10.1172/jci.insight.154191

Figure Lengend Snippet: ( A ) Schematic of acute mast cell activation assay. ( B ) FACS profiles for acute mast cell activation assay measuring TNF and IL6 levels by intracellular staining in response to IgE-OVA. Panels show representative profiles gated on live, single cells. ( C ) Quantification of B . Pooled results from 2 independent experiments are shown ( n = 4 each; mean ± SD; * P < 0.05, unpaired, 2-tailed t test). ( D ) Quantification of acute mast cell activation in control or in response to 10 ng/mL IL10 or 0.5 μg/mL LPS ( n = 4; mean ± SD; * P < 0.05, unpaired, 2-tailed t test). ( E ) Heatmap showing the mean fold change in expression ( n = 4, qPCR) of mast cell activation genes in response to 10 ng/mL IL10 or 0.5 μg/mL LPS compared with control, as in D . Genes associated to HMR in Tet2 –/– KITD816V cells are highlighted in red.

Article Snippet: High variance and the rare number of fresh biopsies from patients with mastocytosis, TET2 mutation, and sufficient mast cell burden in the BM biopsies precluded statistical analysis of RNA-Seq on mast cells sorted from mastocytosis patients with KITD816V and TET2 mutation.

Techniques: Activation Assay, Staining, Control, Expressing

Journal: JCI Insight

Article Title: TET2 regulates immune tolerance in chronically activated mast cells

doi: 10.1172/jci.insight.154191

Figure Lengend Snippet: ( A ) ChIP-qPCR analyses showing enrichment in signal in TET2 ChIP over ChIP using a nonspecific IgG antibody at HMR and at regions 4 kb upstream the HMR in Tet2 +/+ KITD816V (gray) and Tet2 –/– KITD816V cells (blue; negative control for TET2 antibody). Each point is derived from 3 independent mast cell cultures for each genotype that were pooled at the time of assay. Graphs show the combined results from 2 independent experiments (mean ± SEM). ( B ) Expression levels of genes relative to Hprt1 in untreated Tet2 +/+ KITD816V and Tet2 +/– KITD816V mast cells or after 24 hours of treatment with 250 μM vitamin C ( n = 3; mean ± SD; the means of all groups were significantly different by 1-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001 for Dunnett’s multiple-comparison test, each treatment versus control, or unpaired, 2-tailed t test to compare Tet2 +/– KITD816V with or without vitamin C. Only significant differences are shown in the figure). ( C ) Representative methylation profile of HMR associated regions in Tet2 +/+ KITD816V and Tet2 +/– KITD816V mast cells obtained by performing bisulfite sequencing. Samples were untreated or treated for 24 hours with 250 μM vitamin C. Black circles represent methylated CpG dinucleotide, while gray and white circles indicate partially methylated or unmethylated CpGs, respectively (mean methylation of n = 3). ( D ) Enrichment of 5hmC at HMR associated loci in Tet2 +/– KITD816V mast cells with or without vitamin C treatment (250 μM for 24 hours) evaluated by using hydroxy-methyl DNA immunoprecipitation (hMeDIP) ( n = 3; mean ± SD; * P < 0.05, ** P < 0.01 unpaired, 2-tailed t test). ( E ) Quantification analysis for acute mast cell activation assay measuring TNF and IL6 levels by intracellular staining. Cells were pretreated for 24 hours with 250 μM vitamin C, prior to acute mast cell activation assay using 10 ng/mL IL10 or 0.5 μg/mL LPS as in A. Fold change is relative to control for each cell population. Each point is derived from 3 independent mast cell cultures for each genotype that were pooled at the time of assay. Graph show the combined results from 2 independent experiments (mean ± SD; ** P < 0.01 unpaired, 2-tailed t test).

Article Snippet: High variance and the rare number of fresh biopsies from patients with mastocytosis, TET2 mutation, and sufficient mast cell burden in the BM biopsies precluded statistical analysis of RNA-Seq on mast cells sorted from mastocytosis patients with KITD816V and TET2 mutation.

Techniques: ChIP-qPCR, Negative Control, Derivative Assay, Expressing, Comparison, Control, Methylation, Methylation Sequencing, Immunoprecipitation, Activation Assay, Staining