anti bcl11a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti bcl11a
    Loss of Zbtb1 and Cbfa2t3 leads to the T-lineage differentiation arrest in LPs. A , specific depletion of targeted Lmo2, Zbtb1, and Cbfa2t3 proteins. sgRNA against Lmo2 , Zbtb1 , Cbfa2t2 , or Cbfa2t3 was introduced into the Cas9-expressing (GFP + ) LPs. Four days after sgRNA transduction, nuclear lysates from retrovirus infected GFP + hNGFR + cells were subjected to immunoblotting for Lmo2, Zbtb1, Cbfa2t3, <t>Bcl11a,</t> and LaminB. Two independent experiments were performed with similar results. B , an experimental scheme for the deletion of Lmo2 , Zbtb1 , Cbfa2t3 , and Cbfa2t2 using the CRISPR/Cas9 system in Ebf1 -deficient LPs is shown. C , retroviral vectors encoding sgRNAs against luciferase (sgControl), Lmo2 (sgLmo2), Zbtb1 (sgZbtb1), Cbfa2t3 (sgCbfa2t3), or Cbfa2t2 (sgCbfa2t2) were introduced into Cas9-expressing (GFP + ) LPs. Five days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA transduced cells were gated and analyzed for CD44 and CD25 expression. D , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( C ) is shown with standard deviation (SD). E , ten days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. F , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( E ) is shown with SD. Data are representative of two ( A ) or three ( C and E ) independent experiments. Data represent the mean values of three independent biological replicates ( D and F ). ∗∗ p < 0.01 by two-sided Student’s t test. LP, lymphoid progenitor; sgRNA, single-guide RNA.
    Anti Bcl11a, 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/result/anti bcl11a/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
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    1) Product Images from "Transcription factor Zbtb1 interacts with bridging factor Lmo2 and maintains the T-lineage differentiation capacity of lymphoid progenitor cells"

    Article Title: Transcription factor Zbtb1 interacts with bridging factor Lmo2 and maintains the T-lineage differentiation capacity of lymphoid progenitor cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2022.102506

    Loss of Zbtb1 and Cbfa2t3 leads to the T-lineage differentiation arrest in LPs. A , specific depletion of targeted Lmo2, Zbtb1, and Cbfa2t3 proteins. sgRNA against Lmo2 , Zbtb1 , Cbfa2t2 , or Cbfa2t3 was introduced into the Cas9-expressing (GFP + ) LPs. Four days after sgRNA transduction, nuclear lysates from retrovirus infected GFP + hNGFR + cells were subjected to immunoblotting for Lmo2, Zbtb1, Cbfa2t3, Bcl11a, and LaminB. Two independent experiments were performed with similar results. B , an experimental scheme for the deletion of Lmo2 , Zbtb1 , Cbfa2t3 , and Cbfa2t2 using the CRISPR/Cas9 system in Ebf1 -deficient LPs is shown. C , retroviral vectors encoding sgRNAs against luciferase (sgControl), Lmo2 (sgLmo2), Zbtb1 (sgZbtb1), Cbfa2t3 (sgCbfa2t3), or Cbfa2t2 (sgCbfa2t2) were introduced into Cas9-expressing (GFP + ) LPs. Five days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA transduced cells were gated and analyzed for CD44 and CD25 expression. D , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( C ) is shown with standard deviation (SD). E , ten days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. F , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( E ) is shown with SD. Data are representative of two ( A ) or three ( C and E ) independent experiments. Data represent the mean values of three independent biological replicates ( D and F ). ∗∗ p < 0.01 by two-sided Student’s t test. LP, lymphoid progenitor; sgRNA, single-guide RNA.
    Figure Legend Snippet: Loss of Zbtb1 and Cbfa2t3 leads to the T-lineage differentiation arrest in LPs. A , specific depletion of targeted Lmo2, Zbtb1, and Cbfa2t3 proteins. sgRNA against Lmo2 , Zbtb1 , Cbfa2t2 , or Cbfa2t3 was introduced into the Cas9-expressing (GFP + ) LPs. Four days after sgRNA transduction, nuclear lysates from retrovirus infected GFP + hNGFR + cells were subjected to immunoblotting for Lmo2, Zbtb1, Cbfa2t3, Bcl11a, and LaminB. Two independent experiments were performed with similar results. B , an experimental scheme for the deletion of Lmo2 , Zbtb1 , Cbfa2t3 , and Cbfa2t2 using the CRISPR/Cas9 system in Ebf1 -deficient LPs is shown. C , retroviral vectors encoding sgRNAs against luciferase (sgControl), Lmo2 (sgLmo2), Zbtb1 (sgZbtb1), Cbfa2t3 (sgCbfa2t3), or Cbfa2t2 (sgCbfa2t2) were introduced into Cas9-expressing (GFP + ) LPs. Five days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA transduced cells were gated and analyzed for CD44 and CD25 expression. D , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( C ) is shown with standard deviation (SD). E , ten days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. F , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( E ) is shown with SD. Data are representative of two ( A ) or three ( C and E ) independent experiments. Data represent the mean values of three independent biological replicates ( D and F ). ∗∗ p < 0.01 by two-sided Student’s t test. LP, lymphoid progenitor; sgRNA, single-guide RNA.

    Techniques Used: Expressing, Transduction, Infection, Western Blot, CRISPR, Luciferase, Standard Deviation

    Zbtb1 and Cbfa2t3 are involved in the regulation of Tcf7 expression in LPs. A , experimental scheme for the transcriptome analysis is shown. Five days after sgRNA introduction, GFP + hNGFR + LP cells were sorted, and subjected to QuantSeq 3′ mRNA sequencing. B , GO annotation was performed using the DAVID analysis tool. Top five GO terms for DEGs ( p < 0.05, |log2FC| > 1, and TPM > 10) in sgZbtb1-introduced Ebf1 -deficient LPs are shown. The full lists of the DEGs in Lmo2 -, Zbtb1 -, Cbfa2t2 -, or Cbfa2t3 -deficient LPs are shown in <xref ref-type=Table S2 . Data are based on three independent biological replicates. C , heat map showing the expression changes of representative Notch-activated genes in pro-T cell stages in response to the deletion of Lmo2 , Zbtb1 , or Cbfa2t3 . Data are based on the average of three biological replicates. D , five days after sgRNA introduction, GFP + hNGFR + LP cells were sorted ( A ). Relative expression levels of Tcf7 , Bcl11b , Bcl11a , and Gata3 against Actb were determined by RT-qPCR. The relative expression against sgControl-introduced cells is shown with SD. ∗∗ p < 0.01, ∗ p < 0.05 by two-sided Student’s t test. Data are based on three biological replicates. DEG, differentially expressed gene; GO, Gene Ontology; LP, lymphoid progenitor; sgRNA, single-guide RNA; TPM, transcripts per kilobase million. " title="... Relative expression levels of Tcf7 , Bcl11b , Bcl11a , and Gata3 against Actb were determined by ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Zbtb1 and Cbfa2t3 are involved in the regulation of Tcf7 expression in LPs. A , experimental scheme for the transcriptome analysis is shown. Five days after sgRNA introduction, GFP + hNGFR + LP cells were sorted, and subjected to QuantSeq 3′ mRNA sequencing. B , GO annotation was performed using the DAVID analysis tool. Top five GO terms for DEGs ( p < 0.05, |log2FC| > 1, and TPM > 10) in sgZbtb1-introduced Ebf1 -deficient LPs are shown. The full lists of the DEGs in Lmo2 -, Zbtb1 -, Cbfa2t2 -, or Cbfa2t3 -deficient LPs are shown in Table S2 . Data are based on three independent biological replicates. C , heat map showing the expression changes of representative Notch-activated genes in pro-T cell stages in response to the deletion of Lmo2 , Zbtb1 , or Cbfa2t3 . Data are based on the average of three biological replicates. D , five days after sgRNA introduction, GFP + hNGFR + LP cells were sorted ( A ). Relative expression levels of Tcf7 , Bcl11b , Bcl11a , and Gata3 against Actb were determined by RT-qPCR. The relative expression against sgControl-introduced cells is shown with SD. ∗∗ p < 0.01, ∗ p < 0.05 by two-sided Student’s t test. Data are based on three biological replicates. DEG, differentially expressed gene; GO, Gene Ontology; LP, lymphoid progenitor; sgRNA, single-guide RNA; TPM, transcripts per kilobase million.

    Techniques Used: Expressing, Sequencing, Quantitative RT-PCR

    anti bcl11a  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
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    Structured Review

    Cell Signaling Technology Inc anti bcl11a
    Loss of Zbtb1 and Cbfa2t3 leads to the T-lineage differentiation arrest in LPs. A , specific depletion of targeted Lmo2, Zbtb1, and Cbfa2t3 proteins. sgRNA against Lmo2 , Zbtb1 , Cbfa2t2 , or Cbfa2t3 was introduced into the Cas9-expressing (GFP + ) LPs. Four days after sgRNA transduction, nuclear lysates from retrovirus infected GFP + hNGFR + cells were subjected to immunoblotting for Lmo2, Zbtb1, Cbfa2t3, <t>Bcl11a,</t> and LaminB. Two independent experiments were performed with similar results. B , an experimental scheme for the deletion of Lmo2 , Zbtb1 , Cbfa2t3 , and Cbfa2t2 using the CRISPR/Cas9 system in Ebf1 -deficient LPs is shown. C , retroviral vectors encoding sgRNAs against luciferase (sgControl), Lmo2 (sgLmo2), Zbtb1 (sgZbtb1), Cbfa2t3 (sgCbfa2t3), or Cbfa2t2 (sgCbfa2t2) were introduced into Cas9-expressing (GFP + ) LPs. Five days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA transduced cells were gated and analyzed for CD44 and CD25 expression. D , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( C ) is shown with standard deviation (SD). E , ten days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. F , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( E ) is shown with SD. Data are representative of two ( A ) or three ( C and E ) independent experiments. Data represent the mean values of three independent biological replicates ( D and F ). ∗∗ p < 0.01 by two-sided Student’s t test. LP, lymphoid progenitor; sgRNA, single-guide RNA.
    Anti Bcl11a, 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/result/anti bcl11a/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti bcl11a - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Transcription factor Zbtb1 interacts with bridging factor Lmo2 and maintains the T-lineage differentiation capacity of lymphoid progenitor cells"

    Article Title: Transcription factor Zbtb1 interacts with bridging factor Lmo2 and maintains the T-lineage differentiation capacity of lymphoid progenitor cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2022.102506

    Loss of Zbtb1 and Cbfa2t3 leads to the T-lineage differentiation arrest in LPs. A , specific depletion of targeted Lmo2, Zbtb1, and Cbfa2t3 proteins. sgRNA against Lmo2 , Zbtb1 , Cbfa2t2 , or Cbfa2t3 was introduced into the Cas9-expressing (GFP + ) LPs. Four days after sgRNA transduction, nuclear lysates from retrovirus infected GFP + hNGFR + cells were subjected to immunoblotting for Lmo2, Zbtb1, Cbfa2t3, Bcl11a, and LaminB. Two independent experiments were performed with similar results. B , an experimental scheme for the deletion of Lmo2 , Zbtb1 , Cbfa2t3 , and Cbfa2t2 using the CRISPR/Cas9 system in Ebf1 -deficient LPs is shown. C , retroviral vectors encoding sgRNAs against luciferase (sgControl), Lmo2 (sgLmo2), Zbtb1 (sgZbtb1), Cbfa2t3 (sgCbfa2t3), or Cbfa2t2 (sgCbfa2t2) were introduced into Cas9-expressing (GFP + ) LPs. Five days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA transduced cells were gated and analyzed for CD44 and CD25 expression. D , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( C ) is shown with standard deviation (SD). E , ten days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. F , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( E ) is shown with SD. Data are representative of two ( A ) or three ( C and E ) independent experiments. Data represent the mean values of three independent biological replicates ( D and F ). ∗∗ p < 0.01 by two-sided Student’s t test. LP, lymphoid progenitor; sgRNA, single-guide RNA.
    Figure Legend Snippet: Loss of Zbtb1 and Cbfa2t3 leads to the T-lineage differentiation arrest in LPs. A , specific depletion of targeted Lmo2, Zbtb1, and Cbfa2t3 proteins. sgRNA against Lmo2 , Zbtb1 , Cbfa2t2 , or Cbfa2t3 was introduced into the Cas9-expressing (GFP + ) LPs. Four days after sgRNA transduction, nuclear lysates from retrovirus infected GFP + hNGFR + cells were subjected to immunoblotting for Lmo2, Zbtb1, Cbfa2t3, Bcl11a, and LaminB. Two independent experiments were performed with similar results. B , an experimental scheme for the deletion of Lmo2 , Zbtb1 , Cbfa2t3 , and Cbfa2t2 using the CRISPR/Cas9 system in Ebf1 -deficient LPs is shown. C , retroviral vectors encoding sgRNAs against luciferase (sgControl), Lmo2 (sgLmo2), Zbtb1 (sgZbtb1), Cbfa2t3 (sgCbfa2t3), or Cbfa2t2 (sgCbfa2t2) were introduced into Cas9-expressing (GFP + ) LPs. Five days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA transduced cells were gated and analyzed for CD44 and CD25 expression. D , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( C ) is shown with standard deviation (SD). E , ten days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. F , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( E ) is shown with SD. Data are representative of two ( A ) or three ( C and E ) independent experiments. Data represent the mean values of three independent biological replicates ( D and F ). ∗∗ p < 0.01 by two-sided Student’s t test. LP, lymphoid progenitor; sgRNA, single-guide RNA.

    Techniques Used: Expressing, Transduction, Infection, Western Blot, CRISPR, Luciferase, Standard Deviation

    Zbtb1 and Cbfa2t3 are involved in the regulation of Tcf7 expression in LPs. A , experimental scheme for the transcriptome analysis is shown. Five days after sgRNA introduction, GFP + hNGFR + LP cells were sorted, and subjected to QuantSeq 3′ mRNA sequencing. B , GO annotation was performed using the DAVID analysis tool. Top five GO terms for DEGs ( p < 0.05, |log2FC| > 1, and TPM > 10) in sgZbtb1-introduced Ebf1 -deficient LPs are shown. The full lists of the DEGs in Lmo2 -, Zbtb1 -, Cbfa2t2 -, or Cbfa2t3 -deficient LPs are shown in <xref ref-type=Table S2 . Data are based on three independent biological replicates. C , heat map showing the expression changes of representative Notch-activated genes in pro-T cell stages in response to the deletion of Lmo2 , Zbtb1 , or Cbfa2t3 . Data are based on the average of three biological replicates. D , five days after sgRNA introduction, GFP + hNGFR + LP cells were sorted ( A ). Relative expression levels of Tcf7 , Bcl11b , Bcl11a , and Gata3 against Actb were determined by RT-qPCR. The relative expression against sgControl-introduced cells is shown with SD. ∗∗ p < 0.01, ∗ p < 0.05 by two-sided Student’s t test. Data are based on three biological replicates. DEG, differentially expressed gene; GO, Gene Ontology; LP, lymphoid progenitor; sgRNA, single-guide RNA; TPM, transcripts per kilobase million. " title="... Relative expression levels of Tcf7 , Bcl11b , Bcl11a , and Gata3 against Actb were determined by ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Zbtb1 and Cbfa2t3 are involved in the regulation of Tcf7 expression in LPs. A , experimental scheme for the transcriptome analysis is shown. Five days after sgRNA introduction, GFP + hNGFR + LP cells were sorted, and subjected to QuantSeq 3′ mRNA sequencing. B , GO annotation was performed using the DAVID analysis tool. Top five GO terms for DEGs ( p < 0.05, |log2FC| > 1, and TPM > 10) in sgZbtb1-introduced Ebf1 -deficient LPs are shown. The full lists of the DEGs in Lmo2 -, Zbtb1 -, Cbfa2t2 -, or Cbfa2t3 -deficient LPs are shown in Table S2 . Data are based on three independent biological replicates. C , heat map showing the expression changes of representative Notch-activated genes in pro-T cell stages in response to the deletion of Lmo2 , Zbtb1 , or Cbfa2t3 . Data are based on the average of three biological replicates. D , five days after sgRNA introduction, GFP + hNGFR + LP cells were sorted ( A ). Relative expression levels of Tcf7 , Bcl11b , Bcl11a , and Gata3 against Actb were determined by RT-qPCR. The relative expression against sgControl-introduced cells is shown with SD. ∗∗ p < 0.01, ∗ p < 0.05 by two-sided Student’s t test. Data are based on three biological replicates. DEG, differentially expressed gene; GO, Gene Ontology; LP, lymphoid progenitor; sgRNA, single-guide RNA; TPM, transcripts per kilobase million.

    Techniques Used: Expressing, Sequencing, Quantitative RT-PCR

    anti bcl11a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti bcl11a
    Ubiquitous knockdown of target gene expression in HUDEP-2 cells using pZIP-MND-ZsGreen-UltramiR shRNA LVs. ( A ) Schematic map of pZIP-MND-ZsGreen-UltramiR shRNA LV that co-expresses Zoanthus sp. green fluorescence protein (ZsGreen), puromycin resistance (PuroR) and an shRNA from the UltramiR scaffold under the MND promoter. 5′ miR 30 and 3′ miR30: 5′ and 3′ regions of the UltramiR shRNA scaffold, respectively. ( B ) Workflow of the experiment for the target gene knockdown experiment in the HUDEP-2 cells. ( C ) Representative immunoblot analysis to evaluate the knockdown efficiencies of the three shRNAs (labeled 1 to 3) that target <t>BCL11A</t> and compared to the scrambled shRNA (shScr) (data normalized to actin) (left) and the graphical representation of the densitometric data of the immunoblots (right). The highest knockdown efficiencies by sh BCL11A and sh ZBTB7A are indicated in boxes. Full blots are presented in Supplemental Fig. S9 and S10. ( D ) Intracellular HbF analysis by flow cytometry to determine the percentages of HbF + cells in the shRNA transduced HUDEP-2 cells after differentiation (left) and graphical representation of the data (right). Boxes indicate the shRNAs with the highest percentages of HbF + cells. ( E ) HPLC analysis of the percentages of Gγ+Aγ chains in the shRNA transduced cells after differentiation (left) and the graphical representation of the data (right). The shRNAs with the highest elevation in the percentages of Gγ+Aγ are indicated in boxes. All data represent mean ± SD (n = 2).
    Anti Bcl11a, 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/result/anti bcl11a/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti bcl11a - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Erythroid lineage-specific lentiviral RNAi vectors suitable for molecular functional studies and therapeutic applications"

    Article Title: Erythroid lineage-specific lentiviral RNAi vectors suitable for molecular functional studies and therapeutic applications

    Journal: Scientific Reports

    doi: 10.1038/s41598-022-13783-0

    Ubiquitous knockdown of target gene expression in HUDEP-2 cells using pZIP-MND-ZsGreen-UltramiR shRNA LVs. ( A ) Schematic map of pZIP-MND-ZsGreen-UltramiR shRNA LV that co-expresses Zoanthus sp. green fluorescence protein (ZsGreen), puromycin resistance (PuroR) and an shRNA from the UltramiR scaffold under the MND promoter. 5′ miR 30 and 3′ miR30: 5′ and 3′ regions of the UltramiR shRNA scaffold, respectively. ( B ) Workflow of the experiment for the target gene knockdown experiment in the HUDEP-2 cells. ( C ) Representative immunoblot analysis to evaluate the knockdown efficiencies of the three shRNAs (labeled 1 to 3) that target BCL11A and compared to the scrambled shRNA (shScr) (data normalized to actin) (left) and the graphical representation of the densitometric data of the immunoblots (right). The highest knockdown efficiencies by sh BCL11A and sh ZBTB7A are indicated in boxes. Full blots are presented in Supplemental Fig. S9 and S10. ( D ) Intracellular HbF analysis by flow cytometry to determine the percentages of HbF + cells in the shRNA transduced HUDEP-2 cells after differentiation (left) and graphical representation of the data (right). Boxes indicate the shRNAs with the highest percentages of HbF + cells. ( E ) HPLC analysis of the percentages of Gγ+Aγ chains in the shRNA transduced cells after differentiation (left) and the graphical representation of the data (right). The shRNAs with the highest elevation in the percentages of Gγ+Aγ are indicated in boxes. All data represent mean ± SD (n = 2).
    Figure Legend Snippet: Ubiquitous knockdown of target gene expression in HUDEP-2 cells using pZIP-MND-ZsGreen-UltramiR shRNA LVs. ( A ) Schematic map of pZIP-MND-ZsGreen-UltramiR shRNA LV that co-expresses Zoanthus sp. green fluorescence protein (ZsGreen), puromycin resistance (PuroR) and an shRNA from the UltramiR scaffold under the MND promoter. 5′ miR 30 and 3′ miR30: 5′ and 3′ regions of the UltramiR shRNA scaffold, respectively. ( B ) Workflow of the experiment for the target gene knockdown experiment in the HUDEP-2 cells. ( C ) Representative immunoblot analysis to evaluate the knockdown efficiencies of the three shRNAs (labeled 1 to 3) that target BCL11A and compared to the scrambled shRNA (shScr) (data normalized to actin) (left) and the graphical representation of the densitometric data of the immunoblots (right). The highest knockdown efficiencies by sh BCL11A and sh ZBTB7A are indicated in boxes. Full blots are presented in Supplemental Fig. S9 and S10. ( D ) Intracellular HbF analysis by flow cytometry to determine the percentages of HbF + cells in the shRNA transduced HUDEP-2 cells after differentiation (left) and graphical representation of the data (right). Boxes indicate the shRNAs with the highest percentages of HbF + cells. ( E ) HPLC analysis of the percentages of Gγ+Aγ chains in the shRNA transduced cells after differentiation (left) and the graphical representation of the data (right). The shRNAs with the highest elevation in the percentages of Gγ+Aγ are indicated in boxes. All data represent mean ± SD (n = 2).

    Techniques Used: Expressing, shRNA, Fluorescence, Western Blot, Labeling, Flow Cytometry

    Knockdown of target gene expression in HUDEP-2 cells using H23B and H234B Ery-Lin-shRNA LVs. ( A ) Schematic maps of H23B and H234B Ery-Lin-shRNA LVs. HS2, HS3 and HS4: human β-globin LCR hypersensitivity sites 2, 3 and 4, respectively, βp: β-globin promoter and 5' miR30 and 3' miR30: 5′ and 3′ regions of the mir30 scaffold sequence, respectively. ( B ) Workflow of the experiment for target gene knockdown by the Ery-Lin-LVs in HUDEP-2 cells. ( C ) Representative immunoblots to evaluate the knockdown efficiencies of sh BCL11A and sh ZBTB7A in the transduced HUDEP-2 cells (left) and the graphical representation of the densitometric data of the immunoblots (right). Full blots are presented in Supplemental Fig. S9 and S10. ( D ) Intracellular HbF analysis by flow cytometry to determine the percentages of HbF + cells in the shRNA transduced HUDEP-2 cells after differentiation. ( E ) HPLC analysis of the percentages of Gγ+Aγ chains in the shRNA transduced cells after differentiation (left) and graphical representation of the data (right). H23B and H234B represent H23B-Ery-Lin-shRNA and H234B-Ery-Lin-shRNA LVs, respectively. All data represent mean ± SD (n = 2).
    Figure Legend Snippet: Knockdown of target gene expression in HUDEP-2 cells using H23B and H234B Ery-Lin-shRNA LVs. ( A ) Schematic maps of H23B and H234B Ery-Lin-shRNA LVs. HS2, HS3 and HS4: human β-globin LCR hypersensitivity sites 2, 3 and 4, respectively, βp: β-globin promoter and 5' miR30 and 3' miR30: 5′ and 3′ regions of the mir30 scaffold sequence, respectively. ( B ) Workflow of the experiment for target gene knockdown by the Ery-Lin-LVs in HUDEP-2 cells. ( C ) Representative immunoblots to evaluate the knockdown efficiencies of sh BCL11A and sh ZBTB7A in the transduced HUDEP-2 cells (left) and the graphical representation of the densitometric data of the immunoblots (right). Full blots are presented in Supplemental Fig. S9 and S10. ( D ) Intracellular HbF analysis by flow cytometry to determine the percentages of HbF + cells in the shRNA transduced HUDEP-2 cells after differentiation. ( E ) HPLC analysis of the percentages of Gγ+Aγ chains in the shRNA transduced cells after differentiation (left) and graphical representation of the data (right). H23B and H234B represent H23B-Ery-Lin-shRNA and H234B-Ery-Lin-shRNA LVs, respectively. All data represent mean ± SD (n = 2).

    Techniques Used: Expressing, shRNA, Sequencing, Western Blot, Flow Cytometry

    Erythroid-specific downregulation of target gene expression in the erythroid cells differentiated from CD34 + HSPCs transduced with Ery-Lin-shRNA LVs. ( A ) Schematic outline of the experiment. ( B ) Representative immunoblots that illustrate the knockdown efficiencies of sh BCL11A and sh ZBTB7A in the erythroid cells (left) and the densitometric data of the immunoblots (right). ZG represents ZsGreen expression; ‘+’ indicates ZsGreen + cells and ‘–’ indicates ZsGreen - cells. Full blots are presented in Supplemental Fig. S9 and S10. ( C ) Intracellular HbF analysis by flow cytometry to determine the percentages of HbF + cells in the erythroid cells differentiated from the shRNA transduced human CD34 + HSPCs (left) and the graphical representation of the data (right). ( D ) HPLC analysis of the percentages of Gγ+Aγ chains in the shRNA transduced cells after differentiation (left) and the graphical representation of the data (right). H23B and H234B represent H23B-Ery-Lin-shRNA and H234B-Ery-Lin-shRNA LVs, respectively. All data are mean ± SD (n = 2).
    Figure Legend Snippet: Erythroid-specific downregulation of target gene expression in the erythroid cells differentiated from CD34 + HSPCs transduced with Ery-Lin-shRNA LVs. ( A ) Schematic outline of the experiment. ( B ) Representative immunoblots that illustrate the knockdown efficiencies of sh BCL11A and sh ZBTB7A in the erythroid cells (left) and the densitometric data of the immunoblots (right). ZG represents ZsGreen expression; ‘+’ indicates ZsGreen + cells and ‘–’ indicates ZsGreen - cells. Full blots are presented in Supplemental Fig. S9 and S10. ( C ) Intracellular HbF analysis by flow cytometry to determine the percentages of HbF + cells in the erythroid cells differentiated from the shRNA transduced human CD34 + HSPCs (left) and the graphical representation of the data (right). ( D ) HPLC analysis of the percentages of Gγ+Aγ chains in the shRNA transduced cells after differentiation (left) and the graphical representation of the data (right). H23B and H234B represent H23B-Ery-Lin-shRNA and H234B-Ery-Lin-shRNA LVs, respectively. All data are mean ± SD (n = 2).

    Techniques Used: Expressing, Transduction, shRNA, Western Blot, Flow Cytometry

    Analysis of the engrafted H23BW-Ery-Lin-sh BCL11A LV transduced HSPCs in NBSGW mice. ( A ) Schematic representation of the mouse transplantation experiment. CsH: cyclosporin H. ( B ) The percentage engraftment measured in the total BM cells isolated 15 weeks after transplantation. ( C ) Multilineage analysis in the human CD45 positive (hCD45 + ) cells after 15 weeks of transplantation. ( D ) The percentage of CD71 + ZsGreen + cells in the erythroid cells obtained from BM by ex vivo erythropoiesis. ( E ) Representative immunoblots (left) and the densitometric quantitation of BCL11A knockdown (right) in the flow-sorted ZsGreen + erythroid cells (normalized to actin levels). ( F ) Intracellular HbF analysis by flow cytometry to determine the percentages of HbF + cells in the terminally differentiated ZsGreen + erythroid cells (left) and the graphical representation of the data (right). ( G ) HPLC analysis of the percentages of Gγ+Aγ chains in the terminally differentiated ZsGreen + erythroid cells after differentiation (left) and the graphical representation of the data (right). All data are compared to shScr. All data are mean ± SD (n = 3). ns, not significant, * p < 0.05 and ** p < 0.01.
    Figure Legend Snippet: Analysis of the engrafted H23BW-Ery-Lin-sh BCL11A LV transduced HSPCs in NBSGW mice. ( A ) Schematic representation of the mouse transplantation experiment. CsH: cyclosporin H. ( B ) The percentage engraftment measured in the total BM cells isolated 15 weeks after transplantation. ( C ) Multilineage analysis in the human CD45 positive (hCD45 + ) cells after 15 weeks of transplantation. ( D ) The percentage of CD71 + ZsGreen + cells in the erythroid cells obtained from BM by ex vivo erythropoiesis. ( E ) Representative immunoblots (left) and the densitometric quantitation of BCL11A knockdown (right) in the flow-sorted ZsGreen + erythroid cells (normalized to actin levels). ( F ) Intracellular HbF analysis by flow cytometry to determine the percentages of HbF + cells in the terminally differentiated ZsGreen + erythroid cells (left) and the graphical representation of the data (right). ( G ) HPLC analysis of the percentages of Gγ+Aγ chains in the terminally differentiated ZsGreen + erythroid cells after differentiation (left) and the graphical representation of the data (right). All data are compared to shScr. All data are mean ± SD (n = 3). ns, not significant, * p < 0.05 and ** p < 0.01.

    Techniques Used: Transplantation Assay, Isolation, Ex Vivo, Western Blot, Quantitation Assay, Flow Cytometry

    anti bcl11a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti bcl11a
    CARM1 inhibitors activate IFN α/γ pathway genes via inducing the formation of <t>BCL11A/PBAF</t> complex and elevating H3K27Ac levels. ( A ) Venn diagram showing the overlap of DEGs induced by JQ1, CARM1 inhibitor or JQ1 + CARM1 inhibitor in MDA-MB-468 cells ( top ) or HCI-002 ( bottom ). ( B ) Heatmap showing Hallmark gene sets up- (red) or down-regulated (blue) by JQ1, TP-064 or JQ1 + TP-064 treatment in MDA-MB-468 cells. ( C )GSEA of IFN α pathway signature genes induced by JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells. ( D ) Q-RT-PCR analyses of indicated IFNα pathway genes after treatment with JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells ( n = 3). Data are mean ± s.d. ** P < 0.01; * P < 0.05; NS: not significant. ( E ) Increased (red) and decreased (blue) me-BAF155, BRD4, and H3K27Ac ChIP-seq signals after TP-064 or JQ1 treatment in MDA-MB-468 cells on IFN α and γ genes that had increased expressions (magenta) after TP-064 treatment. Many of the IFN genes have expression level decreased (blue) after JQ1 treatment. ( F ) Changes of H3K27Ac ChIP-seq signals on genes from the Hallmark IFNα ( left ) and IFNγ (right) response gene sets induced by JQ1 or TP064. ( G, H ) Scatter plot showing the correlation of mRNA level changes between MDA-MB-468 (x-axis) and HCI-002 tumors (y-axis) by JQ1 (G) or TP-064/EZM2302 (H). IFN α pathway only genes are depicted in blue dots, IFN γ pathway only genes are depicted in yellow dots, and IFN α/γ shared pathway genes are depicted in green dots. ( I ) Enriched transcription factor motifs at H3K27Ac peaks of the promoters and gene bodies of IFN pathway genes. ( J ) Immunoblotting of BCL11A protein levels after treatment of MDA-MB-468 cells with TP-064 for indicated time. ( K, L ) Western blotting of indicated proteins from nuclear extracts of MDA-MB-468 cells by glycerol density gradient (10–45%) after vehicle (K) or TP-064 (L) treatment. ( M ) Summary of ChIP-qPCR data of indicated proteins binding to SEs or IFN pathway genes after JQ1 ( left ) or TP-064 ( right ) treatment in MDA-MB-468 cells. Red depicts high level and blue depicts low level binding of indicated proteins (y-axis), respectively. ( N ) Western blotting of BCL11A in parental or BCL11A knockdown MDA-MB-468 cells. ( O ) Q-RT-PCR analyses of IFNα pathway genes after treatment with DMSO or TP-064 in parental, BCL11A KD and overexpressing MDA-MB-468 cells. ** P < 0.01; * P < 0.05; NS: not significant.
    Anti Bcl11a, 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
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    1) Product Images from "BAF155 methylation drives metastasis by hijacking super-enhancers and subverting anti-tumor immunity"

    Article Title: BAF155 methylation drives metastasis by hijacking super-enhancers and subverting anti-tumor immunity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab1122

    CARM1 inhibitors activate IFN α/γ pathway genes via inducing the formation of BCL11A/PBAF complex and elevating H3K27Ac levels. ( A ) Venn diagram showing the overlap of DEGs induced by JQ1, CARM1 inhibitor or JQ1 + CARM1 inhibitor in MDA-MB-468 cells ( top ) or HCI-002 ( bottom ). ( B ) Heatmap showing Hallmark gene sets up- (red) or down-regulated (blue) by JQ1, TP-064 or JQ1 + TP-064 treatment in MDA-MB-468 cells. ( C )GSEA of IFN α pathway signature genes induced by JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells. ( D ) Q-RT-PCR analyses of indicated IFNα pathway genes after treatment with JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells ( n = 3). Data are mean ± s.d. ** P < 0.01; * P < 0.05; NS: not significant. ( E ) Increased (red) and decreased (blue) me-BAF155, BRD4, and H3K27Ac ChIP-seq signals after TP-064 or JQ1 treatment in MDA-MB-468 cells on IFN α and γ genes that had increased expressions (magenta) after TP-064 treatment. Many of the IFN genes have expression level decreased (blue) after JQ1 treatment. ( F ) Changes of H3K27Ac ChIP-seq signals on genes from the Hallmark IFNα ( left ) and IFNγ (right) response gene sets induced by JQ1 or TP064. ( G, H ) Scatter plot showing the correlation of mRNA level changes between MDA-MB-468 (x-axis) and HCI-002 tumors (y-axis) by JQ1 (G) or TP-064/EZM2302 (H). IFN α pathway only genes are depicted in blue dots, IFN γ pathway only genes are depicted in yellow dots, and IFN α/γ shared pathway genes are depicted in green dots. ( I ) Enriched transcription factor motifs at H3K27Ac peaks of the promoters and gene bodies of IFN pathway genes. ( J ) Immunoblotting of BCL11A protein levels after treatment of MDA-MB-468 cells with TP-064 for indicated time. ( K, L ) Western blotting of indicated proteins from nuclear extracts of MDA-MB-468 cells by glycerol density gradient (10–45%) after vehicle (K) or TP-064 (L) treatment. ( M ) Summary of ChIP-qPCR data of indicated proteins binding to SEs or IFN pathway genes after JQ1 ( left ) or TP-064 ( right ) treatment in MDA-MB-468 cells. Red depicts high level and blue depicts low level binding of indicated proteins (y-axis), respectively. ( N ) Western blotting of BCL11A in parental or BCL11A knockdown MDA-MB-468 cells. ( O ) Q-RT-PCR analyses of IFNα pathway genes after treatment with DMSO or TP-064 in parental, BCL11A KD and overexpressing MDA-MB-468 cells. ** P < 0.01; * P < 0.05; NS: not significant.
    Figure Legend Snippet: CARM1 inhibitors activate IFN α/γ pathway genes via inducing the formation of BCL11A/PBAF complex and elevating H3K27Ac levels. ( A ) Venn diagram showing the overlap of DEGs induced by JQ1, CARM1 inhibitor or JQ1 + CARM1 inhibitor in MDA-MB-468 cells ( top ) or HCI-002 ( bottom ). ( B ) Heatmap showing Hallmark gene sets up- (red) or down-regulated (blue) by JQ1, TP-064 or JQ1 + TP-064 treatment in MDA-MB-468 cells. ( C )GSEA of IFN α pathway signature genes induced by JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells. ( D ) Q-RT-PCR analyses of indicated IFNα pathway genes after treatment with JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells ( n = 3). Data are mean ± s.d. ** P < 0.01; * P < 0.05; NS: not significant. ( E ) Increased (red) and decreased (blue) me-BAF155, BRD4, and H3K27Ac ChIP-seq signals after TP-064 or JQ1 treatment in MDA-MB-468 cells on IFN α and γ genes that had increased expressions (magenta) after TP-064 treatment. Many of the IFN genes have expression level decreased (blue) after JQ1 treatment. ( F ) Changes of H3K27Ac ChIP-seq signals on genes from the Hallmark IFNα ( left ) and IFNγ (right) response gene sets induced by JQ1 or TP064. ( G, H ) Scatter plot showing the correlation of mRNA level changes between MDA-MB-468 (x-axis) and HCI-002 tumors (y-axis) by JQ1 (G) or TP-064/EZM2302 (H). IFN α pathway only genes are depicted in blue dots, IFN γ pathway only genes are depicted in yellow dots, and IFN α/γ shared pathway genes are depicted in green dots. ( I ) Enriched transcription factor motifs at H3K27Ac peaks of the promoters and gene bodies of IFN pathway genes. ( J ) Immunoblotting of BCL11A protein levels after treatment of MDA-MB-468 cells with TP-064 for indicated time. ( K, L ) Western blotting of indicated proteins from nuclear extracts of MDA-MB-468 cells by glycerol density gradient (10–45%) after vehicle (K) or TP-064 (L) treatment. ( M ) Summary of ChIP-qPCR data of indicated proteins binding to SEs or IFN pathway genes after JQ1 ( left ) or TP-064 ( right ) treatment in MDA-MB-468 cells. Red depicts high level and blue depicts low level binding of indicated proteins (y-axis), respectively. ( N ) Western blotting of BCL11A in parental or BCL11A knockdown MDA-MB-468 cells. ( O ) Q-RT-PCR analyses of IFNα pathway genes after treatment with DMSO or TP-064 in parental, BCL11A KD and overexpressing MDA-MB-468 cells. ** P < 0.01; * P < 0.05; NS: not significant.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, ChIP-sequencing, Expressing, Western Blot, Binding Assay

    Immunostaining of me-BAF155 in CTCs of metastatic breast cancer patients. ( A ) Images of me-BAF155 immunostaining in the nucleus of CTCs but not in WBCs from seven breast cancer patients. Representative images of CTCs ( left ) and WBCs ( right ) are shown for each patient (ID listed to the left). Each row of tiles includes an image crop of the intensity distribution of each of the stains included in the panel (Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and Bright Field (BF)) for one single cell, including a merge of all stains. Images were taken at 20x magnification; scale bars represent 10 μm. ( B ) The average me-BAF155 nuclear signal intensities in breast cancer patient CTCs. Each dot represents the average me-BAF155 nuclear staining intensities of each individual CTC from one of the seven patients. The red bars indicate the average me-BAF155 nuclear signals of all CTCs from an individual patient sample. ( C ) The tracing of me-BAF155 nuclear signal intensities measurement in CTCs from breast cancer patient 548. The number of CTCs are depicted in x-axis. ( D ) Representative immunofluorescence images of CTCs stained with Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and merged image of all staining signals for patient 548 in April 2021. Scale bars represent 10 μm. ( E, F ) Models depicting dual functions of me-BAF155 dependent metastasis in TNBC cells and anti-metastasis effects by CARM1 inhibition. Me-BAF155 and BRD4 interact at SEs to activate oncogenes and me-BAF155 containing SWI/SNF complex interacts with HDAC1 to suppress ISGs (E). CARM1 inhibitor treatment leads to increased BCL11A and un-methylated BAF155, which assemble with other SWI/SNF subunits to form PBAF to activate ISGs. Un-methylated BAF155 triggers dissociation of BRD4 from SEs, resulting in inhibition of expression of SE-addicted oncogenes (F).
    Figure Legend Snippet: Immunostaining of me-BAF155 in CTCs of metastatic breast cancer patients. ( A ) Images of me-BAF155 immunostaining in the nucleus of CTCs but not in WBCs from seven breast cancer patients. Representative images of CTCs ( left ) and WBCs ( right ) are shown for each patient (ID listed to the left). Each row of tiles includes an image crop of the intensity distribution of each of the stains included in the panel (Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and Bright Field (BF)) for one single cell, including a merge of all stains. Images were taken at 20x magnification; scale bars represent 10 μm. ( B ) The average me-BAF155 nuclear signal intensities in breast cancer patient CTCs. Each dot represents the average me-BAF155 nuclear staining intensities of each individual CTC from one of the seven patients. The red bars indicate the average me-BAF155 nuclear signals of all CTCs from an individual patient sample. ( C ) The tracing of me-BAF155 nuclear signal intensities measurement in CTCs from breast cancer patient 548. The number of CTCs are depicted in x-axis. ( D ) Representative immunofluorescence images of CTCs stained with Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and merged image of all staining signals for patient 548 in April 2021. Scale bars represent 10 μm. ( E, F ) Models depicting dual functions of me-BAF155 dependent metastasis in TNBC cells and anti-metastasis effects by CARM1 inhibition. Me-BAF155 and BRD4 interact at SEs to activate oncogenes and me-BAF155 containing SWI/SNF complex interacts with HDAC1 to suppress ISGs (E). CARM1 inhibitor treatment leads to increased BCL11A and un-methylated BAF155, which assemble with other SWI/SNF subunits to form PBAF to activate ISGs. Un-methylated BAF155 triggers dissociation of BRD4 from SEs, resulting in inhibition of expression of SE-addicted oncogenes (F).

    Techniques Used: Immunostaining, Staining, Immunofluorescence, Inhibition, Methylation, Expressing

    bcl11a  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc bcl11a
    CARM1 inhibitors activate IFN α/γ pathway genes via inducing the formation of <t>BCL11A/PBAF</t> complex and elevating H3K27Ac levels. ( A ) Venn diagram showing the overlap of DEGs induced by JQ1, CARM1 inhibitor or JQ1 + CARM1 inhibitor in MDA-MB-468 cells ( top ) or HCI-002 ( bottom ). ( B ) Heatmap showing Hallmark gene sets up- (red) or down-regulated (blue) by JQ1, TP-064 or JQ1 + TP-064 treatment in MDA-MB-468 cells. ( C )GSEA of IFN α pathway signature genes induced by JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells. ( D ) Q-RT-PCR analyses of indicated IFNα pathway genes after treatment with JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells ( n = 3). Data are mean ± s.d. ** P < 0.01; * P < 0.05; NS: not significant. ( E ) Increased (red) and decreased (blue) me-BAF155, BRD4, and H3K27Ac ChIP-seq signals after TP-064 or JQ1 treatment in MDA-MB-468 cells on IFN α and γ genes that had increased expressions (magenta) after TP-064 treatment. Many of the IFN genes have expression level decreased (blue) after JQ1 treatment. ( F ) Changes of H3K27Ac ChIP-seq signals on genes from the Hallmark IFNα ( left ) and IFNγ (right) response gene sets induced by JQ1 or TP064. ( G, H ) Scatter plot showing the correlation of mRNA level changes between MDA-MB-468 (x-axis) and HCI-002 tumors (y-axis) by JQ1 (G) or TP-064/EZM2302 (H). IFN α pathway only genes are depicted in blue dots, IFN γ pathway only genes are depicted in yellow dots, and IFN α/γ shared pathway genes are depicted in green dots. ( I ) Enriched transcription factor motifs at H3K27Ac peaks of the promoters and gene bodies of IFN pathway genes. ( J ) Immunoblotting of BCL11A protein levels after treatment of MDA-MB-468 cells with TP-064 for indicated time. ( K, L ) Western blotting of indicated proteins from nuclear extracts of MDA-MB-468 cells by glycerol density gradient (10–45%) after vehicle (K) or TP-064 (L) treatment. ( M ) Summary of ChIP-qPCR data of indicated proteins binding to SEs or IFN pathway genes after JQ1 ( left ) or TP-064 ( right ) treatment in MDA-MB-468 cells. Red depicts high level and blue depicts low level binding of indicated proteins (y-axis), respectively. ( N ) Western blotting of BCL11A in parental or BCL11A knockdown MDA-MB-468 cells. ( O ) Q-RT-PCR analyses of IFNα pathway genes after treatment with DMSO or TP-064 in parental, BCL11A KD and overexpressing MDA-MB-468 cells. ** P < 0.01; * P < 0.05; NS: not significant.
    Bcl11a, 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
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    Images

    1) Product Images from "BAF155 methylation drives metastasis by hijacking super-enhancers and subverting anti-tumor immunity"

    Article Title: BAF155 methylation drives metastasis by hijacking super-enhancers and subverting anti-tumor immunity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab1122

    CARM1 inhibitors activate IFN α/γ pathway genes via inducing the formation of BCL11A/PBAF complex and elevating H3K27Ac levels. ( A ) Venn diagram showing the overlap of DEGs induced by JQ1, CARM1 inhibitor or JQ1 + CARM1 inhibitor in MDA-MB-468 cells ( top ) or HCI-002 ( bottom ). ( B ) Heatmap showing Hallmark gene sets up- (red) or down-regulated (blue) by JQ1, TP-064 or JQ1 + TP-064 treatment in MDA-MB-468 cells. ( C )GSEA of IFN α pathway signature genes induced by JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells. ( D ) Q-RT-PCR analyses of indicated IFNα pathway genes after treatment with JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells ( n = 3). Data are mean ± s.d. ** P < 0.01; * P < 0.05; NS: not significant. ( E ) Increased (red) and decreased (blue) me-BAF155, BRD4, and H3K27Ac ChIP-seq signals after TP-064 or JQ1 treatment in MDA-MB-468 cells on IFN α and γ genes that had increased expressions (magenta) after TP-064 treatment. Many of the IFN genes have expression level decreased (blue) after JQ1 treatment. ( F ) Changes of H3K27Ac ChIP-seq signals on genes from the Hallmark IFNα ( left ) and IFNγ (right) response gene sets induced by JQ1 or TP064. ( G, H ) Scatter plot showing the correlation of mRNA level changes between MDA-MB-468 (x-axis) and HCI-002 tumors (y-axis) by JQ1 (G) or TP-064/EZM2302 (H). IFN α pathway only genes are depicted in blue dots, IFN γ pathway only genes are depicted in yellow dots, and IFN α/γ shared pathway genes are depicted in green dots. ( I ) Enriched transcription factor motifs at H3K27Ac peaks of the promoters and gene bodies of IFN pathway genes. ( J ) Immunoblotting of BCL11A protein levels after treatment of MDA-MB-468 cells with TP-064 for indicated time. ( K, L ) Western blotting of indicated proteins from nuclear extracts of MDA-MB-468 cells by glycerol density gradient (10–45%) after vehicle (K) or TP-064 (L) treatment. ( M ) Summary of ChIP-qPCR data of indicated proteins binding to SEs or IFN pathway genes after JQ1 ( left ) or TP-064 ( right ) treatment in MDA-MB-468 cells. Red depicts high level and blue depicts low level binding of indicated proteins (y-axis), respectively. ( N ) Western blotting of BCL11A in parental or BCL11A knockdown MDA-MB-468 cells. ( O ) Q-RT-PCR analyses of IFNα pathway genes after treatment with DMSO or TP-064 in parental, BCL11A KD and overexpressing MDA-MB-468 cells. ** P < 0.01; * P < 0.05; NS: not significant.
    Figure Legend Snippet: CARM1 inhibitors activate IFN α/γ pathway genes via inducing the formation of BCL11A/PBAF complex and elevating H3K27Ac levels. ( A ) Venn diagram showing the overlap of DEGs induced by JQ1, CARM1 inhibitor or JQ1 + CARM1 inhibitor in MDA-MB-468 cells ( top ) or HCI-002 ( bottom ). ( B ) Heatmap showing Hallmark gene sets up- (red) or down-regulated (blue) by JQ1, TP-064 or JQ1 + TP-064 treatment in MDA-MB-468 cells. ( C )GSEA of IFN α pathway signature genes induced by JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells. ( D ) Q-RT-PCR analyses of indicated IFNα pathway genes after treatment with JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells ( n = 3). Data are mean ± s.d. ** P < 0.01; * P < 0.05; NS: not significant. ( E ) Increased (red) and decreased (blue) me-BAF155, BRD4, and H3K27Ac ChIP-seq signals after TP-064 or JQ1 treatment in MDA-MB-468 cells on IFN α and γ genes that had increased expressions (magenta) after TP-064 treatment. Many of the IFN genes have expression level decreased (blue) after JQ1 treatment. ( F ) Changes of H3K27Ac ChIP-seq signals on genes from the Hallmark IFNα ( left ) and IFNγ (right) response gene sets induced by JQ1 or TP064. ( G, H ) Scatter plot showing the correlation of mRNA level changes between MDA-MB-468 (x-axis) and HCI-002 tumors (y-axis) by JQ1 (G) or TP-064/EZM2302 (H). IFN α pathway only genes are depicted in blue dots, IFN γ pathway only genes are depicted in yellow dots, and IFN α/γ shared pathway genes are depicted in green dots. ( I ) Enriched transcription factor motifs at H3K27Ac peaks of the promoters and gene bodies of IFN pathway genes. ( J ) Immunoblotting of BCL11A protein levels after treatment of MDA-MB-468 cells with TP-064 for indicated time. ( K, L ) Western blotting of indicated proteins from nuclear extracts of MDA-MB-468 cells by glycerol density gradient (10–45%) after vehicle (K) or TP-064 (L) treatment. ( M ) Summary of ChIP-qPCR data of indicated proteins binding to SEs or IFN pathway genes after JQ1 ( left ) or TP-064 ( right ) treatment in MDA-MB-468 cells. Red depicts high level and blue depicts low level binding of indicated proteins (y-axis), respectively. ( N ) Western blotting of BCL11A in parental or BCL11A knockdown MDA-MB-468 cells. ( O ) Q-RT-PCR analyses of IFNα pathway genes after treatment with DMSO or TP-064 in parental, BCL11A KD and overexpressing MDA-MB-468 cells. ** P < 0.01; * P < 0.05; NS: not significant.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, ChIP-sequencing, Expressing, Western Blot, Binding Assay

    Immunostaining of me-BAF155 in CTCs of metastatic breast cancer patients. ( A ) Images of me-BAF155 immunostaining in the nucleus of CTCs but not in WBCs from seven breast cancer patients. Representative images of CTCs ( left ) and WBCs ( right ) are shown for each patient (ID listed to the left). Each row of tiles includes an image crop of the intensity distribution of each of the stains included in the panel (Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and Bright Field (BF)) for one single cell, including a merge of all stains. Images were taken at 20x magnification; scale bars represent 10 μm. ( B ) The average me-BAF155 nuclear signal intensities in breast cancer patient CTCs. Each dot represents the average me-BAF155 nuclear staining intensities of each individual CTC from one of the seven patients. The red bars indicate the average me-BAF155 nuclear signals of all CTCs from an individual patient sample. ( C ) The tracing of me-BAF155 nuclear signal intensities measurement in CTCs from breast cancer patient 548. The number of CTCs are depicted in x-axis. ( D ) Representative immunofluorescence images of CTCs stained with Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and merged image of all staining signals for patient 548 in April 2021. Scale bars represent 10 μm. ( E, F ) Models depicting dual functions of me-BAF155 dependent metastasis in TNBC cells and anti-metastasis effects by CARM1 inhibition. Me-BAF155 and BRD4 interact at SEs to activate oncogenes and me-BAF155 containing SWI/SNF complex interacts with HDAC1 to suppress ISGs (E). CARM1 inhibitor treatment leads to increased BCL11A and un-methylated BAF155, which assemble with other SWI/SNF subunits to form PBAF to activate ISGs. Un-methylated BAF155 triggers dissociation of BRD4 from SEs, resulting in inhibition of expression of SE-addicted oncogenes (F).
    Figure Legend Snippet: Immunostaining of me-BAF155 in CTCs of metastatic breast cancer patients. ( A ) Images of me-BAF155 immunostaining in the nucleus of CTCs but not in WBCs from seven breast cancer patients. Representative images of CTCs ( left ) and WBCs ( right ) are shown for each patient (ID listed to the left). Each row of tiles includes an image crop of the intensity distribution of each of the stains included in the panel (Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and Bright Field (BF)) for one single cell, including a merge of all stains. Images were taken at 20x magnification; scale bars represent 10 μm. ( B ) The average me-BAF155 nuclear signal intensities in breast cancer patient CTCs. Each dot represents the average me-BAF155 nuclear staining intensities of each individual CTC from one of the seven patients. The red bars indicate the average me-BAF155 nuclear signals of all CTCs from an individual patient sample. ( C ) The tracing of me-BAF155 nuclear signal intensities measurement in CTCs from breast cancer patient 548. The number of CTCs are depicted in x-axis. ( D ) Representative immunofluorescence images of CTCs stained with Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and merged image of all staining signals for patient 548 in April 2021. Scale bars represent 10 μm. ( E, F ) Models depicting dual functions of me-BAF155 dependent metastasis in TNBC cells and anti-metastasis effects by CARM1 inhibition. Me-BAF155 and BRD4 interact at SEs to activate oncogenes and me-BAF155 containing SWI/SNF complex interacts with HDAC1 to suppress ISGs (E). CARM1 inhibitor treatment leads to increased BCL11A and un-methylated BAF155, which assemble with other SWI/SNF subunits to form PBAF to activate ISGs. Un-methylated BAF155 triggers dissociation of BRD4 from SEs, resulting in inhibition of expression of SE-addicted oncogenes (F).

    Techniques Used: Immunostaining, Staining, Immunofluorescence, Inhibition, Methylation, Expressing

    anti bcl11a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti bcl11a
    CARM1 inhibitors activate IFN α/γ pathway genes via inducing the formation of <t>BCL11A/PBAF</t> complex and elevating H3K27Ac levels. ( A ) Venn diagram showing the overlap of DEGs induced by JQ1, CARM1 inhibitor or JQ1 + CARM1 inhibitor in MDA-MB-468 cells ( top ) or HCI-002 ( bottom ). ( B ) Heatmap showing Hallmark gene sets up- (red) or down-regulated (blue) by JQ1, TP-064 or JQ1 + TP-064 treatment in MDA-MB-468 cells. ( C )GSEA of IFN α pathway signature genes induced by JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells. ( D ) Q-RT-PCR analyses of indicated IFNα pathway genes after treatment with JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells ( n = 3). Data are mean ± s.d. ** P < 0.01; * P < 0.05; NS: not significant. ( E ) Increased (red) and decreased (blue) me-BAF155, BRD4, and H3K27Ac ChIP-seq signals after TP-064 or JQ1 treatment in MDA-MB-468 cells on IFN α and γ genes that had increased expressions (magenta) after TP-064 treatment. Many of the IFN genes have expression level decreased (blue) after JQ1 treatment. ( F ) Changes of H3K27Ac ChIP-seq signals on genes from the Hallmark IFNα ( left ) and IFNγ (right) response gene sets induced by JQ1 or TP064. ( G, H ) Scatter plot showing the correlation of mRNA level changes between MDA-MB-468 (x-axis) and HCI-002 tumors (y-axis) by JQ1 (G) or TP-064/EZM2302 (H). IFN α pathway only genes are depicted in blue dots, IFN γ pathway only genes are depicted in yellow dots, and IFN α/γ shared pathway genes are depicted in green dots. ( I ) Enriched transcription factor motifs at H3K27Ac peaks of the promoters and gene bodies of IFN pathway genes. ( J ) Immunoblotting of BCL11A protein levels after treatment of MDA-MB-468 cells with TP-064 for indicated time. ( K, L ) Western blotting of indicated proteins from nuclear extracts of MDA-MB-468 cells by glycerol density gradient (10–45%) after vehicle (K) or TP-064 (L) treatment. ( M ) Summary of ChIP-qPCR data of indicated proteins binding to SEs or IFN pathway genes after JQ1 ( left ) or TP-064 ( right ) treatment in MDA-MB-468 cells. Red depicts high level and blue depicts low level binding of indicated proteins (y-axis), respectively. ( N ) Western blotting of BCL11A in parental or BCL11A knockdown MDA-MB-468 cells. ( O ) Q-RT-PCR analyses of IFNα pathway genes after treatment with DMSO or TP-064 in parental, BCL11A KD and overexpressing MDA-MB-468 cells. ** P < 0.01; * P < 0.05; NS: not significant.
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    1) Product Images from "BAF155 methylation drives metastasis by hijacking super-enhancers and subverting anti-tumor immunity"

    Article Title: BAF155 methylation drives metastasis by hijacking super-enhancers and subverting anti-tumor immunity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab1122

    CARM1 inhibitors activate IFN α/γ pathway genes via inducing the formation of BCL11A/PBAF complex and elevating H3K27Ac levels. ( A ) Venn diagram showing the overlap of DEGs induced by JQ1, CARM1 inhibitor or JQ1 + CARM1 inhibitor in MDA-MB-468 cells ( top ) or HCI-002 ( bottom ). ( B ) Heatmap showing Hallmark gene sets up- (red) or down-regulated (blue) by JQ1, TP-064 or JQ1 + TP-064 treatment in MDA-MB-468 cells. ( C )GSEA of IFN α pathway signature genes induced by JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells. ( D ) Q-RT-PCR analyses of indicated IFNα pathway genes after treatment with JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells ( n = 3). Data are mean ± s.d. ** P < 0.01; * P < 0.05; NS: not significant. ( E ) Increased (red) and decreased (blue) me-BAF155, BRD4, and H3K27Ac ChIP-seq signals after TP-064 or JQ1 treatment in MDA-MB-468 cells on IFN α and γ genes that had increased expressions (magenta) after TP-064 treatment. Many of the IFN genes have expression level decreased (blue) after JQ1 treatment. ( F ) Changes of H3K27Ac ChIP-seq signals on genes from the Hallmark IFNα ( left ) and IFNγ (right) response gene sets induced by JQ1 or TP064. ( G, H ) Scatter plot showing the correlation of mRNA level changes between MDA-MB-468 (x-axis) and HCI-002 tumors (y-axis) by JQ1 (G) or TP-064/EZM2302 (H). IFN α pathway only genes are depicted in blue dots, IFN γ pathway only genes are depicted in yellow dots, and IFN α/γ shared pathway genes are depicted in green dots. ( I ) Enriched transcription factor motifs at H3K27Ac peaks of the promoters and gene bodies of IFN pathway genes. ( J ) Immunoblotting of BCL11A protein levels after treatment of MDA-MB-468 cells with TP-064 for indicated time. ( K, L ) Western blotting of indicated proteins from nuclear extracts of MDA-MB-468 cells by glycerol density gradient (10–45%) after vehicle (K) or TP-064 (L) treatment. ( M ) Summary of ChIP-qPCR data of indicated proteins binding to SEs or IFN pathway genes after JQ1 ( left ) or TP-064 ( right ) treatment in MDA-MB-468 cells. Red depicts high level and blue depicts low level binding of indicated proteins (y-axis), respectively. ( N ) Western blotting of BCL11A in parental or BCL11A knockdown MDA-MB-468 cells. ( O ) Q-RT-PCR analyses of IFNα pathway genes after treatment with DMSO or TP-064 in parental, BCL11A KD and overexpressing MDA-MB-468 cells. ** P < 0.01; * P < 0.05; NS: not significant.
    Figure Legend Snippet: CARM1 inhibitors activate IFN α/γ pathway genes via inducing the formation of BCL11A/PBAF complex and elevating H3K27Ac levels. ( A ) Venn diagram showing the overlap of DEGs induced by JQ1, CARM1 inhibitor or JQ1 + CARM1 inhibitor in MDA-MB-468 cells ( top ) or HCI-002 ( bottom ). ( B ) Heatmap showing Hallmark gene sets up- (red) or down-regulated (blue) by JQ1, TP-064 or JQ1 + TP-064 treatment in MDA-MB-468 cells. ( C )GSEA of IFN α pathway signature genes induced by JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells. ( D ) Q-RT-PCR analyses of indicated IFNα pathway genes after treatment with JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells ( n = 3). Data are mean ± s.d. ** P < 0.01; * P < 0.05; NS: not significant. ( E ) Increased (red) and decreased (blue) me-BAF155, BRD4, and H3K27Ac ChIP-seq signals after TP-064 or JQ1 treatment in MDA-MB-468 cells on IFN α and γ genes that had increased expressions (magenta) after TP-064 treatment. Many of the IFN genes have expression level decreased (blue) after JQ1 treatment. ( F ) Changes of H3K27Ac ChIP-seq signals on genes from the Hallmark IFNα ( left ) and IFNγ (right) response gene sets induced by JQ1 or TP064. ( G, H ) Scatter plot showing the correlation of mRNA level changes between MDA-MB-468 (x-axis) and HCI-002 tumors (y-axis) by JQ1 (G) or TP-064/EZM2302 (H). IFN α pathway only genes are depicted in blue dots, IFN γ pathway only genes are depicted in yellow dots, and IFN α/γ shared pathway genes are depicted in green dots. ( I ) Enriched transcription factor motifs at H3K27Ac peaks of the promoters and gene bodies of IFN pathway genes. ( J ) Immunoblotting of BCL11A protein levels after treatment of MDA-MB-468 cells with TP-064 for indicated time. ( K, L ) Western blotting of indicated proteins from nuclear extracts of MDA-MB-468 cells by glycerol density gradient (10–45%) after vehicle (K) or TP-064 (L) treatment. ( M ) Summary of ChIP-qPCR data of indicated proteins binding to SEs or IFN pathway genes after JQ1 ( left ) or TP-064 ( right ) treatment in MDA-MB-468 cells. Red depicts high level and blue depicts low level binding of indicated proteins (y-axis), respectively. ( N ) Western blotting of BCL11A in parental or BCL11A knockdown MDA-MB-468 cells. ( O ) Q-RT-PCR analyses of IFNα pathway genes after treatment with DMSO or TP-064 in parental, BCL11A KD and overexpressing MDA-MB-468 cells. ** P < 0.01; * P < 0.05; NS: not significant.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, ChIP-sequencing, Expressing, Western Blot, Binding Assay

    Immunostaining of me-BAF155 in CTCs of metastatic breast cancer patients. ( A ) Images of me-BAF155 immunostaining in the nucleus of CTCs but not in WBCs from seven breast cancer patients. Representative images of CTCs ( left ) and WBCs ( right ) are shown for each patient (ID listed to the left). Each row of tiles includes an image crop of the intensity distribution of each of the stains included in the panel (Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and Bright Field (BF)) for one single cell, including a merge of all stains. Images were taken at 20x magnification; scale bars represent 10 μm. ( B ) The average me-BAF155 nuclear signal intensities in breast cancer patient CTCs. Each dot represents the average me-BAF155 nuclear staining intensities of each individual CTC from one of the seven patients. The red bars indicate the average me-BAF155 nuclear signals of all CTCs from an individual patient sample. ( C ) The tracing of me-BAF155 nuclear signal intensities measurement in CTCs from breast cancer patient 548. The number of CTCs are depicted in x-axis. ( D ) Representative immunofluorescence images of CTCs stained with Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and merged image of all staining signals for patient 548 in April 2021. Scale bars represent 10 μm. ( E, F ) Models depicting dual functions of me-BAF155 dependent metastasis in TNBC cells and anti-metastasis effects by CARM1 inhibition. Me-BAF155 and BRD4 interact at SEs to activate oncogenes and me-BAF155 containing SWI/SNF complex interacts with HDAC1 to suppress ISGs (E). CARM1 inhibitor treatment leads to increased BCL11A and un-methylated BAF155, which assemble with other SWI/SNF subunits to form PBAF to activate ISGs. Un-methylated BAF155 triggers dissociation of BRD4 from SEs, resulting in inhibition of expression of SE-addicted oncogenes (F).
    Figure Legend Snippet: Immunostaining of me-BAF155 in CTCs of metastatic breast cancer patients. ( A ) Images of me-BAF155 immunostaining in the nucleus of CTCs but not in WBCs from seven breast cancer patients. Representative images of CTCs ( left ) and WBCs ( right ) are shown for each patient (ID listed to the left). Each row of tiles includes an image crop of the intensity distribution of each of the stains included in the panel (Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and Bright Field (BF)) for one single cell, including a merge of all stains. Images were taken at 20x magnification; scale bars represent 10 μm. ( B ) The average me-BAF155 nuclear signal intensities in breast cancer patient CTCs. Each dot represents the average me-BAF155 nuclear staining intensities of each individual CTC from one of the seven patients. The red bars indicate the average me-BAF155 nuclear signals of all CTCs from an individual patient sample. ( C ) The tracing of me-BAF155 nuclear signal intensities measurement in CTCs from breast cancer patient 548. The number of CTCs are depicted in x-axis. ( D ) Representative immunofluorescence images of CTCs stained with Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and merged image of all staining signals for patient 548 in April 2021. Scale bars represent 10 μm. ( E, F ) Models depicting dual functions of me-BAF155 dependent metastasis in TNBC cells and anti-metastasis effects by CARM1 inhibition. Me-BAF155 and BRD4 interact at SEs to activate oncogenes and me-BAF155 containing SWI/SNF complex interacts with HDAC1 to suppress ISGs (E). CARM1 inhibitor treatment leads to increased BCL11A and un-methylated BAF155, which assemble with other SWI/SNF subunits to form PBAF to activate ISGs. Un-methylated BAF155 triggers dissociation of BRD4 from SEs, resulting in inhibition of expression of SE-addicted oncogenes (F).

    Techniques Used: Immunostaining, Staining, Immunofluorescence, Inhibition, Methylation, Expressing

    anti bcl11a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti bcl11a
    Knockdown of <t>BCL11A</t> in PBiEPC-1. ( A ) Schematic representation of the experimental setup for BCL11A knockdown in PBiEPC-1. ( B ) Western blot analysis of BCL11A knockdown in PBiEPC-1. Data were compared with the cells transduced with the scrambled shRNA. ( C ) (Left) An HPLC chromatogram showing β, α, Gγ, and Aγ globins in the cells transduced with shBCL11A and shScrambled lentiviruses. (Right) Gγ + Aγ% in the differentiated erythroid cells transduced with shBCL11A and shScrambled lentiviruses. ( D ) Flow cytometry analysis of F-cells after knockdown of BCL11A . Numbers indicate mean ± standard deviation (SD) from two independent experiments.
    Anti Bcl11a, 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
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    Images

    1) Product Images from "Direct Generation of Immortalized Erythroid Progenitor Cell Lines from Peripheral Blood Mononuclear Cells"

    Article Title: Direct Generation of Immortalized Erythroid Progenitor Cell Lines from Peripheral Blood Mononuclear Cells

    Journal: Cells

    doi: 10.3390/cells10030523

    Knockdown of BCL11A in PBiEPC-1. ( A ) Schematic representation of the experimental setup for BCL11A knockdown in PBiEPC-1. ( B ) Western blot analysis of BCL11A knockdown in PBiEPC-1. Data were compared with the cells transduced with the scrambled shRNA. ( C ) (Left) An HPLC chromatogram showing β, α, Gγ, and Aγ globins in the cells transduced with shBCL11A and shScrambled lentiviruses. (Right) Gγ + Aγ% in the differentiated erythroid cells transduced with shBCL11A and shScrambled lentiviruses. ( D ) Flow cytometry analysis of F-cells after knockdown of BCL11A . Numbers indicate mean ± standard deviation (SD) from two independent experiments.
    Figure Legend Snippet: Knockdown of BCL11A in PBiEPC-1. ( A ) Schematic representation of the experimental setup for BCL11A knockdown in PBiEPC-1. ( B ) Western blot analysis of BCL11A knockdown in PBiEPC-1. Data were compared with the cells transduced with the scrambled shRNA. ( C ) (Left) An HPLC chromatogram showing β, α, Gγ, and Aγ globins in the cells transduced with shBCL11A and shScrambled lentiviruses. (Right) Gγ + Aγ% in the differentiated erythroid cells transduced with shBCL11A and shScrambled lentiviruses. ( D ) Flow cytometry analysis of F-cells after knockdown of BCL11A . Numbers indicate mean ± standard deviation (SD) from two independent experiments.

    Techniques Used: Western Blot, Transduction, shRNA, Flow Cytometry, Standard Deviation

    CRISPR-Cas9 mediated gene editing of BCL11A enhancer and exon 2 in PBiEPC-1. ( A ) Schematic representation of the CRISPR/Cas9 experiment in PBiEPC-1. ( B ) Inference of CRISPR Edits (ICE) analysis results of gene editing at the BCL11A enhancer and exon 2. The percentages of different types of mutations are shown. “+” indicates insertions, and “–“ indicates deletions of nucleotides. “WT” indicates wild type sequence, and “others” indicates rare insertions and deletions. ( C ) Flow cytometry analysis of F-cells in the cells edited at BCL11A enhancer and AAVS1 (control genomic region). Numbers indicate mean ± standard deviation (SD) from two independent experiments. ( D ) Flow cytometry analysis of F-cells after editing at BCL11A exon 2 in PBiEPC-1 and HUDEP-2. Numbers indicate mean ± standard deviation (SD) from two independent experiments. RNP: ribonucleoprotein complex containing Cas9 protein and gRNA.
    Figure Legend Snippet: CRISPR-Cas9 mediated gene editing of BCL11A enhancer and exon 2 in PBiEPC-1. ( A ) Schematic representation of the CRISPR/Cas9 experiment in PBiEPC-1. ( B ) Inference of CRISPR Edits (ICE) analysis results of gene editing at the BCL11A enhancer and exon 2. The percentages of different types of mutations are shown. “+” indicates insertions, and “–“ indicates deletions of nucleotides. “WT” indicates wild type sequence, and “others” indicates rare insertions and deletions. ( C ) Flow cytometry analysis of F-cells in the cells edited at BCL11A enhancer and AAVS1 (control genomic region). Numbers indicate mean ± standard deviation (SD) from two independent experiments. ( D ) Flow cytometry analysis of F-cells after editing at BCL11A exon 2 in PBiEPC-1 and HUDEP-2. Numbers indicate mean ± standard deviation (SD) from two independent experiments. RNP: ribonucleoprotein complex containing Cas9 protein and gRNA.

    Techniques Used: CRISPR, Sequencing, Flow Cytometry, Standard Deviation

    bcl11a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc bcl11a
    KEY RESOURCES TABLE
    Bcl11a, 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
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    1) Product Images from "BCL2 Amplicon Loss and Transcriptional Remodeling Drives ABT-199 Resistance in B Cell Lymphoma Models"

    Article Title: BCL2 Amplicon Loss and Transcriptional Remodeling Drives ABT-199 Resistance in B Cell Lymphoma Models

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2019.04.005

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Protease Inhibitor, Immunoprecipitation, SYBR Green Assay, Expressing, Software

    bcl11a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc bcl11a
    IGF2BP1-OE regulates <t>BCL11A</t> and HMGA2 in human adult erythroblasts. IGF2BP1-OE and control transductions in adult CD34(+) cells were investigated for the mRNA levels of (A) BCL11A, (B) HMGA2, (C) ZBTB7A, (D) KLF1, and (E) SOX6. RT-qPCRs were performed at culture day 14. Open bars represent empty vector control and black bars represent IGF2BP1-OE. Mean value ± SD of three independent donors for each condition is shown. P values were calculated using two-tailed Student’s t test. *P < 0.05. Western blot analyses of (F) BCL11A, HMGA2, and ZBTB7A and (G) KLF1 and SOX6 expression using protein extracts at culture day 14 of empty vector control and IGF2BP1 overexpressed CD34(+) cells are shown. Histone H3 and lamin B1 were used as loading controls. Molecular weight is shown in kilodaltons (kDa). C, empty vector control transduction; OE, IGF2BP1 overexpression.
    Bcl11a, 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
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    1) Product Images from "IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts"

    Article Title: IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1609552114

    IGF2BP1-OE regulates BCL11A and HMGA2 in human adult erythroblasts. IGF2BP1-OE and control transductions in adult CD34(+) cells were investigated for the mRNA levels of (A) BCL11A, (B) HMGA2, (C) ZBTB7A, (D) KLF1, and (E) SOX6. RT-qPCRs were performed at culture day 14. Open bars represent empty vector control and black bars represent IGF2BP1-OE. Mean value ± SD of three independent donors for each condition is shown. P values were calculated using two-tailed Student’s t test. *P < 0.05. Western blot analyses of (F) BCL11A, HMGA2, and ZBTB7A and (G) KLF1 and SOX6 expression using protein extracts at culture day 14 of empty vector control and IGF2BP1 overexpressed CD34(+) cells are shown. Histone H3 and lamin B1 were used as loading controls. Molecular weight is shown in kilodaltons (kDa). C, empty vector control transduction; OE, IGF2BP1 overexpression.
    Figure Legend Snippet: IGF2BP1-OE regulates BCL11A and HMGA2 in human adult erythroblasts. IGF2BP1-OE and control transductions in adult CD34(+) cells were investigated for the mRNA levels of (A) BCL11A, (B) HMGA2, (C) ZBTB7A, (D) KLF1, and (E) SOX6. RT-qPCRs were performed at culture day 14. Open bars represent empty vector control and black bars represent IGF2BP1-OE. Mean value ± SD of three independent donors for each condition is shown. P values were calculated using two-tailed Student’s t test. *P < 0.05. Western blot analyses of (F) BCL11A, HMGA2, and ZBTB7A and (G) KLF1 and SOX6 expression using protein extracts at culture day 14 of empty vector control and IGF2BP1 overexpressed CD34(+) cells are shown. Histone H3 and lamin B1 were used as loading controls. Molecular weight is shown in kilodaltons (kDa). C, empty vector control transduction; OE, IGF2BP1 overexpression.

    Techniques Used: Plasmid Preparation, Two Tailed Test, Western Blot, Expressing, Molecular Weight, Transduction, Over Expression

    Protein analyses of IGF2BP1 knockdown or overexpression in cord blood erythroblasts. Western images for IGF2BP1, IGF2BP3, LIN28B, BCL11A, HMGA2, and ZBTB7A expression using protein extracts at culture day 14. Lamin B1, histone H3, and beta-actin were used as loading controls. Molecular weight is shown in kilodaltons (kDa). Percentage of fetal hemoglobin (%HbF) shown corresponds to the representative HPLC profiles shown in Fig. S7. Control 1, empty vector control transduction for knockdown vector; control 2, empty vector control transduction for overexpression vector; KD, knockdown; OE, overexpression.
    Figure Legend Snippet: Protein analyses of IGF2BP1 knockdown or overexpression in cord blood erythroblasts. Western images for IGF2BP1, IGF2BP3, LIN28B, BCL11A, HMGA2, and ZBTB7A expression using protein extracts at culture day 14. Lamin B1, histone H3, and beta-actin were used as loading controls. Molecular weight is shown in kilodaltons (kDa). Percentage of fetal hemoglobin (%HbF) shown corresponds to the representative HPLC profiles shown in Fig. S7. Control 1, empty vector control transduction for knockdown vector; control 2, empty vector control transduction for overexpression vector; KD, knockdown; OE, overexpression.

    Techniques Used: Over Expression, Western Blot, Expressing, Molecular Weight, Plasmid Preparation, Transduction

    IGF2BP1 colocalizes with BCL11A in the ribosomes. RNA immunoprecipitation (RIP) using antibody against IGF2BP1 was performed to assess binding of RNAs to IGF2BP1 protein followed by RT-qPCR quantitation for (A) BCL11A transcripts. RIP was performed at culture day 14. Mean value ± SD of three independent donors for each condition is shown. (B) BCL11A stability analysis in IGF2BP1-OE versus empty vector control after actinomycin D treatment at culture day 14. Time course for RNA stability started by adding the transcription inhibitor actinomycin D (10 µg/mL) and cells were harvested at the indicated time points. Expression levels (percentage) were calculated considering the time point “0 h” as 100%. Mean value ± SD of two independent donors for each condition is shown. (C–H) Polysome profiling analysis of IGF2BP1-OE versus empty vector control at culture day 12. A representative polysome gradient (control versus IGF2BP1-OE) is shown in C. BCL11A mRNA was measured by RT-qPCR quantitation on the polysome fractions and mean value ± SD of two independent donors for each condition is shown in D. Western blot analysis of IGF2BP1 on representative polysome fractions from one representative donor is shown in E. Beta-globin (F), gamma-globin (G), and beta-globin + gamma-globin (H) mRNAs were measured by RT-qPCR quantitation on representative polysome profiling fractions. Results are shown from one representative donor. Molecular weight is shown in kilodaltons (kDa). P value was calculated using two-tailed Student’s t test. *P < 0.05. Experiments were performed in adult CD34(+) cells. BP1-RIP, IGF2BP1 RNA immunoprecipitation; IgG, immunoprecipitation with isotype control; Input, RNA sample before immunoprecipitation; OE, overexpression.
    Figure Legend Snippet: IGF2BP1 colocalizes with BCL11A in the ribosomes. RNA immunoprecipitation (RIP) using antibody against IGF2BP1 was performed to assess binding of RNAs to IGF2BP1 protein followed by RT-qPCR quantitation for (A) BCL11A transcripts. RIP was performed at culture day 14. Mean value ± SD of three independent donors for each condition is shown. (B) BCL11A stability analysis in IGF2BP1-OE versus empty vector control after actinomycin D treatment at culture day 14. Time course for RNA stability started by adding the transcription inhibitor actinomycin D (10 µg/mL) and cells were harvested at the indicated time points. Expression levels (percentage) were calculated considering the time point “0 h” as 100%. Mean value ± SD of two independent donors for each condition is shown. (C–H) Polysome profiling analysis of IGF2BP1-OE versus empty vector control at culture day 12. A representative polysome gradient (control versus IGF2BP1-OE) is shown in C. BCL11A mRNA was measured by RT-qPCR quantitation on the polysome fractions and mean value ± SD of two independent donors for each condition is shown in D. Western blot analysis of IGF2BP1 on representative polysome fractions from one representative donor is shown in E. Beta-globin (F), gamma-globin (G), and beta-globin + gamma-globin (H) mRNAs were measured by RT-qPCR quantitation on representative polysome profiling fractions. Results are shown from one representative donor. Molecular weight is shown in kilodaltons (kDa). P value was calculated using two-tailed Student’s t test. *P < 0.05. Experiments were performed in adult CD34(+) cells. BP1-RIP, IGF2BP1 RNA immunoprecipitation; IgG, immunoprecipitation with isotype control; Input, RNA sample before immunoprecipitation; OE, overexpression.

    Techniques Used: Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Quantitation Assay, Plasmid Preparation, Expressing, Western Blot, Molecular Weight, Two Tailed Test, Over Expression

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    Cell Signaling Technology Inc anti bcl11a
    Loss of Zbtb1 and Cbfa2t3 leads to the T-lineage differentiation arrest in LPs. A , specific depletion of targeted Lmo2, Zbtb1, and Cbfa2t3 proteins. sgRNA against Lmo2 , Zbtb1 , Cbfa2t2 , or Cbfa2t3 was introduced into the Cas9-expressing (GFP + ) LPs. Four days after sgRNA transduction, nuclear lysates from retrovirus infected GFP + hNGFR + cells were subjected to immunoblotting for Lmo2, Zbtb1, Cbfa2t3, <t>Bcl11a,</t> and LaminB. Two independent experiments were performed with similar results. B , an experimental scheme for the deletion of Lmo2 , Zbtb1 , Cbfa2t3 , and Cbfa2t2 using the CRISPR/Cas9 system in Ebf1 -deficient LPs is shown. C , retroviral vectors encoding sgRNAs against luciferase (sgControl), Lmo2 (sgLmo2), Zbtb1 (sgZbtb1), Cbfa2t3 (sgCbfa2t3), or Cbfa2t2 (sgCbfa2t2) were introduced into Cas9-expressing (GFP + ) LPs. Five days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA transduced cells were gated and analyzed for CD44 and CD25 expression. D , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( C ) is shown with standard deviation (SD). E , ten days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. F , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( E ) is shown with SD. Data are representative of two ( A ) or three ( C and E ) independent experiments. Data represent the mean values of three independent biological replicates ( D and F ). ∗∗ p < 0.01 by two-sided Student’s t test. LP, lymphoid progenitor; sgRNA, single-guide RNA.
    Anti Bcl11a, 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
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    94
    Cell Signaling Technology Inc bcl11a
    CARM1 inhibitors activate IFN α/γ pathway genes via inducing the formation of <t>BCL11A/PBAF</t> complex and elevating H3K27Ac levels. ( A ) Venn diagram showing the overlap of DEGs induced by JQ1, CARM1 inhibitor or JQ1 + CARM1 inhibitor in MDA-MB-468 cells ( top ) or HCI-002 ( bottom ). ( B ) Heatmap showing Hallmark gene sets up- (red) or down-regulated (blue) by JQ1, TP-064 or JQ1 + TP-064 treatment in MDA-MB-468 cells. ( C )GSEA of IFN α pathway signature genes induced by JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells. ( D ) Q-RT-PCR analyses of indicated IFNα pathway genes after treatment with JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells ( n = 3). Data are mean ± s.d. ** P < 0.01; * P < 0.05; NS: not significant. ( E ) Increased (red) and decreased (blue) me-BAF155, BRD4, and H3K27Ac ChIP-seq signals after TP-064 or JQ1 treatment in MDA-MB-468 cells on IFN α and γ genes that had increased expressions (magenta) after TP-064 treatment. Many of the IFN genes have expression level decreased (blue) after JQ1 treatment. ( F ) Changes of H3K27Ac ChIP-seq signals on genes from the Hallmark IFNα ( left ) and IFNγ (right) response gene sets induced by JQ1 or TP064. ( G, H ) Scatter plot showing the correlation of mRNA level changes between MDA-MB-468 (x-axis) and HCI-002 tumors (y-axis) by JQ1 (G) or TP-064/EZM2302 (H). IFN α pathway only genes are depicted in blue dots, IFN γ pathway only genes are depicted in yellow dots, and IFN α/γ shared pathway genes are depicted in green dots. ( I ) Enriched transcription factor motifs at H3K27Ac peaks of the promoters and gene bodies of IFN pathway genes. ( J ) Immunoblotting of BCL11A protein levels after treatment of MDA-MB-468 cells with TP-064 for indicated time. ( K, L ) Western blotting of indicated proteins from nuclear extracts of MDA-MB-468 cells by glycerol density gradient (10–45%) after vehicle (K) or TP-064 (L) treatment. ( M ) Summary of ChIP-qPCR data of indicated proteins binding to SEs or IFN pathway genes after JQ1 ( left ) or TP-064 ( right ) treatment in MDA-MB-468 cells. Red depicts high level and blue depicts low level binding of indicated proteins (y-axis), respectively. ( N ) Western blotting of BCL11A in parental or BCL11A knockdown MDA-MB-468 cells. ( O ) Q-RT-PCR analyses of IFNα pathway genes after treatment with DMSO or TP-064 in parental, BCL11A KD and overexpressing MDA-MB-468 cells. ** P < 0.01; * P < 0.05; NS: not significant.
    Bcl11a, 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/result/bcl11a/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
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    Image Search Results


    Loss of Zbtb1 and Cbfa2t3 leads to the T-lineage differentiation arrest in LPs. A , specific depletion of targeted Lmo2, Zbtb1, and Cbfa2t3 proteins. sgRNA against Lmo2 , Zbtb1 , Cbfa2t2 , or Cbfa2t3 was introduced into the Cas9-expressing (GFP + ) LPs. Four days after sgRNA transduction, nuclear lysates from retrovirus infected GFP + hNGFR + cells were subjected to immunoblotting for Lmo2, Zbtb1, Cbfa2t3, Bcl11a, and LaminB. Two independent experiments were performed with similar results. B , an experimental scheme for the deletion of Lmo2 , Zbtb1 , Cbfa2t3 , and Cbfa2t2 using the CRISPR/Cas9 system in Ebf1 -deficient LPs is shown. C , retroviral vectors encoding sgRNAs against luciferase (sgControl), Lmo2 (sgLmo2), Zbtb1 (sgZbtb1), Cbfa2t3 (sgCbfa2t3), or Cbfa2t2 (sgCbfa2t2) were introduced into Cas9-expressing (GFP + ) LPs. Five days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA transduced cells were gated and analyzed for CD44 and CD25 expression. D , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( C ) is shown with standard deviation (SD). E , ten days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. F , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( E ) is shown with SD. Data are representative of two ( A ) or three ( C and E ) independent experiments. Data represent the mean values of three independent biological replicates ( D and F ). ∗∗ p < 0.01 by two-sided Student’s t test. LP, lymphoid progenitor; sgRNA, single-guide RNA.

    Journal: The Journal of Biological Chemistry

    Article Title: Transcription factor Zbtb1 interacts with bridging factor Lmo2 and maintains the T-lineage differentiation capacity of lymphoid progenitor cells

    doi: 10.1016/j.jbc.2022.102506

    Figure Lengend Snippet: Loss of Zbtb1 and Cbfa2t3 leads to the T-lineage differentiation arrest in LPs. A , specific depletion of targeted Lmo2, Zbtb1, and Cbfa2t3 proteins. sgRNA against Lmo2 , Zbtb1 , Cbfa2t2 , or Cbfa2t3 was introduced into the Cas9-expressing (GFP + ) LPs. Four days after sgRNA transduction, nuclear lysates from retrovirus infected GFP + hNGFR + cells were subjected to immunoblotting for Lmo2, Zbtb1, Cbfa2t3, Bcl11a, and LaminB. Two independent experiments were performed with similar results. B , an experimental scheme for the deletion of Lmo2 , Zbtb1 , Cbfa2t3 , and Cbfa2t2 using the CRISPR/Cas9 system in Ebf1 -deficient LPs is shown. C , retroviral vectors encoding sgRNAs against luciferase (sgControl), Lmo2 (sgLmo2), Zbtb1 (sgZbtb1), Cbfa2t3 (sgCbfa2t3), or Cbfa2t2 (sgCbfa2t2) were introduced into Cas9-expressing (GFP + ) LPs. Five days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA transduced cells were gated and analyzed for CD44 and CD25 expression. D , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( C ) is shown with standard deviation (SD). E , ten days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP + hNGFR + sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. F , the percentage of CD25 + cells among GFP + hNGFR + sgRNA transduced cells ( E ) is shown with SD. Data are representative of two ( A ) or three ( C and E ) independent experiments. Data represent the mean values of three independent biological replicates ( D and F ). ∗∗ p < 0.01 by two-sided Student’s t test. LP, lymphoid progenitor; sgRNA, single-guide RNA.

    Article Snippet: The antibodies used for the immunoblot analysis were anti-LaminB (CST, 13435), anti-Myc (MBL; M192-3), anti-Zbtb1 (Bethyl; S303-242A), anti-Lmo2 (Novus, NB110-78626), anti-Bcl11a (CST; 75432), and anti-Cbfa2t3 (ProteinTech; 17190-1-AP).

    Techniques: Expressing, Transduction, Infection, Western Blot, CRISPR, Luciferase, Standard Deviation

    Zbtb1 and Cbfa2t3 are involved in the regulation of Tcf7 expression in LPs. A , experimental scheme for the transcriptome analysis is shown. Five days after sgRNA introduction, GFP + hNGFR + LP cells were sorted, and subjected to QuantSeq 3′ mRNA sequencing. B , GO annotation was performed using the DAVID analysis tool. Top five GO terms for DEGs ( p < 0.05, |log2FC| > 1, and TPM > 10) in sgZbtb1-introduced Ebf1 -deficient LPs are shown. The full lists of the DEGs in Lmo2 -, Zbtb1 -, Cbfa2t2 -, or Cbfa2t3 -deficient LPs are shown in <xref ref-type=Table S2 . Data are based on three independent biological replicates. C , heat map showing the expression changes of representative Notch-activated genes in pro-T cell stages in response to the deletion of Lmo2 , Zbtb1 , or Cbfa2t3 . Data are based on the average of three biological replicates. D , five days after sgRNA introduction, GFP + hNGFR + LP cells were sorted ( A ). Relative expression levels of Tcf7 , Bcl11b , Bcl11a , and Gata3 against Actb were determined by RT-qPCR. The relative expression against sgControl-introduced cells is shown with SD. ∗∗ p < 0.01, ∗ p < 0.05 by two-sided Student’s t test. Data are based on three biological replicates. DEG, differentially expressed gene; GO, Gene Ontology; LP, lymphoid progenitor; sgRNA, single-guide RNA; TPM, transcripts per kilobase million. " width="100%" height="100%">

    Journal: The Journal of Biological Chemistry

    Article Title: Transcription factor Zbtb1 interacts with bridging factor Lmo2 and maintains the T-lineage differentiation capacity of lymphoid progenitor cells

    doi: 10.1016/j.jbc.2022.102506

    Figure Lengend Snippet: Zbtb1 and Cbfa2t3 are involved in the regulation of Tcf7 expression in LPs. A , experimental scheme for the transcriptome analysis is shown. Five days after sgRNA introduction, GFP + hNGFR + LP cells were sorted, and subjected to QuantSeq 3′ mRNA sequencing. B , GO annotation was performed using the DAVID analysis tool. Top five GO terms for DEGs ( p < 0.05, |log2FC| > 1, and TPM > 10) in sgZbtb1-introduced Ebf1 -deficient LPs are shown. The full lists of the DEGs in Lmo2 -, Zbtb1 -, Cbfa2t2 -, or Cbfa2t3 -deficient LPs are shown in Table S2 . Data are based on three independent biological replicates. C , heat map showing the expression changes of representative Notch-activated genes in pro-T cell stages in response to the deletion of Lmo2 , Zbtb1 , or Cbfa2t3 . Data are based on the average of three biological replicates. D , five days after sgRNA introduction, GFP + hNGFR + LP cells were sorted ( A ). Relative expression levels of Tcf7 , Bcl11b , Bcl11a , and Gata3 against Actb were determined by RT-qPCR. The relative expression against sgControl-introduced cells is shown with SD. ∗∗ p < 0.01, ∗ p < 0.05 by two-sided Student’s t test. Data are based on three biological replicates. DEG, differentially expressed gene; GO, Gene Ontology; LP, lymphoid progenitor; sgRNA, single-guide RNA; TPM, transcripts per kilobase million.

    Article Snippet: The antibodies used for the immunoblot analysis were anti-LaminB (CST, 13435), anti-Myc (MBL; M192-3), anti-Zbtb1 (Bethyl; S303-242A), anti-Lmo2 (Novus, NB110-78626), anti-Bcl11a (CST; 75432), and anti-Cbfa2t3 (ProteinTech; 17190-1-AP).

    Techniques: Expressing, Sequencing, Quantitative RT-PCR

    CARM1 inhibitors activate IFN α/γ pathway genes via inducing the formation of BCL11A/PBAF complex and elevating H3K27Ac levels. ( A ) Venn diagram showing the overlap of DEGs induced by JQ1, CARM1 inhibitor or JQ1 + CARM1 inhibitor in MDA-MB-468 cells ( top ) or HCI-002 ( bottom ). ( B ) Heatmap showing Hallmark gene sets up- (red) or down-regulated (blue) by JQ1, TP-064 or JQ1 + TP-064 treatment in MDA-MB-468 cells. ( C )GSEA of IFN α pathway signature genes induced by JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells. ( D ) Q-RT-PCR analyses of indicated IFNα pathway genes after treatment with JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells ( n = 3). Data are mean ± s.d. ** P < 0.01; * P < 0.05; NS: not significant. ( E ) Increased (red) and decreased (blue) me-BAF155, BRD4, and H3K27Ac ChIP-seq signals after TP-064 or JQ1 treatment in MDA-MB-468 cells on IFN α and γ genes that had increased expressions (magenta) after TP-064 treatment. Many of the IFN genes have expression level decreased (blue) after JQ1 treatment. ( F ) Changes of H3K27Ac ChIP-seq signals on genes from the Hallmark IFNα ( left ) and IFNγ (right) response gene sets induced by JQ1 or TP064. ( G, H ) Scatter plot showing the correlation of mRNA level changes between MDA-MB-468 (x-axis) and HCI-002 tumors (y-axis) by JQ1 (G) or TP-064/EZM2302 (H). IFN α pathway only genes are depicted in blue dots, IFN γ pathway only genes are depicted in yellow dots, and IFN α/γ shared pathway genes are depicted in green dots. ( I ) Enriched transcription factor motifs at H3K27Ac peaks of the promoters and gene bodies of IFN pathway genes. ( J ) Immunoblotting of BCL11A protein levels after treatment of MDA-MB-468 cells with TP-064 for indicated time. ( K, L ) Western blotting of indicated proteins from nuclear extracts of MDA-MB-468 cells by glycerol density gradient (10–45%) after vehicle (K) or TP-064 (L) treatment. ( M ) Summary of ChIP-qPCR data of indicated proteins binding to SEs or IFN pathway genes after JQ1 ( left ) or TP-064 ( right ) treatment in MDA-MB-468 cells. Red depicts high level and blue depicts low level binding of indicated proteins (y-axis), respectively. ( N ) Western blotting of BCL11A in parental or BCL11A knockdown MDA-MB-468 cells. ( O ) Q-RT-PCR analyses of IFNα pathway genes after treatment with DMSO or TP-064 in parental, BCL11A KD and overexpressing MDA-MB-468 cells. ** P < 0.01; * P < 0.05; NS: not significant.

    Journal: Nucleic Acids Research

    Article Title: BAF155 methylation drives metastasis by hijacking super-enhancers and subverting anti-tumor immunity

    doi: 10.1093/nar/gkab1122

    Figure Lengend Snippet: CARM1 inhibitors activate IFN α/γ pathway genes via inducing the formation of BCL11A/PBAF complex and elevating H3K27Ac levels. ( A ) Venn diagram showing the overlap of DEGs induced by JQ1, CARM1 inhibitor or JQ1 + CARM1 inhibitor in MDA-MB-468 cells ( top ) or HCI-002 ( bottom ). ( B ) Heatmap showing Hallmark gene sets up- (red) or down-regulated (blue) by JQ1, TP-064 or JQ1 + TP-064 treatment in MDA-MB-468 cells. ( C )GSEA of IFN α pathway signature genes induced by JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells. ( D ) Q-RT-PCR analyses of indicated IFNα pathway genes after treatment with JQ1, TP-064 or JQ1 and TP-064 in combination in MDA-MB-468 cells ( n = 3). Data are mean ± s.d. ** P < 0.01; * P < 0.05; NS: not significant. ( E ) Increased (red) and decreased (blue) me-BAF155, BRD4, and H3K27Ac ChIP-seq signals after TP-064 or JQ1 treatment in MDA-MB-468 cells on IFN α and γ genes that had increased expressions (magenta) after TP-064 treatment. Many of the IFN genes have expression level decreased (blue) after JQ1 treatment. ( F ) Changes of H3K27Ac ChIP-seq signals on genes from the Hallmark IFNα ( left ) and IFNγ (right) response gene sets induced by JQ1 or TP064. ( G, H ) Scatter plot showing the correlation of mRNA level changes between MDA-MB-468 (x-axis) and HCI-002 tumors (y-axis) by JQ1 (G) or TP-064/EZM2302 (H). IFN α pathway only genes are depicted in blue dots, IFN γ pathway only genes are depicted in yellow dots, and IFN α/γ shared pathway genes are depicted in green dots. ( I ) Enriched transcription factor motifs at H3K27Ac peaks of the promoters and gene bodies of IFN pathway genes. ( J ) Immunoblotting of BCL11A protein levels after treatment of MDA-MB-468 cells with TP-064 for indicated time. ( K, L ) Western blotting of indicated proteins from nuclear extracts of MDA-MB-468 cells by glycerol density gradient (10–45%) after vehicle (K) or TP-064 (L) treatment. ( M ) Summary of ChIP-qPCR data of indicated proteins binding to SEs or IFN pathway genes after JQ1 ( left ) or TP-064 ( right ) treatment in MDA-MB-468 cells. Red depicts high level and blue depicts low level binding of indicated proteins (y-axis), respectively. ( N ) Western blotting of BCL11A in parental or BCL11A knockdown MDA-MB-468 cells. ( O ) Q-RT-PCR analyses of IFNα pathway genes after treatment with DMSO or TP-064 in parental, BCL11A KD and overexpressing MDA-MB-468 cells. ** P < 0.01; * P < 0.05; NS: not significant.

    Article Snippet: Solubilized chromatin was diluted and incubated incubated at 4°C overnight with the following antibodies: anti-me-BAF155 , anti-BAF155 (D7F8S, Cell Signaling Technology), anti-H3K4me1 (Cell Signaling Technology), anti-ARID1A (D2A8U, Cell Signaling Technology), anti-ARID1B (E9J4T, Cell Signaling Technology), anti-ARID2 (D8D8U, Cell Signaling Technology), BRG1 (D1Q7F, Cell Signaling Technology), BCL11A (D4E3P, Cell Signaling Technology), anti-Ac-CBP/p300 (Cell Signaling Technology), anti-p300 (D8Z4E, Cell Signaling Technology), anti-HDAC1 (D5C6U, Cell Signaling Technology), anti-PBRM1 (D3F7O, Cell Signaling Technology), anti-H3K27Ac (Abcam), anti-BRD7 (Abcam), anti-BRD9 (Abcam), anti-BRD4 (Bethyl).

    Techniques: Reverse Transcription Polymerase Chain Reaction, ChIP-sequencing, Expressing, Western Blot, Binding Assay

    Immunostaining of me-BAF155 in CTCs of metastatic breast cancer patients. ( A ) Images of me-BAF155 immunostaining in the nucleus of CTCs but not in WBCs from seven breast cancer patients. Representative images of CTCs ( left ) and WBCs ( right ) are shown for each patient (ID listed to the left). Each row of tiles includes an image crop of the intensity distribution of each of the stains included in the panel (Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and Bright Field (BF)) for one single cell, including a merge of all stains. Images were taken at 20x magnification; scale bars represent 10 μm. ( B ) The average me-BAF155 nuclear signal intensities in breast cancer patient CTCs. Each dot represents the average me-BAF155 nuclear staining intensities of each individual CTC from one of the seven patients. The red bars indicate the average me-BAF155 nuclear signals of all CTCs from an individual patient sample. ( C ) The tracing of me-BAF155 nuclear signal intensities measurement in CTCs from breast cancer patient 548. The number of CTCs are depicted in x-axis. ( D ) Representative immunofluorescence images of CTCs stained with Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and merged image of all staining signals for patient 548 in April 2021. Scale bars represent 10 μm. ( E, F ) Models depicting dual functions of me-BAF155 dependent metastasis in TNBC cells and anti-metastasis effects by CARM1 inhibition. Me-BAF155 and BRD4 interact at SEs to activate oncogenes and me-BAF155 containing SWI/SNF complex interacts with HDAC1 to suppress ISGs (E). CARM1 inhibitor treatment leads to increased BCL11A and un-methylated BAF155, which assemble with other SWI/SNF subunits to form PBAF to activate ISGs. Un-methylated BAF155 triggers dissociation of BRD4 from SEs, resulting in inhibition of expression of SE-addicted oncogenes (F).

    Journal: Nucleic Acids Research

    Article Title: BAF155 methylation drives metastasis by hijacking super-enhancers and subverting anti-tumor immunity

    doi: 10.1093/nar/gkab1122

    Figure Lengend Snippet: Immunostaining of me-BAF155 in CTCs of metastatic breast cancer patients. ( A ) Images of me-BAF155 immunostaining in the nucleus of CTCs but not in WBCs from seven breast cancer patients. Representative images of CTCs ( left ) and WBCs ( right ) are shown for each patient (ID listed to the left). Each row of tiles includes an image crop of the intensity distribution of each of the stains included in the panel (Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and Bright Field (BF)) for one single cell, including a merge of all stains. Images were taken at 20x magnification; scale bars represent 10 μm. ( B ) The average me-BAF155 nuclear signal intensities in breast cancer patient CTCs. Each dot represents the average me-BAF155 nuclear staining intensities of each individual CTC from one of the seven patients. The red bars indicate the average me-BAF155 nuclear signals of all CTCs from an individual patient sample. ( C ) The tracing of me-BAF155 nuclear signal intensities measurement in CTCs from breast cancer patient 548. The number of CTCs are depicted in x-axis. ( D ) Representative immunofluorescence images of CTCs stained with Hoechst, me-BAF155, Exclusion (CD45/CD34/CD66b), pCK and merged image of all staining signals for patient 548 in April 2021. Scale bars represent 10 μm. ( E, F ) Models depicting dual functions of me-BAF155 dependent metastasis in TNBC cells and anti-metastasis effects by CARM1 inhibition. Me-BAF155 and BRD4 interact at SEs to activate oncogenes and me-BAF155 containing SWI/SNF complex interacts with HDAC1 to suppress ISGs (E). CARM1 inhibitor treatment leads to increased BCL11A and un-methylated BAF155, which assemble with other SWI/SNF subunits to form PBAF to activate ISGs. Un-methylated BAF155 triggers dissociation of BRD4 from SEs, resulting in inhibition of expression of SE-addicted oncogenes (F).

    Article Snippet: Solubilized chromatin was diluted and incubated incubated at 4°C overnight with the following antibodies: anti-me-BAF155 , anti-BAF155 (D7F8S, Cell Signaling Technology), anti-H3K4me1 (Cell Signaling Technology), anti-ARID1A (D2A8U, Cell Signaling Technology), anti-ARID1B (E9J4T, Cell Signaling Technology), anti-ARID2 (D8D8U, Cell Signaling Technology), BRG1 (D1Q7F, Cell Signaling Technology), BCL11A (D4E3P, Cell Signaling Technology), anti-Ac-CBP/p300 (Cell Signaling Technology), anti-p300 (D8Z4E, Cell Signaling Technology), anti-HDAC1 (D5C6U, Cell Signaling Technology), anti-PBRM1 (D3F7O, Cell Signaling Technology), anti-H3K27Ac (Abcam), anti-BRD7 (Abcam), anti-BRD9 (Abcam), anti-BRD4 (Bethyl).

    Techniques: Immunostaining, Staining, Immunofluorescence, Inhibition, Methylation, Expressing