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bap1 catalog 13271 antibodies  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc bap1 catalog 13271 antibodies
    ( A ) The protein levels of <t>BAP1</t> in 4 uveal melanoma cell lines MP41, MP38, MP46, and MP65 were determined by Western blot. Representative blot from 2 biological repeats. ( B ) The Venn diagram shows the overlap between WT and catalytically dead BAP1 rescued genes in BAP1-null MP65 cell line. ( C ) The track example shows the expression levels of MHC-II cluster genes in MP65 BAP1 null cells restored by GFP, BAP1-WT, or BAP1-C91S. ( D ) The mRNA levels of MHC-II cluster genes were determined by real-time PCR in MP41, MP38, MP46, and MP65 cells. n = 3 technical replicates. Data are represented as mean ± SD. ( E ) Pathway analysis was performed with the genes that are selectively upregulated by WT BAP1 but not catalytically dead BAP1 in BAP1-KO HEK293T cells. ( F ) The mRNA levels of MHC-II cluster genes were determined by RNA-Seq in BAP1-WT and catalytic-dead KI HEK293T cells. BAP1 was depleted by CRISPR with 2 distinct sgRNAs in MDA-MB-231, KNS42, and MDA-MB-435s cell lines. ( G ) The protein levels of BAP1 were determined by Western blot. Representative blot from 2 biological repeats. ( H ) The mRNA levels of MHC-II cluster genes HLA-DMA , HLA-DMB , HLA-DPA1 , and HLA-DRB1 were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD. ( I ) The track example shows the mRNA levels of MHC-II gene loci in BAP1-WT and BAP1-KO A20 cells. ( J ) The protein levels of cell-surface MHC-II molecule in BAP1-WT and -KO A20 cells were determined by FACS and analyzed by FlowJo software. ( K ) The protein levels of histone H2AK119Ub were determined by Western blot in both the WT A20 and 2 independent BAP1-KO clones. Representative blot from 2 biological repeats.
    Bap1 Catalog 13271 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/bap1+catalog+13271+antibodies/pmc11735100-187-29-36?v=Cell+Signaling+Technology+Inc
    Average 94 stars, based on 48 article reviews
    bap1 catalog 13271 antibodies - by Bioz Stars, 2026-07
    94/100 stars

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    1) Product Images from "An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models"

    Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI179703

    ( A ) The protein levels of BAP1 in 4 uveal melanoma cell lines MP41, MP38, MP46, and MP65 were determined by Western blot. Representative blot from 2 biological repeats. ( B ) The Venn diagram shows the overlap between WT and catalytically dead BAP1 rescued genes in BAP1-null MP65 cell line. ( C ) The track example shows the expression levels of MHC-II cluster genes in MP65 BAP1 null cells restored by GFP, BAP1-WT, or BAP1-C91S. ( D ) The mRNA levels of MHC-II cluster genes were determined by real-time PCR in MP41, MP38, MP46, and MP65 cells. n = 3 technical replicates. Data are represented as mean ± SD. ( E ) Pathway analysis was performed with the genes that are selectively upregulated by WT BAP1 but not catalytically dead BAP1 in BAP1-KO HEK293T cells. ( F ) The mRNA levels of MHC-II cluster genes were determined by RNA-Seq in BAP1-WT and catalytic-dead KI HEK293T cells. BAP1 was depleted by CRISPR with 2 distinct sgRNAs in MDA-MB-231, KNS42, and MDA-MB-435s cell lines. ( G ) The protein levels of BAP1 were determined by Western blot. Representative blot from 2 biological repeats. ( H ) The mRNA levels of MHC-II cluster genes HLA-DMA , HLA-DMB , HLA-DPA1 , and HLA-DRB1 were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD. ( I ) The track example shows the mRNA levels of MHC-II gene loci in BAP1-WT and BAP1-KO A20 cells. ( J ) The protein levels of cell-surface MHC-II molecule in BAP1-WT and -KO A20 cells were determined by FACS and analyzed by FlowJo software. ( K ) The protein levels of histone H2AK119Ub were determined by Western blot in both the WT A20 and 2 independent BAP1-KO clones. Representative blot from 2 biological repeats.
    Figure Legend Snippet: ( A ) The protein levels of BAP1 in 4 uveal melanoma cell lines MP41, MP38, MP46, and MP65 were determined by Western blot. Representative blot from 2 biological repeats. ( B ) The Venn diagram shows the overlap between WT and catalytically dead BAP1 rescued genes in BAP1-null MP65 cell line. ( C ) The track example shows the expression levels of MHC-II cluster genes in MP65 BAP1 null cells restored by GFP, BAP1-WT, or BAP1-C91S. ( D ) The mRNA levels of MHC-II cluster genes were determined by real-time PCR in MP41, MP38, MP46, and MP65 cells. n = 3 technical replicates. Data are represented as mean ± SD. ( E ) Pathway analysis was performed with the genes that are selectively upregulated by WT BAP1 but not catalytically dead BAP1 in BAP1-KO HEK293T cells. ( F ) The mRNA levels of MHC-II cluster genes were determined by RNA-Seq in BAP1-WT and catalytic-dead KI HEK293T cells. BAP1 was depleted by CRISPR with 2 distinct sgRNAs in MDA-MB-231, KNS42, and MDA-MB-435s cell lines. ( G ) The protein levels of BAP1 were determined by Western blot. Representative blot from 2 biological repeats. ( H ) The mRNA levels of MHC-II cluster genes HLA-DMA , HLA-DMB , HLA-DPA1 , and HLA-DRB1 were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD. ( I ) The track example shows the mRNA levels of MHC-II gene loci in BAP1-WT and BAP1-KO A20 cells. ( J ) The protein levels of cell-surface MHC-II molecule in BAP1-WT and -KO A20 cells were determined by FACS and analyzed by FlowJo software. ( K ) The protein levels of histone H2AK119Ub were determined by Western blot in both the WT A20 and 2 independent BAP1-KO clones. Representative blot from 2 biological repeats.

    Techniques Used: Western Blot, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing, CRISPR, Software, Clone Assay

    ( A ) The track example shows BAP1 occupancy at the MHC-II gene loci in MHC-II–positive cell line A20 cells and MHC-II–negative cell line LLC cells. ( B ) The heatmap shows the log 2 fold change of H2AK119Ub levels in BAP1-WT and 2 independent KO clones. All of the ChIP-Seq signals were centered on BAP1 peaks in BAP1-WT cells. ( C ) The track example shows the H2AK119Ub and H3K27me3 levels between BAP1-WT and -KO A20 cells (upper panel). The bottom panel shows the ATAC-Seq signal from both BAP1-WT and 2 independent KO clones at MHC-II gene loci. ( D ) Pathway analysis with the genes that are downregulated in BAP1-KO A20 cells. ( E ). The heatmap shows the log 2 fold change of UTX occupancy and H3K27me3 levels in BAP1-WT and -KO A20 cells centered on BAP1 peaks. ( F and G ) The track examples show the reduction of UTX occupancy and the increase of H3K27me3 levels at MHC-II cluster genes in BAP1-WT and -KO A20 cells.
    Figure Legend Snippet: ( A ) The track example shows BAP1 occupancy at the MHC-II gene loci in MHC-II–positive cell line A20 cells and MHC-II–negative cell line LLC cells. ( B ) The heatmap shows the log 2 fold change of H2AK119Ub levels in BAP1-WT and 2 independent KO clones. All of the ChIP-Seq signals were centered on BAP1 peaks in BAP1-WT cells. ( C ) The track example shows the H2AK119Ub and H3K27me3 levels between BAP1-WT and -KO A20 cells (upper panel). The bottom panel shows the ATAC-Seq signal from both BAP1-WT and 2 independent KO clones at MHC-II gene loci. ( D ) Pathway analysis with the genes that are downregulated in BAP1-KO A20 cells. ( E ). The heatmap shows the log 2 fold change of UTX occupancy and H3K27me3 levels in BAP1-WT and -KO A20 cells centered on BAP1 peaks. ( F and G ) The track examples show the reduction of UTX occupancy and the increase of H3K27me3 levels at MHC-II cluster genes in BAP1-WT and -KO A20 cells.

    Techniques Used: Clone Assay, ChIP-sequencing

    ( A ) The MA plot (Bland-Altman plot) shows the up- or downregulated ATAC-Seq signal comparing BAP1-WT/KO cells. ( B ) The motif analysis with the downregulated ATAC-Seq peaks in BAP1-KO cells. ( C ) The heatmap shows the log 2 fold change of IRF1 chromatin binding in BAP1-WT and -KO cells. ( D ) The track example shows the chromatin occupancy of BAP1 and the protein levels of H2AK119Ub and H3K27me3 at the Irf1 gene locus. ( E ) The mRNA levels of Irf1 in A20 BAP1-WT and -KO cells were determined by RNA-Seq. ( F ) The protein levels of IRF1 in A20 BAP1-WT and -KO cells were determined by Western blot. Representative blot from 2 biological repeats. ( G ) The track example shows the chromatin occupancy of IRF1 at the Ciita promoter in both BAP1-WT and -KO cells. ( H ) The occupancy of IRF1 at the Ciita promoter region was quantified by ChIP-QPCR in BAP1-WT and -KO cells. n = 3 technical replicates. Data are represented as mean ± SD. ( I ) The track example shows the Ciita levels in BAP1-WT and -KO cells. ( J ) The track example shows the occupancy of CIITA at MHC-II cluster gene loci in BAP1-WT and -KO cells. ( K ) The Venn diagram shows the genes that are rescued by IRF1 in BAP1-KO cells. ( L ) The protein levels of MHC-II molecules in BAP1-WT, BAP1-KO, BAP1-KO-GFP, and BAP1-KO-Flag-IRF1 cells were determined by FACS analysis. ( M ) The pathway analysis with the total upregulated genes by IRF1 in A20 BAP1-KO cells. The BAP1-KO A20 cells were transduced with either GFP, WT BAP1, or catalytically dead BAP1. ( N ) The protein levels of BAP1 were determined by Western blot. Representative blot from 2 biological repeats. ( O ) The mRNA levels of Irf1 and Ciita were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD.
    Figure Legend Snippet: ( A ) The MA plot (Bland-Altman plot) shows the up- or downregulated ATAC-Seq signal comparing BAP1-WT/KO cells. ( B ) The motif analysis with the downregulated ATAC-Seq peaks in BAP1-KO cells. ( C ) The heatmap shows the log 2 fold change of IRF1 chromatin binding in BAP1-WT and -KO cells. ( D ) The track example shows the chromatin occupancy of BAP1 and the protein levels of H2AK119Ub and H3K27me3 at the Irf1 gene locus. ( E ) The mRNA levels of Irf1 in A20 BAP1-WT and -KO cells were determined by RNA-Seq. ( F ) The protein levels of IRF1 in A20 BAP1-WT and -KO cells were determined by Western blot. Representative blot from 2 biological repeats. ( G ) The track example shows the chromatin occupancy of IRF1 at the Ciita promoter in both BAP1-WT and -KO cells. ( H ) The occupancy of IRF1 at the Ciita promoter region was quantified by ChIP-QPCR in BAP1-WT and -KO cells. n = 3 technical replicates. Data are represented as mean ± SD. ( I ) The track example shows the Ciita levels in BAP1-WT and -KO cells. ( J ) The track example shows the occupancy of CIITA at MHC-II cluster gene loci in BAP1-WT and -KO cells. ( K ) The Venn diagram shows the genes that are rescued by IRF1 in BAP1-KO cells. ( L ) The protein levels of MHC-II molecules in BAP1-WT, BAP1-KO, BAP1-KO-GFP, and BAP1-KO-Flag-IRF1 cells were determined by FACS analysis. ( M ) The pathway analysis with the total upregulated genes by IRF1 in A20 BAP1-KO cells. The BAP1-KO A20 cells were transduced with either GFP, WT BAP1, or catalytically dead BAP1. ( N ) The protein levels of BAP1 were determined by Western blot. Representative blot from 2 biological repeats. ( O ) The mRNA levels of Irf1 and Ciita were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD.

    Techniques Used: Binding Assay, RNA Sequencing, Western Blot, ChIP-qPCR, Transduction, Real-time Polymerase Chain Reaction

    ( A ) The A20 BAP1-KO cells were treated with various concentrations of PRC1 inhibitor RB-3 for 6 days. The protein levels of H2AK119Ub and RING1B were determined by Western blot. Representative blot from 2 biological repeats. ( B ) The log 2 fold change heatmap shows the H2AK119Ub levels in A20 BAP1-KO cells treated with either DMSO or RB-3. ( C ) The A20 BAP1-WT or -KO cells were treated with either DMSO or RB-3 for 6 days. The gene-expression profiles for each condition were determined by RNA-Seq. ( D ) The Venn diagram shows the overlap between BAP1 targeted genes and RB-3 rescued genes. ( E ) The pathway analysis with the 860 BAP1 targeted genes that were restored by RB-3 treatment. ( F ) The A20 BAP1-WT cells were treated with DMSO for 6 days, and the BAP1-KO cells were treated with either DMSO or RB-3 (5 μM) for 6 days. The mRNA levels of MHC-II cluster genes in each treatment were determined by RNA-Seq. ( G ) The BAP1-KO A20 cells were treated with either DMSO or RB-3 (10 μM) for 6 days. The protein levels of MHC-II were determined by FACS analysis. ( H ) The A20 BAP1-WT cells were treated with DMSO for 6 days, and the BAP1-KO cells were treated with either DMSO or various concentrations of RB-3 for 6 days. The IRF1 protein levels were determined by Western blot. Representative blot from 2 biological repeats. ( I and J ) The mRNA levels of Irf1 ( I ) and Ciita mRNA ( J ) were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD. ( K and L ) The BAP1-KO cells were treated with RB-3 (10 μM) or/and GSK126 (5 μM) for 6 days. The protein levels of H2AK119Ub and H3K27me3 were determined by Western blot. ( K ) Representative blot from 2 biological repeats. The protein levels of cell surface MHC-II molecules were determined by FACS analysis ( L ).
    Figure Legend Snippet: ( A ) The A20 BAP1-KO cells were treated with various concentrations of PRC1 inhibitor RB-3 for 6 days. The protein levels of H2AK119Ub and RING1B were determined by Western blot. Representative blot from 2 biological repeats. ( B ) The log 2 fold change heatmap shows the H2AK119Ub levels in A20 BAP1-KO cells treated with either DMSO or RB-3. ( C ) The A20 BAP1-WT or -KO cells were treated with either DMSO or RB-3 for 6 days. The gene-expression profiles for each condition were determined by RNA-Seq. ( D ) The Venn diagram shows the overlap between BAP1 targeted genes and RB-3 rescued genes. ( E ) The pathway analysis with the 860 BAP1 targeted genes that were restored by RB-3 treatment. ( F ) The A20 BAP1-WT cells were treated with DMSO for 6 days, and the BAP1-KO cells were treated with either DMSO or RB-3 (5 μM) for 6 days. The mRNA levels of MHC-II cluster genes in each treatment were determined by RNA-Seq. ( G ) The BAP1-KO A20 cells were treated with either DMSO or RB-3 (10 μM) for 6 days. The protein levels of MHC-II were determined by FACS analysis. ( H ) The A20 BAP1-WT cells were treated with DMSO for 6 days, and the BAP1-KO cells were treated with either DMSO or various concentrations of RB-3 for 6 days. The IRF1 protein levels were determined by Western blot. Representative blot from 2 biological repeats. ( I and J ) The mRNA levels of Irf1 ( I ) and Ciita mRNA ( J ) were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD. ( K and L ) The BAP1-KO cells were treated with RB-3 (10 μM) or/and GSK126 (5 μM) for 6 days. The protein levels of H2AK119Ub and H3K27me3 were determined by Western blot. ( K ) Representative blot from 2 biological repeats. The protein levels of cell surface MHC-II molecules were determined by FACS analysis ( L ).

    Techniques Used: Western Blot, Gene Expression, RNA Sequencing, Real-time Polymerase Chain Reaction

    ( A ) The cell growth rate of A20 BAP1-WT and -KO cells was determined by cell counting assay. ( B ) 2 × 10 6 of BAP1-WT and BAP1-KO clones were inoculated into the right flank of 6-week-old nude mice. The tumor size was measured every day for 1 week after inoculation. ( C ) Images of representative tumor tissue samples from each mouse were taken at the end of the experiment. ( D ) 5 × 10 6 of BAP1-WT and BAP1-KO clones were inoculated into the right flank of 6-week-old BALB/c mice. The tumor size was measured every day for 2 weeks after the inoculation. Data are represented as mean ± SEM. A 2-tailed unpaired Student’s t test was used for statistical analysis. ** P < 0.01; * P < 0.05. ( E ) When each tumor reached 1 cm 3 , the mouse was euthanized, and the survival probability was shown. ( F ) Tumor tissue from both BAP1-WT and BAP1-KO was harvested and subjected to scRNA-Seq. The UMAP analysis identifies different clusters of cell populations within the tumor tissue based on the gene-expression profiles. ( G ) The bar plot shows the percentage of each cell cluster in the tumor tissues. ( H ) The violin plot shows the expression levels of Irf1 , Ciita , and MHC-II cluster genes in BAP1-WT and -KO tumor cell population. ( I ) Immune cells from the first clustering were isolated (T cells and macrophages) and reclustered with UMAP algorithm. ( J ) The percentages of each type of immune cells were calculated in BAP1-WT and -KO A20 tumors and are shown in the bar plot. ( K ) The IHC staining was performed with CD4-specific antibody in BAP1-WT and -KO tumor tissues. Scale bars: 10 μm.
    Figure Legend Snippet: ( A ) The cell growth rate of A20 BAP1-WT and -KO cells was determined by cell counting assay. ( B ) 2 × 10 6 of BAP1-WT and BAP1-KO clones were inoculated into the right flank of 6-week-old nude mice. The tumor size was measured every day for 1 week after inoculation. ( C ) Images of representative tumor tissue samples from each mouse were taken at the end of the experiment. ( D ) 5 × 10 6 of BAP1-WT and BAP1-KO clones were inoculated into the right flank of 6-week-old BALB/c mice. The tumor size was measured every day for 2 weeks after the inoculation. Data are represented as mean ± SEM. A 2-tailed unpaired Student’s t test was used for statistical analysis. ** P < 0.01; * P < 0.05. ( E ) When each tumor reached 1 cm 3 , the mouse was euthanized, and the survival probability was shown. ( F ) Tumor tissue from both BAP1-WT and BAP1-KO was harvested and subjected to scRNA-Seq. The UMAP analysis identifies different clusters of cell populations within the tumor tissue based on the gene-expression profiles. ( G ) The bar plot shows the percentage of each cell cluster in the tumor tissues. ( H ) The violin plot shows the expression levels of Irf1 , Ciita , and MHC-II cluster genes in BAP1-WT and -KO tumor cell population. ( I ) Immune cells from the first clustering were isolated (T cells and macrophages) and reclustered with UMAP algorithm. ( J ) The percentages of each type of immune cells were calculated in BAP1-WT and -KO A20 tumors and are shown in the bar plot. ( K ) The IHC staining was performed with CD4-specific antibody in BAP1-WT and -KO tumor tissues. Scale bars: 10 μm.

    Techniques Used: Cell Counting, Clone Assay, Gene Expression, Expressing, Isolation, Immunohistochemistry

    ( A ) The box plot shows the Spearman’s correlation between the expression levels of human MHC-II genes and BAP1, EZH2, RNF2, PCGF6, or KDM2B in B cell lymphoma patients (TCGA, PanCancer Atlas). ( B – E ) The scatter plot shows the positive correlation between HLA-DMA and ( B ), EZH2 ( C ), PCGF6 ( D ), or KDM2B ( E ) in B cell lymphoma patient samples. ( F ) The schematic model shows how the BAP1 complex and the PRC1 establish a dynamic epigenetic state at MHC-II cluster gene loci by precisely regulating histone H2AK119Ub level and IRF1/CIITA axis which is critical for MHC-II gene expression. ( G ) The epigenetic balance between BAP1 and PRC1 plays an essential role in “hot” and “cold” tumor switching and tumor immune response.
    Figure Legend Snippet: ( A ) The box plot shows the Spearman’s correlation between the expression levels of human MHC-II genes and BAP1, EZH2, RNF2, PCGF6, or KDM2B in B cell lymphoma patients (TCGA, PanCancer Atlas). ( B – E ) The scatter plot shows the positive correlation between HLA-DMA and ( B ), EZH2 ( C ), PCGF6 ( D ), or KDM2B ( E ) in B cell lymphoma patient samples. ( F ) The schematic model shows how the BAP1 complex and the PRC1 establish a dynamic epigenetic state at MHC-II cluster gene loci by precisely regulating histone H2AK119Ub level and IRF1/CIITA axis which is critical for MHC-II gene expression. ( G ) The epigenetic balance between BAP1 and PRC1 plays an essential role in “hot” and “cold” tumor switching and tumor immune response.

    Techniques Used: Expressing, Gene Expression



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    bap1 catalog 13271 antibodies  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc bap1 catalog 13271 antibodies
    ( A ) The protein levels of <t>BAP1</t> in 4 uveal melanoma cell lines MP41, MP38, MP46, and MP65 were determined by Western blot. Representative blot from 2 biological repeats. ( B ) The Venn diagram shows the overlap between WT and catalytically dead BAP1 rescued genes in BAP1-null MP65 cell line. ( C ) The track example shows the expression levels of MHC-II cluster genes in MP65 BAP1 null cells restored by GFP, BAP1-WT, or BAP1-C91S. ( D ) The mRNA levels of MHC-II cluster genes were determined by real-time PCR in MP41, MP38, MP46, and MP65 cells. n = 3 technical replicates. Data are represented as mean ± SD. ( E ) Pathway analysis was performed with the genes that are selectively upregulated by WT BAP1 but not catalytically dead BAP1 in BAP1-KO HEK293T cells. ( F ) The mRNA levels of MHC-II cluster genes were determined by RNA-Seq in BAP1-WT and catalytic-dead KI HEK293T cells. BAP1 was depleted by CRISPR with 2 distinct sgRNAs in MDA-MB-231, KNS42, and MDA-MB-435s cell lines. ( G ) The protein levels of BAP1 were determined by Western blot. Representative blot from 2 biological repeats. ( H ) The mRNA levels of MHC-II cluster genes HLA-DMA , HLA-DMB , HLA-DPA1 , and HLA-DRB1 were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD. ( I ) The track example shows the mRNA levels of MHC-II gene loci in BAP1-WT and BAP1-KO A20 cells. ( J ) The protein levels of cell-surface MHC-II molecule in BAP1-WT and -KO A20 cells were determined by FACS and analyzed by FlowJo software. ( K ) The protein levels of histone H2AK119Ub were determined by Western blot in both the WT A20 and 2 independent BAP1-KO clones. Representative blot from 2 biological repeats.
    Bap1 Catalog 13271 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/bap1+catalog+13271+antibodies/pmc11735100-187-29-36?v=Cell+Signaling+Technology+Inc
    Average 94 stars, based on 1 article reviews
    bap1 catalog 13271 antibodies - by Bioz Stars, 2026-07
    94/100 stars
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    ( A ) The protein levels of BAP1 in 4 uveal melanoma cell lines MP41, MP38, MP46, and MP65 were determined by Western blot. Representative blot from 2 biological repeats. ( B ) The Venn diagram shows the overlap between WT and catalytically dead BAP1 rescued genes in BAP1-null MP65 cell line. ( C ) The track example shows the expression levels of MHC-II cluster genes in MP65 BAP1 null cells restored by GFP, BAP1-WT, or BAP1-C91S. ( D ) The mRNA levels of MHC-II cluster genes were determined by real-time PCR in MP41, MP38, MP46, and MP65 cells. n = 3 technical replicates. Data are represented as mean ± SD. ( E ) Pathway analysis was performed with the genes that are selectively upregulated by WT BAP1 but not catalytically dead BAP1 in BAP1-KO HEK293T cells. ( F ) The mRNA levels of MHC-II cluster genes were determined by RNA-Seq in BAP1-WT and catalytic-dead KI HEK293T cells. BAP1 was depleted by CRISPR with 2 distinct sgRNAs in MDA-MB-231, KNS42, and MDA-MB-435s cell lines. ( G ) The protein levels of BAP1 were determined by Western blot. Representative blot from 2 biological repeats. ( H ) The mRNA levels of MHC-II cluster genes HLA-DMA , HLA-DMB , HLA-DPA1 , and HLA-DRB1 were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD. ( I ) The track example shows the mRNA levels of MHC-II gene loci in BAP1-WT and BAP1-KO A20 cells. ( J ) The protein levels of cell-surface MHC-II molecule in BAP1-WT and -KO A20 cells were determined by FACS and analyzed by FlowJo software. ( K ) The protein levels of histone H2AK119Ub were determined by Western blot in both the WT A20 and 2 independent BAP1-KO clones. Representative blot from 2 biological repeats.

    Journal: The Journal of Clinical Investigation

    Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

    doi: 10.1172/JCI179703

    Figure Lengend Snippet: ( A ) The protein levels of BAP1 in 4 uveal melanoma cell lines MP41, MP38, MP46, and MP65 were determined by Western blot. Representative blot from 2 biological repeats. ( B ) The Venn diagram shows the overlap between WT and catalytically dead BAP1 rescued genes in BAP1-null MP65 cell line. ( C ) The track example shows the expression levels of MHC-II cluster genes in MP65 BAP1 null cells restored by GFP, BAP1-WT, or BAP1-C91S. ( D ) The mRNA levels of MHC-II cluster genes were determined by real-time PCR in MP41, MP38, MP46, and MP65 cells. n = 3 technical replicates. Data are represented as mean ± SD. ( E ) Pathway analysis was performed with the genes that are selectively upregulated by WT BAP1 but not catalytically dead BAP1 in BAP1-KO HEK293T cells. ( F ) The mRNA levels of MHC-II cluster genes were determined by RNA-Seq in BAP1-WT and catalytic-dead KI HEK293T cells. BAP1 was depleted by CRISPR with 2 distinct sgRNAs in MDA-MB-231, KNS42, and MDA-MB-435s cell lines. ( G ) The protein levels of BAP1 were determined by Western blot. Representative blot from 2 biological repeats. ( H ) The mRNA levels of MHC-II cluster genes HLA-DMA , HLA-DMB , HLA-DPA1 , and HLA-DRB1 were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD. ( I ) The track example shows the mRNA levels of MHC-II gene loci in BAP1-WT and BAP1-KO A20 cells. ( J ) The protein levels of cell-surface MHC-II molecule in BAP1-WT and -KO A20 cells were determined by FACS and analyzed by FlowJo software. ( K ) The protein levels of histone H2AK119Ub were determined by Western blot in both the WT A20 and 2 independent BAP1-KO clones. Representative blot from 2 biological repeats.

    Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

    Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, RNA Sequencing, CRISPR, Software, Clone Assay

    ( A ) The track example shows BAP1 occupancy at the MHC-II gene loci in MHC-II–positive cell line A20 cells and MHC-II–negative cell line LLC cells. ( B ) The heatmap shows the log 2 fold change of H2AK119Ub levels in BAP1-WT and 2 independent KO clones. All of the ChIP-Seq signals were centered on BAP1 peaks in BAP1-WT cells. ( C ) The track example shows the H2AK119Ub and H3K27me3 levels between BAP1-WT and -KO A20 cells (upper panel). The bottom panel shows the ATAC-Seq signal from both BAP1-WT and 2 independent KO clones at MHC-II gene loci. ( D ) Pathway analysis with the genes that are downregulated in BAP1-KO A20 cells. ( E ). The heatmap shows the log 2 fold change of UTX occupancy and H3K27me3 levels in BAP1-WT and -KO A20 cells centered on BAP1 peaks. ( F and G ) The track examples show the reduction of UTX occupancy and the increase of H3K27me3 levels at MHC-II cluster genes in BAP1-WT and -KO A20 cells.

    Journal: The Journal of Clinical Investigation

    Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

    doi: 10.1172/JCI179703

    Figure Lengend Snippet: ( A ) The track example shows BAP1 occupancy at the MHC-II gene loci in MHC-II–positive cell line A20 cells and MHC-II–negative cell line LLC cells. ( B ) The heatmap shows the log 2 fold change of H2AK119Ub levels in BAP1-WT and 2 independent KO clones. All of the ChIP-Seq signals were centered on BAP1 peaks in BAP1-WT cells. ( C ) The track example shows the H2AK119Ub and H3K27me3 levels between BAP1-WT and -KO A20 cells (upper panel). The bottom panel shows the ATAC-Seq signal from both BAP1-WT and 2 independent KO clones at MHC-II gene loci. ( D ) Pathway analysis with the genes that are downregulated in BAP1-KO A20 cells. ( E ). The heatmap shows the log 2 fold change of UTX occupancy and H3K27me3 levels in BAP1-WT and -KO A20 cells centered on BAP1 peaks. ( F and G ) The track examples show the reduction of UTX occupancy and the increase of H3K27me3 levels at MHC-II cluster genes in BAP1-WT and -KO A20 cells.

    Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

    Techniques: Clone Assay, ChIP-sequencing

    ( A ) The MA plot (Bland-Altman plot) shows the up- or downregulated ATAC-Seq signal comparing BAP1-WT/KO cells. ( B ) The motif analysis with the downregulated ATAC-Seq peaks in BAP1-KO cells. ( C ) The heatmap shows the log 2 fold change of IRF1 chromatin binding in BAP1-WT and -KO cells. ( D ) The track example shows the chromatin occupancy of BAP1 and the protein levels of H2AK119Ub and H3K27me3 at the Irf1 gene locus. ( E ) The mRNA levels of Irf1 in A20 BAP1-WT and -KO cells were determined by RNA-Seq. ( F ) The protein levels of IRF1 in A20 BAP1-WT and -KO cells were determined by Western blot. Representative blot from 2 biological repeats. ( G ) The track example shows the chromatin occupancy of IRF1 at the Ciita promoter in both BAP1-WT and -KO cells. ( H ) The occupancy of IRF1 at the Ciita promoter region was quantified by ChIP-QPCR in BAP1-WT and -KO cells. n = 3 technical replicates. Data are represented as mean ± SD. ( I ) The track example shows the Ciita levels in BAP1-WT and -KO cells. ( J ) The track example shows the occupancy of CIITA at MHC-II cluster gene loci in BAP1-WT and -KO cells. ( K ) The Venn diagram shows the genes that are rescued by IRF1 in BAP1-KO cells. ( L ) The protein levels of MHC-II molecules in BAP1-WT, BAP1-KO, BAP1-KO-GFP, and BAP1-KO-Flag-IRF1 cells were determined by FACS analysis. ( M ) The pathway analysis with the total upregulated genes by IRF1 in A20 BAP1-KO cells. The BAP1-KO A20 cells were transduced with either GFP, WT BAP1, or catalytically dead BAP1. ( N ) The protein levels of BAP1 were determined by Western blot. Representative blot from 2 biological repeats. ( O ) The mRNA levels of Irf1 and Ciita were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD.

    Journal: The Journal of Clinical Investigation

    Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

    doi: 10.1172/JCI179703

    Figure Lengend Snippet: ( A ) The MA plot (Bland-Altman plot) shows the up- or downregulated ATAC-Seq signal comparing BAP1-WT/KO cells. ( B ) The motif analysis with the downregulated ATAC-Seq peaks in BAP1-KO cells. ( C ) The heatmap shows the log 2 fold change of IRF1 chromatin binding in BAP1-WT and -KO cells. ( D ) The track example shows the chromatin occupancy of BAP1 and the protein levels of H2AK119Ub and H3K27me3 at the Irf1 gene locus. ( E ) The mRNA levels of Irf1 in A20 BAP1-WT and -KO cells were determined by RNA-Seq. ( F ) The protein levels of IRF1 in A20 BAP1-WT and -KO cells were determined by Western blot. Representative blot from 2 biological repeats. ( G ) The track example shows the chromatin occupancy of IRF1 at the Ciita promoter in both BAP1-WT and -KO cells. ( H ) The occupancy of IRF1 at the Ciita promoter region was quantified by ChIP-QPCR in BAP1-WT and -KO cells. n = 3 technical replicates. Data are represented as mean ± SD. ( I ) The track example shows the Ciita levels in BAP1-WT and -KO cells. ( J ) The track example shows the occupancy of CIITA at MHC-II cluster gene loci in BAP1-WT and -KO cells. ( K ) The Venn diagram shows the genes that are rescued by IRF1 in BAP1-KO cells. ( L ) The protein levels of MHC-II molecules in BAP1-WT, BAP1-KO, BAP1-KO-GFP, and BAP1-KO-Flag-IRF1 cells were determined by FACS analysis. ( M ) The pathway analysis with the total upregulated genes by IRF1 in A20 BAP1-KO cells. The BAP1-KO A20 cells were transduced with either GFP, WT BAP1, or catalytically dead BAP1. ( N ) The protein levels of BAP1 were determined by Western blot. Representative blot from 2 biological repeats. ( O ) The mRNA levels of Irf1 and Ciita were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD.

    Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

    Techniques: Binding Assay, RNA Sequencing, Western Blot, ChIP-qPCR, Transduction, Real-time Polymerase Chain Reaction

    ( A ) The A20 BAP1-KO cells were treated with various concentrations of PRC1 inhibitor RB-3 for 6 days. The protein levels of H2AK119Ub and RING1B were determined by Western blot. Representative blot from 2 biological repeats. ( B ) The log 2 fold change heatmap shows the H2AK119Ub levels in A20 BAP1-KO cells treated with either DMSO or RB-3. ( C ) The A20 BAP1-WT or -KO cells were treated with either DMSO or RB-3 for 6 days. The gene-expression profiles for each condition were determined by RNA-Seq. ( D ) The Venn diagram shows the overlap between BAP1 targeted genes and RB-3 rescued genes. ( E ) The pathway analysis with the 860 BAP1 targeted genes that were restored by RB-3 treatment. ( F ) The A20 BAP1-WT cells were treated with DMSO for 6 days, and the BAP1-KO cells were treated with either DMSO or RB-3 (5 μM) for 6 days. The mRNA levels of MHC-II cluster genes in each treatment were determined by RNA-Seq. ( G ) The BAP1-KO A20 cells were treated with either DMSO or RB-3 (10 μM) for 6 days. The protein levels of MHC-II were determined by FACS analysis. ( H ) The A20 BAP1-WT cells were treated with DMSO for 6 days, and the BAP1-KO cells were treated with either DMSO or various concentrations of RB-3 for 6 days. The IRF1 protein levels were determined by Western blot. Representative blot from 2 biological repeats. ( I and J ) The mRNA levels of Irf1 ( I ) and Ciita mRNA ( J ) were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD. ( K and L ) The BAP1-KO cells were treated with RB-3 (10 μM) or/and GSK126 (5 μM) for 6 days. The protein levels of H2AK119Ub and H3K27me3 were determined by Western blot. ( K ) Representative blot from 2 biological repeats. The protein levels of cell surface MHC-II molecules were determined by FACS analysis ( L ).

    Journal: The Journal of Clinical Investigation

    Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

    doi: 10.1172/JCI179703

    Figure Lengend Snippet: ( A ) The A20 BAP1-KO cells were treated with various concentrations of PRC1 inhibitor RB-3 for 6 days. The protein levels of H2AK119Ub and RING1B were determined by Western blot. Representative blot from 2 biological repeats. ( B ) The log 2 fold change heatmap shows the H2AK119Ub levels in A20 BAP1-KO cells treated with either DMSO or RB-3. ( C ) The A20 BAP1-WT or -KO cells were treated with either DMSO or RB-3 for 6 days. The gene-expression profiles for each condition were determined by RNA-Seq. ( D ) The Venn diagram shows the overlap between BAP1 targeted genes and RB-3 rescued genes. ( E ) The pathway analysis with the 860 BAP1 targeted genes that were restored by RB-3 treatment. ( F ) The A20 BAP1-WT cells were treated with DMSO for 6 days, and the BAP1-KO cells were treated with either DMSO or RB-3 (5 μM) for 6 days. The mRNA levels of MHC-II cluster genes in each treatment were determined by RNA-Seq. ( G ) The BAP1-KO A20 cells were treated with either DMSO or RB-3 (10 μM) for 6 days. The protein levels of MHC-II were determined by FACS analysis. ( H ) The A20 BAP1-WT cells were treated with DMSO for 6 days, and the BAP1-KO cells were treated with either DMSO or various concentrations of RB-3 for 6 days. The IRF1 protein levels were determined by Western blot. Representative blot from 2 biological repeats. ( I and J ) The mRNA levels of Irf1 ( I ) and Ciita mRNA ( J ) were determined by real-time PCR. n = 3 technical replicates. Data are represented as mean ± SD. ( K and L ) The BAP1-KO cells were treated with RB-3 (10 μM) or/and GSK126 (5 μM) for 6 days. The protein levels of H2AK119Ub and H3K27me3 were determined by Western blot. ( K ) Representative blot from 2 biological repeats. The protein levels of cell surface MHC-II molecules were determined by FACS analysis ( L ).

    Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

    Techniques: Western Blot, Gene Expression, RNA Sequencing, Real-time Polymerase Chain Reaction

    ( A ) The cell growth rate of A20 BAP1-WT and -KO cells was determined by cell counting assay. ( B ) 2 × 10 6 of BAP1-WT and BAP1-KO clones were inoculated into the right flank of 6-week-old nude mice. The tumor size was measured every day for 1 week after inoculation. ( C ) Images of representative tumor tissue samples from each mouse were taken at the end of the experiment. ( D ) 5 × 10 6 of BAP1-WT and BAP1-KO clones were inoculated into the right flank of 6-week-old BALB/c mice. The tumor size was measured every day for 2 weeks after the inoculation. Data are represented as mean ± SEM. A 2-tailed unpaired Student’s t test was used for statistical analysis. ** P < 0.01; * P < 0.05. ( E ) When each tumor reached 1 cm 3 , the mouse was euthanized, and the survival probability was shown. ( F ) Tumor tissue from both BAP1-WT and BAP1-KO was harvested and subjected to scRNA-Seq. The UMAP analysis identifies different clusters of cell populations within the tumor tissue based on the gene-expression profiles. ( G ) The bar plot shows the percentage of each cell cluster in the tumor tissues. ( H ) The violin plot shows the expression levels of Irf1 , Ciita , and MHC-II cluster genes in BAP1-WT and -KO tumor cell population. ( I ) Immune cells from the first clustering were isolated (T cells and macrophages) and reclustered with UMAP algorithm. ( J ) The percentages of each type of immune cells were calculated in BAP1-WT and -KO A20 tumors and are shown in the bar plot. ( K ) The IHC staining was performed with CD4-specific antibody in BAP1-WT and -KO tumor tissues. Scale bars: 10 μm.

    Journal: The Journal of Clinical Investigation

    Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

    doi: 10.1172/JCI179703

    Figure Lengend Snippet: ( A ) The cell growth rate of A20 BAP1-WT and -KO cells was determined by cell counting assay. ( B ) 2 × 10 6 of BAP1-WT and BAP1-KO clones were inoculated into the right flank of 6-week-old nude mice. The tumor size was measured every day for 1 week after inoculation. ( C ) Images of representative tumor tissue samples from each mouse were taken at the end of the experiment. ( D ) 5 × 10 6 of BAP1-WT and BAP1-KO clones were inoculated into the right flank of 6-week-old BALB/c mice. The tumor size was measured every day for 2 weeks after the inoculation. Data are represented as mean ± SEM. A 2-tailed unpaired Student’s t test was used for statistical analysis. ** P < 0.01; * P < 0.05. ( E ) When each tumor reached 1 cm 3 , the mouse was euthanized, and the survival probability was shown. ( F ) Tumor tissue from both BAP1-WT and BAP1-KO was harvested and subjected to scRNA-Seq. The UMAP analysis identifies different clusters of cell populations within the tumor tissue based on the gene-expression profiles. ( G ) The bar plot shows the percentage of each cell cluster in the tumor tissues. ( H ) The violin plot shows the expression levels of Irf1 , Ciita , and MHC-II cluster genes in BAP1-WT and -KO tumor cell population. ( I ) Immune cells from the first clustering were isolated (T cells and macrophages) and reclustered with UMAP algorithm. ( J ) The percentages of each type of immune cells were calculated in BAP1-WT and -KO A20 tumors and are shown in the bar plot. ( K ) The IHC staining was performed with CD4-specific antibody in BAP1-WT and -KO tumor tissues. Scale bars: 10 μm.

    Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

    Techniques: Cell Counting, Clone Assay, Gene Expression, Expressing, Isolation, Immunohistochemistry

    ( A ) The box plot shows the Spearman’s correlation between the expression levels of human MHC-II genes and BAP1, EZH2, RNF2, PCGF6, or KDM2B in B cell lymphoma patients (TCGA, PanCancer Atlas). ( B – E ) The scatter plot shows the positive correlation between HLA-DMA and ( B ), EZH2 ( C ), PCGF6 ( D ), or KDM2B ( E ) in B cell lymphoma patient samples. ( F ) The schematic model shows how the BAP1 complex and the PRC1 establish a dynamic epigenetic state at MHC-II cluster gene loci by precisely regulating histone H2AK119Ub level and IRF1/CIITA axis which is critical for MHC-II gene expression. ( G ) The epigenetic balance between BAP1 and PRC1 plays an essential role in “hot” and “cold” tumor switching and tumor immune response.

    Journal: The Journal of Clinical Investigation

    Article Title: An epigenetic pathway regulates MHC-II expression and function in B cell lymphoma models

    doi: 10.1172/JCI179703

    Figure Lengend Snippet: ( A ) The box plot shows the Spearman’s correlation between the expression levels of human MHC-II genes and BAP1, EZH2, RNF2, PCGF6, or KDM2B in B cell lymphoma patients (TCGA, PanCancer Atlas). ( B – E ) The scatter plot shows the positive correlation between HLA-DMA and ( B ), EZH2 ( C ), PCGF6 ( D ), or KDM2B ( E ) in B cell lymphoma patient samples. ( F ) The schematic model shows how the BAP1 complex and the PRC1 establish a dynamic epigenetic state at MHC-II cluster gene loci by precisely regulating histone H2AK119Ub level and IRF1/CIITA axis which is critical for MHC-II gene expression. ( G ) The epigenetic balance between BAP1 and PRC1 plays an essential role in “hot” and “cold” tumor switching and tumor immune response.

    Article Snippet: H3K27me3 (catalog 9733), Histone H3 (catalog 4499S), H2AK119Ub (catalog 8240), IRF1 (catalog 8478S), RING1B (catalog 5694S), UTX (catalog 33510S), CD4 (catalog 25229S), MHC-II-FITC (catalog 42594S), MHC-II-APC (catalog 64776), and BAP1 (catalog 13271) antibodies were purchased from Cell Signaling Technology.

    Techniques: Expressing, Gene Expression