anti h2bub1 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti h2bub1 antibody
    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) <t>H2Bub1</t> and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).
    Anti H2bub1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti h2bub1 antibody/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti h2bub1 antibody - by Bioz Stars, 2023-09
    95/100 stars

    Images

    1) Product Images from "BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis"

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2023.02.014

    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).
    Figure Legend Snippet: The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).

    Techniques Used: RNA Sequencing Assay, Infection, Expressing

    BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, Infection, Staining, Immunohistochemistry

    H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, ChIP-sequencing, Infection, Binding Assay

    TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, Western Blot, Immunofluorescence, Staining

    Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.
    Figure Legend Snippet: Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.

    Techniques Used: Injection, Western Blot, Expressing, Fluorescence, Immunofluorescence, Staining, Micro-CT

    Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis
    Figure Legend Snippet: Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis

    Techniques Used:

    anti h2bub1 antibody  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc anti h2bub1 antibody
    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) <t>H2Bub1</t> and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).
    Anti H2bub1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti h2bub1 antibody/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti h2bub1 antibody - by Bioz Stars, 2023-09
    95/100 stars

    Images

    1) Product Images from "BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis"

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2023.02.014

    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).
    Figure Legend Snippet: The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).

    Techniques Used: RNA Sequencing Assay, Infection, Expressing

    BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, Infection, Staining, Immunohistochemistry

    H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, ChIP-sequencing, Infection, Binding Assay

    TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, Western Blot, Immunofluorescence, Staining

    Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.
    Figure Legend Snippet: Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.

    Techniques Used: Injection, Western Blot, Expressing, Fluorescence, Immunofluorescence, Staining, Micro-CT

    Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis
    Figure Legend Snippet: Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis

    Techniques Used:

    anti h2bub1 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti h2bub1 antibody
    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) <t>H2Bub1</t> and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).
    Anti H2bub1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti h2bub1 antibody/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti h2bub1 antibody - by Bioz Stars, 2023-09
    95/100 stars

    Images

    1) Product Images from "BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis"

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2023.02.014

    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).
    Figure Legend Snippet: The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).

    Techniques Used: RNA Sequencing Assay, Infection, Expressing

    BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, Infection, Staining, Immunohistochemistry

    H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, ChIP-sequencing, Infection, Binding Assay

    TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, Western Blot, Immunofluorescence, Staining

    Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.
    Figure Legend Snippet: Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.

    Techniques Used: Injection, Western Blot, Expressing, Fluorescence, Immunofluorescence, Staining, Micro-CT

    Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis
    Figure Legend Snippet: Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis

    Techniques Used:

    anti h2bub1 antibody  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc anti h2bub1 antibody
    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) <t>H2Bub1</t> and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).
    Anti H2bub1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti h2bub1 antibody/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti h2bub1 antibody - by Bioz Stars, 2023-09
    95/100 stars

    Images

    1) Product Images from "BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis"

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2023.02.014

    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).
    Figure Legend Snippet: The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).

    Techniques Used: RNA Sequencing Assay, Infection, Expressing

    BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, Infection, Staining, Immunohistochemistry

    H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, ChIP-sequencing, Infection, Binding Assay

    TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, Western Blot, Immunofluorescence, Staining

    Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.
    Figure Legend Snippet: Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.

    Techniques Used: Injection, Western Blot, Expressing, Fluorescence, Immunofluorescence, Staining, Micro-CT

    Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis
    Figure Legend Snippet: Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis

    Techniques Used:

    anti h2bub1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti h2bub1
    (A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of H2B, <t>H2Bub1</t> and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.
    Anti H2bub1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications"

    Article Title: Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061447

    (A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of H2B, H2Bub1 and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.
    Figure Legend Snippet: (A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of H2B, H2Bub1 and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.

    Techniques Used: Expressing, Amplification, Positive Control, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot

    The figure summarizes the regulations of the genes described in this study, highlighting a central position of NKX2-1. Chromosomal aberrations activate expression of MLL (11q23) and histones including H2B (6p22). MLL together with H2Bub1 generate an activatory chromatin structure at NKX2-1. This structure is reinforced by reduced expression of USP46 and E2F6 and elevated expression of RNF20/40, JMJD3 and HOPX, and mediates activation of NKX2-1. NKX2-1 in turn activates directly expression of HEY1 which performs inhibition of B-cell differentiation. Both, NKX2-1 and HEY1 contribute to the activatory chromatin structure by regulating RNF40 and E2F6, respectively. IL4/STAT3-signaling enhances expression of NKX2-1. TNFa, cGMP, cAMP and PRKCE support NFkB which activates both NKX2-1 and HEY1. Reduced expression of PDE6D and enhanced expression of NOS1 contribute to elevated cGMP. NOS1 and PRKCE are activated by NKX2-1. HEY1 mediates reduced expression of PDE4A resulting in elevated cAMP levels. Finally, NOTCH-signaling and TGFb-signaling (via SMAD) activate HEY1 expression. SMAD and NKX2-1 interact and coactivate HEY1 transcription.
    Figure Legend Snippet: The figure summarizes the regulations of the genes described in this study, highlighting a central position of NKX2-1. Chromosomal aberrations activate expression of MLL (11q23) and histones including H2B (6p22). MLL together with H2Bub1 generate an activatory chromatin structure at NKX2-1. This structure is reinforced by reduced expression of USP46 and E2F6 and elevated expression of RNF20/40, JMJD3 and HOPX, and mediates activation of NKX2-1. NKX2-1 in turn activates directly expression of HEY1 which performs inhibition of B-cell differentiation. Both, NKX2-1 and HEY1 contribute to the activatory chromatin structure by regulating RNF40 and E2F6, respectively. IL4/STAT3-signaling enhances expression of NKX2-1. TNFa, cGMP, cAMP and PRKCE support NFkB which activates both NKX2-1 and HEY1. Reduced expression of PDE6D and enhanced expression of NOS1 contribute to elevated cGMP. NOS1 and PRKCE are activated by NKX2-1. HEY1 mediates reduced expression of PDE4A resulting in elevated cAMP levels. Finally, NOTCH-signaling and TGFb-signaling (via SMAD) activate HEY1 expression. SMAD and NKX2-1 interact and coactivate HEY1 transcription.

    Techniques Used: Expressing, Activation Assay, Inhibition, Cell Differentiation

    h2bub1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc h2bub1
    Downregulated expression of target genes by OsUBR7-mediated reduction of <t>H2Bub1</t> results in suppression of cell-cycle progression. (A) Diagrammatic representation of ChIP-seq maps (using an anti-H2Bub1 antibody; n = 2 biological repeats) for four gene loci at which the H2Bub1 level was completely suppressed or decreased in osubr7 seedlings . (B) ChIP–qPCR analysis to determine H2Bub1 levels in candidate target genes, including the two pleiotropic genes and two cell-cycle-related genes shown in (A) . P1 to P2 represent regions covered by the primers used to assess the level of H2Bub1 by qPCR following ChIP. Data were normalized to the input chromatin, OsNRAMP2 was used as the reference, and the values of H2Bub1 levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test). (C) qRT–PCR analysis of five pleiotropic genes selected from the ChIP-seq and transcriptome data in 14-day-old WT and osubr7 seedlings. Actin1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 (Student’s t -test). (D) Relative expression of six cell-cycle marker genes in 14-day-old WT and osubr7 seedlings. UFC1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t- test). NS, not significant. (E) Cell numbers during cell-cycle phases with 2C and 4C nuclei in WT and osubr7 seedlings (7 days old) measured by flow cytometry. (F) Percentage of cells in different phases of the cell cycle in WT and osubr7 seedlings. Data are means ± SD ( n = 4 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test).
    H2bub1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Rice OsUBR7 modulates plant height by regulating histone H2B monoubiquitination and cell proliferation"

    Article Title: Rice OsUBR7 modulates plant height by regulating histone H2B monoubiquitination and cell proliferation

    Journal: Plant Communications

    doi: 10.1016/j.xplc.2022.100412

    Downregulated expression of target genes by OsUBR7-mediated reduction of H2Bub1 results in suppression of cell-cycle progression. (A) Diagrammatic representation of ChIP-seq maps (using an anti-H2Bub1 antibody; n = 2 biological repeats) for four gene loci at which the H2Bub1 level was completely suppressed or decreased in osubr7 seedlings . (B) ChIP–qPCR analysis to determine H2Bub1 levels in candidate target genes, including the two pleiotropic genes and two cell-cycle-related genes shown in (A) . P1 to P2 represent regions covered by the primers used to assess the level of H2Bub1 by qPCR following ChIP. Data were normalized to the input chromatin, OsNRAMP2 was used as the reference, and the values of H2Bub1 levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test). (C) qRT–PCR analysis of five pleiotropic genes selected from the ChIP-seq and transcriptome data in 14-day-old WT and osubr7 seedlings. Actin1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 (Student’s t -test). (D) Relative expression of six cell-cycle marker genes in 14-day-old WT and osubr7 seedlings. UFC1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t- test). NS, not significant. (E) Cell numbers during cell-cycle phases with 2C and 4C nuclei in WT and osubr7 seedlings (7 days old) measured by flow cytometry. (F) Percentage of cells in different phases of the cell cycle in WT and osubr7 seedlings. Data are means ± SD ( n = 4 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test).
    Figure Legend Snippet: Downregulated expression of target genes by OsUBR7-mediated reduction of H2Bub1 results in suppression of cell-cycle progression. (A) Diagrammatic representation of ChIP-seq maps (using an anti-H2Bub1 antibody; n = 2 biological repeats) for four gene loci at which the H2Bub1 level was completely suppressed or decreased in osubr7 seedlings . (B) ChIP–qPCR analysis to determine H2Bub1 levels in candidate target genes, including the two pleiotropic genes and two cell-cycle-related genes shown in (A) . P1 to P2 represent regions covered by the primers used to assess the level of H2Bub1 by qPCR following ChIP. Data were normalized to the input chromatin, OsNRAMP2 was used as the reference, and the values of H2Bub1 levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test). (C) qRT–PCR analysis of five pleiotropic genes selected from the ChIP-seq and transcriptome data in 14-day-old WT and osubr7 seedlings. Actin1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 (Student’s t -test). (D) Relative expression of six cell-cycle marker genes in 14-day-old WT and osubr7 seedlings. UFC1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t- test). NS, not significant. (E) Cell numbers during cell-cycle phases with 2C and 4C nuclei in WT and osubr7 seedlings (7 days old) measured by flow cytometry. (F) Percentage of cells in different phases of the cell cycle in WT and osubr7 seedlings. Data are means ± SD ( n = 4 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test).

    Techniques Used: Expressing, ChIP-sequencing, Quantitative RT-PCR, Marker, Flow Cytometry

    OsUBR7 monoubiquitinates H2B at K148, and OsUBC18 serves as its specific E2. (A) In vitro GST pull-down assay to examine the interaction between OsUBR7 and OsUBC18. OsUBR7 was fused with GST, OsUBC18 was fused with His, and GST served as a control. Precipitated His-OsUBC18 associated with immobilized GST-OsUBR7 was detected by western blotting with anti-His or anti-GST antibody. (B) BiFC assays in rice sheath protoplasts demonstrating that OsUBR7 interacted with OsUBC18. Scale bars, 10 μm. (C) Thioester assay for the activity of recombinant His-OsUBC18. Binding of OsUBC18 with Ub was detected with anti-Ub antibody. Thioester binding was disrupted in the presence of the reducing agent DTT. (D) In vitro ubiquitination assay using recombinant His-AtUBA2 as E1, His-OsUBC18 as E2, His-OsUBR7 as E3, and MBP-H2B as substrate. Ubiquitinated MBP-H2B was detected with anti-H2Bub1 or anti-MBP antibody. RT, reaction time (h). (E) Schematic of the C-terminal region of the MBP-tagged H2B (OsH2B.1) native construct (H2B_N) and its mutant forms, in which K136, K144, or K148 was replaced by R (H2B_K136R, H2B_K144R, and H2B_ K148R, respectively). (F) In vitro ubiquitination assay using the H2B native construct (H2B_N) or a mutant variant (H2B_K136R, H2B_K144R, or H2B_K148R) as a substrate. Ubiquitinated MBP-H2B was detected with the anti-H2Bub1 antibody. (G) Comparison of H2Bub1 levels between WT and osubr7 plants by western blotting. H3 protein was detected as a loading control. Values of relative band intensity (RBI) to the first lanes from the left are given below the bands. ∗∗ p < 0.01 (Student’s t -test). NS, not significant.
    Figure Legend Snippet: OsUBR7 monoubiquitinates H2B at K148, and OsUBC18 serves as its specific E2. (A) In vitro GST pull-down assay to examine the interaction between OsUBR7 and OsUBC18. OsUBR7 was fused with GST, OsUBC18 was fused with His, and GST served as a control. Precipitated His-OsUBC18 associated with immobilized GST-OsUBR7 was detected by western blotting with anti-His or anti-GST antibody. (B) BiFC assays in rice sheath protoplasts demonstrating that OsUBR7 interacted with OsUBC18. Scale bars, 10 μm. (C) Thioester assay for the activity of recombinant His-OsUBC18. Binding of OsUBC18 with Ub was detected with anti-Ub antibody. Thioester binding was disrupted in the presence of the reducing agent DTT. (D) In vitro ubiquitination assay using recombinant His-AtUBA2 as E1, His-OsUBC18 as E2, His-OsUBR7 as E3, and MBP-H2B as substrate. Ubiquitinated MBP-H2B was detected with anti-H2Bub1 or anti-MBP antibody. RT, reaction time (h). (E) Schematic of the C-terminal region of the MBP-tagged H2B (OsH2B.1) native construct (H2B_N) and its mutant forms, in which K136, K144, or K148 was replaced by R (H2B_K136R, H2B_K144R, and H2B_ K148R, respectively). (F) In vitro ubiquitination assay using the H2B native construct (H2B_N) or a mutant variant (H2B_K136R, H2B_K144R, or H2B_K148R) as a substrate. Ubiquitinated MBP-H2B was detected with the anti-H2Bub1 antibody. (G) Comparison of H2Bub1 levels between WT and osubr7 plants by western blotting. H3 protein was detected as a loading control. Values of relative band intensity (RBI) to the first lanes from the left are given below the bands. ∗∗ p < 0.01 (Student’s t -test). NS, not significant.

    Techniques Used: In Vitro, Pull Down Assay, Western Blot, Activity Assay, Recombinant, Binding Assay, Ubiquitin Assay, Construct, Mutagenesis, Variant Assay

    A working model for the molecular function of OsUBR7 in rice development. (A) In WT plants, OsUBR7 functions as an E3 ligase. It binds to an E2 conjugase (OsUBC18) and a substrate (H2B) in the target chromatin regions and transfers ubiquitin from OsUBC18 to K148 of H2B. This E1-OsUBC18-OsUBR7 H2Bub1 pathway, possibly together with the E1-E2-OsHUB1/2 H2Bub1 pathway, results in normal H2Bub1 levels at target gene loci (including cell-cycle-related and pleiotropic genes) for their normal transcription, ultimately properly controlling cell-cycle progression and normal organ development. (B) In osubr7 plants, the lack of OsUBR7 results in reduced H2Bub1 levels (basal H2Bub1 levels may depend on the OsHUB1/2-mediated pathway) and decreased expression of the target genes. The suppressed cell-cycle progression leads to semi-dwarfing and other trait variations of osubr7 plants.
    Figure Legend Snippet: A working model for the molecular function of OsUBR7 in rice development. (A) In WT plants, OsUBR7 functions as an E3 ligase. It binds to an E2 conjugase (OsUBC18) and a substrate (H2B) in the target chromatin regions and transfers ubiquitin from OsUBC18 to K148 of H2B. This E1-OsUBC18-OsUBR7 H2Bub1 pathway, possibly together with the E1-E2-OsHUB1/2 H2Bub1 pathway, results in normal H2Bub1 levels at target gene loci (including cell-cycle-related and pleiotropic genes) for their normal transcription, ultimately properly controlling cell-cycle progression and normal organ development. (B) In osubr7 plants, the lack of OsUBR7 results in reduced H2Bub1 levels (basal H2Bub1 levels may depend on the OsHUB1/2-mediated pathway) and decreased expression of the target genes. The suppressed cell-cycle progression leads to semi-dwarfing and other trait variations of osubr7 plants.

    Techniques Used: Expressing

    anti h2bub1 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti h2bub1 antibody
    Downregulated expression of target genes by OsUBR7-mediated reduction of <t>H2Bub1</t> results in suppression of cell-cycle progression. (A) Diagrammatic representation of ChIP-seq maps (using an anti-H2Bub1 antibody; n = 2 biological repeats) for four gene loci at which the H2Bub1 level was completely suppressed or decreased in osubr7 seedlings . (B) ChIP–qPCR analysis to determine H2Bub1 levels in candidate target genes, including the two pleiotropic genes and two cell-cycle-related genes shown in (A) . P1 to P2 represent regions covered by the primers used to assess the level of H2Bub1 by qPCR following ChIP. Data were normalized to the input chromatin, OsNRAMP2 was used as the reference, and the values of H2Bub1 levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test). (C) qRT–PCR analysis of five pleiotropic genes selected from the ChIP-seq and transcriptome data in 14-day-old WT and osubr7 seedlings. Actin1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 (Student’s t -test). (D) Relative expression of six cell-cycle marker genes in 14-day-old WT and osubr7 seedlings. UFC1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t- test). NS, not significant. (E) Cell numbers during cell-cycle phases with 2C and 4C nuclei in WT and osubr7 seedlings (7 days old) measured by flow cytometry. (F) Percentage of cells in different phases of the cell cycle in WT and osubr7 seedlings. Data are means ± SD ( n = 4 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test).
    Anti H2bub1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Rice OsUBR7 modulates plant height by regulating histone H2B monoubiquitination and cell proliferation"

    Article Title: Rice OsUBR7 modulates plant height by regulating histone H2B monoubiquitination and cell proliferation

    Journal: Plant Communications

    doi: 10.1016/j.xplc.2022.100412

    Downregulated expression of target genes by OsUBR7-mediated reduction of H2Bub1 results in suppression of cell-cycle progression. (A) Diagrammatic representation of ChIP-seq maps (using an anti-H2Bub1 antibody; n = 2 biological repeats) for four gene loci at which the H2Bub1 level was completely suppressed or decreased in osubr7 seedlings . (B) ChIP–qPCR analysis to determine H2Bub1 levels in candidate target genes, including the two pleiotropic genes and two cell-cycle-related genes shown in (A) . P1 to P2 represent regions covered by the primers used to assess the level of H2Bub1 by qPCR following ChIP. Data were normalized to the input chromatin, OsNRAMP2 was used as the reference, and the values of H2Bub1 levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test). (C) qRT–PCR analysis of five pleiotropic genes selected from the ChIP-seq and transcriptome data in 14-day-old WT and osubr7 seedlings. Actin1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 (Student’s t -test). (D) Relative expression of six cell-cycle marker genes in 14-day-old WT and osubr7 seedlings. UFC1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t- test). NS, not significant. (E) Cell numbers during cell-cycle phases with 2C and 4C nuclei in WT and osubr7 seedlings (7 days old) measured by flow cytometry. (F) Percentage of cells in different phases of the cell cycle in WT and osubr7 seedlings. Data are means ± SD ( n = 4 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test).
    Figure Legend Snippet: Downregulated expression of target genes by OsUBR7-mediated reduction of H2Bub1 results in suppression of cell-cycle progression. (A) Diagrammatic representation of ChIP-seq maps (using an anti-H2Bub1 antibody; n = 2 biological repeats) for four gene loci at which the H2Bub1 level was completely suppressed or decreased in osubr7 seedlings . (B) ChIP–qPCR analysis to determine H2Bub1 levels in candidate target genes, including the two pleiotropic genes and two cell-cycle-related genes shown in (A) . P1 to P2 represent regions covered by the primers used to assess the level of H2Bub1 by qPCR following ChIP. Data were normalized to the input chromatin, OsNRAMP2 was used as the reference, and the values of H2Bub1 levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test). (C) qRT–PCR analysis of five pleiotropic genes selected from the ChIP-seq and transcriptome data in 14-day-old WT and osubr7 seedlings. Actin1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 (Student’s t -test). (D) Relative expression of six cell-cycle marker genes in 14-day-old WT and osubr7 seedlings. UFC1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t- test). NS, not significant. (E) Cell numbers during cell-cycle phases with 2C and 4C nuclei in WT and osubr7 seedlings (7 days old) measured by flow cytometry. (F) Percentage of cells in different phases of the cell cycle in WT and osubr7 seedlings. Data are means ± SD ( n = 4 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test).

    Techniques Used: Expressing, ChIP-sequencing, Quantitative RT-PCR, Marker, Flow Cytometry

    OsUBR7 monoubiquitinates H2B at K148, and OsUBC18 serves as its specific E2. (A) In vitro GST pull-down assay to examine the interaction between OsUBR7 and OsUBC18. OsUBR7 was fused with GST, OsUBC18 was fused with His, and GST served as a control. Precipitated His-OsUBC18 associated with immobilized GST-OsUBR7 was detected by western blotting with anti-His or anti-GST antibody. (B) BiFC assays in rice sheath protoplasts demonstrating that OsUBR7 interacted with OsUBC18. Scale bars, 10 μm. (C) Thioester assay for the activity of recombinant His-OsUBC18. Binding of OsUBC18 with Ub was detected with anti-Ub antibody. Thioester binding was disrupted in the presence of the reducing agent DTT. (D) In vitro ubiquitination assay using recombinant His-AtUBA2 as E1, His-OsUBC18 as E2, His-OsUBR7 as E3, and MBP-H2B as substrate. Ubiquitinated MBP-H2B was detected with anti-H2Bub1 or anti-MBP antibody. RT, reaction time (h). (E) Schematic of the C-terminal region of the MBP-tagged H2B (OsH2B.1) native construct (H2B_N) and its mutant forms, in which K136, K144, or K148 was replaced by R (H2B_K136R, H2B_K144R, and H2B_ K148R, respectively). (F) In vitro ubiquitination assay using the H2B native construct (H2B_N) or a mutant variant (H2B_K136R, H2B_K144R, or H2B_K148R) as a substrate. Ubiquitinated MBP-H2B was detected with the anti-H2Bub1 antibody. (G) Comparison of H2Bub1 levels between WT and osubr7 plants by western blotting. H3 protein was detected as a loading control. Values of relative band intensity (RBI) to the first lanes from the left are given below the bands. ∗∗ p < 0.01 (Student’s t -test). NS, not significant.
    Figure Legend Snippet: OsUBR7 monoubiquitinates H2B at K148, and OsUBC18 serves as its specific E2. (A) In vitro GST pull-down assay to examine the interaction between OsUBR7 and OsUBC18. OsUBR7 was fused with GST, OsUBC18 was fused with His, and GST served as a control. Precipitated His-OsUBC18 associated with immobilized GST-OsUBR7 was detected by western blotting with anti-His or anti-GST antibody. (B) BiFC assays in rice sheath protoplasts demonstrating that OsUBR7 interacted with OsUBC18. Scale bars, 10 μm. (C) Thioester assay for the activity of recombinant His-OsUBC18. Binding of OsUBC18 with Ub was detected with anti-Ub antibody. Thioester binding was disrupted in the presence of the reducing agent DTT. (D) In vitro ubiquitination assay using recombinant His-AtUBA2 as E1, His-OsUBC18 as E2, His-OsUBR7 as E3, and MBP-H2B as substrate. Ubiquitinated MBP-H2B was detected with anti-H2Bub1 or anti-MBP antibody. RT, reaction time (h). (E) Schematic of the C-terminal region of the MBP-tagged H2B (OsH2B.1) native construct (H2B_N) and its mutant forms, in which K136, K144, or K148 was replaced by R (H2B_K136R, H2B_K144R, and H2B_ K148R, respectively). (F) In vitro ubiquitination assay using the H2B native construct (H2B_N) or a mutant variant (H2B_K136R, H2B_K144R, or H2B_K148R) as a substrate. Ubiquitinated MBP-H2B was detected with the anti-H2Bub1 antibody. (G) Comparison of H2Bub1 levels between WT and osubr7 plants by western blotting. H3 protein was detected as a loading control. Values of relative band intensity (RBI) to the first lanes from the left are given below the bands. ∗∗ p < 0.01 (Student’s t -test). NS, not significant.

    Techniques Used: In Vitro, Pull Down Assay, Western Blot, Activity Assay, Recombinant, Binding Assay, Ubiquitin Assay, Construct, Mutagenesis, Variant Assay

    A working model for the molecular function of OsUBR7 in rice development. (A) In WT plants, OsUBR7 functions as an E3 ligase. It binds to an E2 conjugase (OsUBC18) and a substrate (H2B) in the target chromatin regions and transfers ubiquitin from OsUBC18 to K148 of H2B. This E1-OsUBC18-OsUBR7 H2Bub1 pathway, possibly together with the E1-E2-OsHUB1/2 H2Bub1 pathway, results in normal H2Bub1 levels at target gene loci (including cell-cycle-related and pleiotropic genes) for their normal transcription, ultimately properly controlling cell-cycle progression and normal organ development. (B) In osubr7 plants, the lack of OsUBR7 results in reduced H2Bub1 levels (basal H2Bub1 levels may depend on the OsHUB1/2-mediated pathway) and decreased expression of the target genes. The suppressed cell-cycle progression leads to semi-dwarfing and other trait variations of osubr7 plants.
    Figure Legend Snippet: A working model for the molecular function of OsUBR7 in rice development. (A) In WT plants, OsUBR7 functions as an E3 ligase. It binds to an E2 conjugase (OsUBC18) and a substrate (H2B) in the target chromatin regions and transfers ubiquitin from OsUBC18 to K148 of H2B. This E1-OsUBC18-OsUBR7 H2Bub1 pathway, possibly together with the E1-E2-OsHUB1/2 H2Bub1 pathway, results in normal H2Bub1 levels at target gene loci (including cell-cycle-related and pleiotropic genes) for their normal transcription, ultimately properly controlling cell-cycle progression and normal organ development. (B) In osubr7 plants, the lack of OsUBR7 results in reduced H2Bub1 levels (basal H2Bub1 levels may depend on the OsHUB1/2-mediated pathway) and decreased expression of the target genes. The suppressed cell-cycle progression leads to semi-dwarfing and other trait variations of osubr7 plants.

    Techniques Used: Expressing

    h2bub1 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc h2bub1 antibody
    a–d Neonatal pups were treated with CASAAV-RNF20/40 (RNF Cas-KO) at a dose that transduced most CMs. Western blotting of ventricular apexes ( a ) was performed to measure protein levels of RNF20, RNF40, <t>H2Bub1,</t> and NPPA, with GAPDH internal control. RNF20, RNF40, and H2Bub1 were depleted, whereas NPPA was upregulated. n = 4 mice per group. Quantification of H2Bub1 ( b ) showed significant 74% reduction ( P = 0.0007), consistent with reduced RNF20/40 ubiquitin ligase activity. Reduction in tissue samples with multiple cell types underestimates changes in cardiomyocytes. Survival curve ( c ) demonstrated death of juvenile RNF Cas-KO mice in the majority of CMs. At P7, before the onset of lethality, echocardiography ( d ) showed that a subset of mice (arrowheads) exhibited cardiac dysfunction (reduced fractional shortening percentage and increased LV internal diameter at end systole). e Sections of RNF Cas-KO hearts ( n = 3 mice) at P7 showed dramatic upregulation of Myh7 YFP compared with controls ( n = 3 mice). Scale bars = 200 μm. f – n In order to avoid non-cell-autonomous secondary effects of heart failure, CASAAV-RNF20/40 was administered to newborn R26 Cas9-GFP/+ ; Myh7 YFP/+ pups at a mosaic dose. Analysis was performed at P28. f Representative flow cytometry analysis. Cells were gated on GFP (transduction marker) and then on YFP. g Quantification of YFP + transduced CMs. RNF Cas-KO markedly increased the fraction of transduced CMs that retained Myh7 YFP expression at P28 (92% of CASAAV-RNF20/40-transduced CMs, compared with 13.5% of AAV-Cre; P < 0.0001). h – n Analysis of CM maturation. RNF Cas-KO CMs were markedly smaller than controls ( h ; P < 0.0001), with a moderate increase in length-to-width ratio ( i; P = 0.0002). j These RNF20/40-depleted CMs had increased mononucleation ( P = 0.0207). k – l However, sarcomere organization appeared unperturbed. In total 89 YFP- and 93 YFP + CMs were randomly selected from three mice in equal proportions for quantification. m , n T-tubules were markedly disrupted, as determined by optical sectioning of freshly isolated, FM4–64-perfused hearts followed by quantitative analysis of T-tubule transverse element (TE) density ( P < 0.0001). In total 79 YFP- and 69 YFP + CMs were randomly selected from three mice in equal proportions for quantification. Collectively, these phenotypes observed in RNF Cas-KO CMs indicate that maturation is impaired. Source data are provided as a Source Data file. Scale bars in k and m = 5 μm. Violin plots: shape indicates data distribution; point, median; whiskers, starts at quartile and extends 1.5 times the interquartile distance. Bar plots: error bars denote standard error. Two-tailed t-test: * P < 0.05, ** P < 0.001.
    H2bub1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h2bub1 antibody/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    h2bub1 antibody - by Bioz Stars, 2023-09
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    Images

    1) Product Images from "Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation"

    Article Title: Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

    Journal: Nature Communications

    doi: 10.1038/s41467-021-24743-z

    a–d Neonatal pups were treated with CASAAV-RNF20/40 (RNF Cas-KO) at a dose that transduced most CMs. Western blotting of ventricular apexes ( a ) was performed to measure protein levels of RNF20, RNF40, H2Bub1, and NPPA, with GAPDH internal control. RNF20, RNF40, and H2Bub1 were depleted, whereas NPPA was upregulated. n = 4 mice per group. Quantification of H2Bub1 ( b ) showed significant 74% reduction ( P = 0.0007), consistent with reduced RNF20/40 ubiquitin ligase activity. Reduction in tissue samples with multiple cell types underestimates changes in cardiomyocytes. Survival curve ( c ) demonstrated death of juvenile RNF Cas-KO mice in the majority of CMs. At P7, before the onset of lethality, echocardiography ( d ) showed that a subset of mice (arrowheads) exhibited cardiac dysfunction (reduced fractional shortening percentage and increased LV internal diameter at end systole). e Sections of RNF Cas-KO hearts ( n = 3 mice) at P7 showed dramatic upregulation of Myh7 YFP compared with controls ( n = 3 mice). Scale bars = 200 μm. f – n In order to avoid non-cell-autonomous secondary effects of heart failure, CASAAV-RNF20/40 was administered to newborn R26 Cas9-GFP/+ ; Myh7 YFP/+ pups at a mosaic dose. Analysis was performed at P28. f Representative flow cytometry analysis. Cells were gated on GFP (transduction marker) and then on YFP. g Quantification of YFP + transduced CMs. RNF Cas-KO markedly increased the fraction of transduced CMs that retained Myh7 YFP expression at P28 (92% of CASAAV-RNF20/40-transduced CMs, compared with 13.5% of AAV-Cre; P < 0.0001). h – n Analysis of CM maturation. RNF Cas-KO CMs were markedly smaller than controls ( h ; P < 0.0001), with a moderate increase in length-to-width ratio ( i; P = 0.0002). j These RNF20/40-depleted CMs had increased mononucleation ( P = 0.0207). k – l However, sarcomere organization appeared unperturbed. In total 89 YFP- and 93 YFP + CMs were randomly selected from three mice in equal proportions for quantification. m , n T-tubules were markedly disrupted, as determined by optical sectioning of freshly isolated, FM4–64-perfused hearts followed by quantitative analysis of T-tubule transverse element (TE) density ( P < 0.0001). In total 79 YFP- and 69 YFP + CMs were randomly selected from three mice in equal proportions for quantification. Collectively, these phenotypes observed in RNF Cas-KO CMs indicate that maturation is impaired. Source data are provided as a Source Data file. Scale bars in k and m = 5 μm. Violin plots: shape indicates data distribution; point, median; whiskers, starts at quartile and extends 1.5 times the interquartile distance. Bar plots: error bars denote standard error. Two-tailed t-test: * P < 0.05, ** P < 0.001.
    Figure Legend Snippet: a–d Neonatal pups were treated with CASAAV-RNF20/40 (RNF Cas-KO) at a dose that transduced most CMs. Western blotting of ventricular apexes ( a ) was performed to measure protein levels of RNF20, RNF40, H2Bub1, and NPPA, with GAPDH internal control. RNF20, RNF40, and H2Bub1 were depleted, whereas NPPA was upregulated. n = 4 mice per group. Quantification of H2Bub1 ( b ) showed significant 74% reduction ( P = 0.0007), consistent with reduced RNF20/40 ubiquitin ligase activity. Reduction in tissue samples with multiple cell types underestimates changes in cardiomyocytes. Survival curve ( c ) demonstrated death of juvenile RNF Cas-KO mice in the majority of CMs. At P7, before the onset of lethality, echocardiography ( d ) showed that a subset of mice (arrowheads) exhibited cardiac dysfunction (reduced fractional shortening percentage and increased LV internal diameter at end systole). e Sections of RNF Cas-KO hearts ( n = 3 mice) at P7 showed dramatic upregulation of Myh7 YFP compared with controls ( n = 3 mice). Scale bars = 200 μm. f – n In order to avoid non-cell-autonomous secondary effects of heart failure, CASAAV-RNF20/40 was administered to newborn R26 Cas9-GFP/+ ; Myh7 YFP/+ pups at a mosaic dose. Analysis was performed at P28. f Representative flow cytometry analysis. Cells were gated on GFP (transduction marker) and then on YFP. g Quantification of YFP + transduced CMs. RNF Cas-KO markedly increased the fraction of transduced CMs that retained Myh7 YFP expression at P28 (92% of CASAAV-RNF20/40-transduced CMs, compared with 13.5% of AAV-Cre; P < 0.0001). h – n Analysis of CM maturation. RNF Cas-KO CMs were markedly smaller than controls ( h ; P < 0.0001), with a moderate increase in length-to-width ratio ( i; P = 0.0002). j These RNF20/40-depleted CMs had increased mononucleation ( P = 0.0207). k – l However, sarcomere organization appeared unperturbed. In total 89 YFP- and 93 YFP + CMs were randomly selected from three mice in equal proportions for quantification. m , n T-tubules were markedly disrupted, as determined by optical sectioning of freshly isolated, FM4–64-perfused hearts followed by quantitative analysis of T-tubule transverse element (TE) density ( P < 0.0001). In total 79 YFP- and 69 YFP + CMs were randomly selected from three mice in equal proportions for quantification. Collectively, these phenotypes observed in RNF Cas-KO CMs indicate that maturation is impaired. Source data are provided as a Source Data file. Scale bars in k and m = 5 μm. Violin plots: shape indicates data distribution; point, median; whiskers, starts at quartile and extends 1.5 times the interquartile distance. Bar plots: error bars denote standard error. Two-tailed t-test: * P < 0.05, ** P < 0.001.

    Techniques Used: Western Blot, Activity Assay, Flow Cytometry, Transduction, Marker, Expressing, Isolation, Two Tailed Test

    a Representative genome browser view of neonatal and adult H2Bub1 ChIP-seq replicates. Dashed red box highlights a gene with dynamic H2Bub1 occupancy. b Profile plot showing average H2Bub1 signal at gene bodies. Plot shows gene bodies and 50 kb upstream of the TSS or downstream of the TSE. c Genes with detectable H2Bub1 ChIP-seq signal at P1, P28, or both timepoints. d Distribution of changes in H2Bub1 signal intensity during maturation, for all genes marked by H2Bub1 in at least one timepoint. e H2Bub1 P1 ChIP signal (top) or the change of H2Bub1 ChIP signal during maturation (bottom) as a function of the gene’s maturational change in RNA expression. The change in RNA was binned to contain the indicated range of log2 fold-change values. The change in gene expression correlated with the change in H2Bub1 occupancy, but not with the absolute H2Bub1 occupancy. Numbers at the top of plot (n) indicate genes per bin. Center lines in box plots indicate the median, while boxes show 25th and 75th percentiles. Whiskers denote the maximum observation within the 75th percentile +1.5 times the interquartile range, or the minimum observation within the 25th percentile −1.5 times the interquartile range. P -value: Steel–Dwass vs. H2Bub1 P1 ChIP signal in the (−1,0] bin; *** P < 0.001. f Maturational change in expression versus maturational change in H2Bub1 signal plotted for genes differentially expressed in RNF20/40-depleted CMs. Color indicates the direction of differential expression in RNF20/40-depleted CMs. Genes with maturational gain in H2Bub1 and RNA expression were predominantly downregulated in RNF20/40 depletion (blue, upper-right quadrant), whereas genes with maturational loss in H2Bub1 and RNA expression were mostly upregulated in RNF20/40 depeletion (red, lower-left quadrant). g Ingenuity pathway analysis of functional terms enriched amongst the indicated genes. Left: Red-colored genes in the lower-left quadrant of panel f (i.e., genes that decrease H2Bub1 and gene expression during maturation and are upregulated in RNF20/40 depletion). Right: Blue-colored genes in the upper-right quadrant of panel f (i.e., genes that increase H2Bub1 and gene expression during maturation and are downregulated in RNF20/40 depletion). P -values were calculated by IPA Core Analysis. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Representative genome browser view of neonatal and adult H2Bub1 ChIP-seq replicates. Dashed red box highlights a gene with dynamic H2Bub1 occupancy. b Profile plot showing average H2Bub1 signal at gene bodies. Plot shows gene bodies and 50 kb upstream of the TSS or downstream of the TSE. c Genes with detectable H2Bub1 ChIP-seq signal at P1, P28, or both timepoints. d Distribution of changes in H2Bub1 signal intensity during maturation, for all genes marked by H2Bub1 in at least one timepoint. e H2Bub1 P1 ChIP signal (top) or the change of H2Bub1 ChIP signal during maturation (bottom) as a function of the gene’s maturational change in RNA expression. The change in RNA was binned to contain the indicated range of log2 fold-change values. The change in gene expression correlated with the change in H2Bub1 occupancy, but not with the absolute H2Bub1 occupancy. Numbers at the top of plot (n) indicate genes per bin. Center lines in box plots indicate the median, while boxes show 25th and 75th percentiles. Whiskers denote the maximum observation within the 75th percentile +1.5 times the interquartile range, or the minimum observation within the 25th percentile −1.5 times the interquartile range. P -value: Steel–Dwass vs. H2Bub1 P1 ChIP signal in the (−1,0] bin; *** P < 0.001. f Maturational change in expression versus maturational change in H2Bub1 signal plotted for genes differentially expressed in RNF20/40-depleted CMs. Color indicates the direction of differential expression in RNF20/40-depleted CMs. Genes with maturational gain in H2Bub1 and RNA expression were predominantly downregulated in RNF20/40 depletion (blue, upper-right quadrant), whereas genes with maturational loss in H2Bub1 and RNA expression were mostly upregulated in RNF20/40 depeletion (red, lower-left quadrant). g Ingenuity pathway analysis of functional terms enriched amongst the indicated genes. Left: Red-colored genes in the lower-left quadrant of panel f (i.e., genes that decrease H2Bub1 and gene expression during maturation and are upregulated in RNF20/40 depletion). Right: Blue-colored genes in the upper-right quadrant of panel f (i.e., genes that increase H2Bub1 and gene expression during maturation and are downregulated in RNF20/40 depletion). P -values were calculated by IPA Core Analysis. Source data are provided as a Source Data file.

    Techniques Used: ChIP-sequencing, RNA Expression, Expressing, Functional Assay

    anti h2bub1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti h2bub1
    Anti H2bub1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti h2bub1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti h2bub1
    ( A ) Transgenic Vav1-Cre mice were crossed with mice carrying a floxed Usp22 allele to obtain mice with pan-hematopoietic Usp22 deficiency ( Usp22 KO). ( B ) Usp22 genotype of individual cells within the indicated cell populations isolated from Usp22 KO mice determined by single-cell PCR (n=67-142 cells) (delta/delta = KO). ( C ) Quantitative reverse transcription (qRT-)PCR analysis for Usp22 mRNA expression in long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), multipotent progenitors (MPP) and common myeloid progenitors (CMP) from bone marrow, and B cells, CD4 + T cells, CD8a + T cells and granulocytes from the spleens of wild type and Usp22 KO mice (n=3-6). ( D ) Western Blot analysis for H2B and <t>H2Bub1</t> in bone marrow HSPC, and splenic B (CD19 + B220 + ), T (both CD4 + and CD8a + cells) and myeloid cells (CD11b + ) from wild type and Usp22 KO mice (n=8 mice per genotype). ( E ) GO term analysis of DEG upregulated in Usp22 KO versus wild type HSPC. ( F ) Heatmap showing the expression of genes differentially expressed between wild type and Usp22 KO HSPC within selected GO terms identified in ( E ). ( G ) Interferon serum levels in wild type and Usp22 KO mice (n=9-12). Geometric mean ± geometric s.d. ( C ), mean ± s.d. ( G ). * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; n.s., not significant by unpaired, two-tailed Student’s t-test.
    Anti H2bub1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti h2bub1/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
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    1) Product Images from "Loss of Usp22 enhances histone H2B monoubiquitination and stimulates intracellular and systemic interferon immunity"

    Article Title: Loss of Usp22 enhances histone H2B monoubiquitination and stimulates intracellular and systemic interferon immunity

    Journal: bioRxiv

    doi: 10.1101/2021.04.09.439190

    ( A ) Transgenic Vav1-Cre mice were crossed with mice carrying a floxed Usp22 allele to obtain mice with pan-hematopoietic Usp22 deficiency ( Usp22 KO). ( B ) Usp22 genotype of individual cells within the indicated cell populations isolated from Usp22 KO mice determined by single-cell PCR (n=67-142 cells) (delta/delta = KO). ( C ) Quantitative reverse transcription (qRT-)PCR analysis for Usp22 mRNA expression in long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), multipotent progenitors (MPP) and common myeloid progenitors (CMP) from bone marrow, and B cells, CD4 + T cells, CD8a + T cells and granulocytes from the spleens of wild type and Usp22 KO mice (n=3-6). ( D ) Western Blot analysis for H2B and H2Bub1 in bone marrow HSPC, and splenic B (CD19 + B220 + ), T (both CD4 + and CD8a + cells) and myeloid cells (CD11b + ) from wild type and Usp22 KO mice (n=8 mice per genotype). ( E ) GO term analysis of DEG upregulated in Usp22 KO versus wild type HSPC. ( F ) Heatmap showing the expression of genes differentially expressed between wild type and Usp22 KO HSPC within selected GO terms identified in ( E ). ( G ) Interferon serum levels in wild type and Usp22 KO mice (n=9-12). Geometric mean ± geometric s.d. ( C ), mean ± s.d. ( G ). * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; n.s., not significant by unpaired, two-tailed Student’s t-test.
    Figure Legend Snippet: ( A ) Transgenic Vav1-Cre mice were crossed with mice carrying a floxed Usp22 allele to obtain mice with pan-hematopoietic Usp22 deficiency ( Usp22 KO). ( B ) Usp22 genotype of individual cells within the indicated cell populations isolated from Usp22 KO mice determined by single-cell PCR (n=67-142 cells) (delta/delta = KO). ( C ) Quantitative reverse transcription (qRT-)PCR analysis for Usp22 mRNA expression in long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), multipotent progenitors (MPP) and common myeloid progenitors (CMP) from bone marrow, and B cells, CD4 + T cells, CD8a + T cells and granulocytes from the spleens of wild type and Usp22 KO mice (n=3-6). ( D ) Western Blot analysis for H2B and H2Bub1 in bone marrow HSPC, and splenic B (CD19 + B220 + ), T (both CD4 + and CD8a + cells) and myeloid cells (CD11b + ) from wild type and Usp22 KO mice (n=8 mice per genotype). ( E ) GO term analysis of DEG upregulated in Usp22 KO versus wild type HSPC. ( F ) Heatmap showing the expression of genes differentially expressed between wild type and Usp22 KO HSPC within selected GO terms identified in ( E ). ( G ) Interferon serum levels in wild type and Usp22 KO mice (n=9-12). Geometric mean ± geometric s.d. ( C ), mean ± s.d. ( G ). * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; n.s., not significant by unpaired, two-tailed Student’s t-test.

    Techniques Used: Transgenic Assay, Isolation, Quantitative RT-PCR, Expressing, Western Blot, Two Tailed Test

    ( A ) Western Blot for H2B and H2Bub1 at day 4 post transduction of wild type and Usp22 KO HSPC with empty pMSCV-IRES-GFP (pMIG) or pMIG- Usp22 . ( B ) CD11b and B220 expression in cells differentiated in vitro for 1.5 weeks from Usp22 KO HSPC transduced with the indicated vectors. Transduced cells can be identified based on the expression of GFP. ( C ) Relative proportions of B220 + B cells and CD11b + myeloid cells (gated as in B ) within cells differentiated for 1.5 weeks in vitro from wild type and Usp22 KO HSPC transduced with the indicated gamma-retroviral expression vectors (n=3). The partial myeloid cell production in these experiments also from wild type progenitors presumably results from the cellular pre-expansion conditions required for viral transduction prior to the differentiation assay. Mean ± s.d.
    Figure Legend Snippet: ( A ) Western Blot for H2B and H2Bub1 at day 4 post transduction of wild type and Usp22 KO HSPC with empty pMSCV-IRES-GFP (pMIG) or pMIG- Usp22 . ( B ) CD11b and B220 expression in cells differentiated in vitro for 1.5 weeks from Usp22 KO HSPC transduced with the indicated vectors. Transduced cells can be identified based on the expression of GFP. ( C ) Relative proportions of B220 + B cells and CD11b + myeloid cells (gated as in B ) within cells differentiated for 1.5 weeks in vitro from wild type and Usp22 KO HSPC transduced with the indicated gamma-retroviral expression vectors (n=3). The partial myeloid cell production in these experiments also from wild type progenitors presumably results from the cellular pre-expansion conditions required for viral transduction prior to the differentiation assay. Mean ± s.d.

    Techniques Used: Western Blot, Transduction, Expressing, In Vitro, Differentiation Assay

    ( A - C ) Average H2Bub1 ChIP sequencing signal in wild type and Usp22 KO HSPC across gene bodies of all genes ( A ), of non-expressed genes ( B , top panels), of genes highly expressed in HSPC ( B , bottom panels), of all immune cell ISG ( C , top panels), and of the immune cell core ISG ( C , bottom panels). ( D ) Average ATAC sequencing signal around the TSS of genes upregulated in Usp22 KO HSPC (top panels), and of genes downregulated in Usp22 KO HSPC (bottom panels) comparing wild type and Usp22 KO HSPC. ( E ) Correlation between changes in TSS accessibility (measured by ATAC sequencing) and changes in mRNA expression comparing Usp22 KO and wild type HSPC. Each dot represents an individual gene, left panel shows all genes, and right panel only genes differentially expressed between wild type and Usp22 KO HSPC. ( F ) Average ATAC sequencing signal around the TSS of differentially expressed and ubiquitinated genes (top panel), and differentially expressed (DE) and differentially ubiquitinated (DU) ISG (bottom panel) in wild type and Usp22 KO HSPC. TSS = Transcription start site; TES = Transcription end site.
    Figure Legend Snippet: ( A - C ) Average H2Bub1 ChIP sequencing signal in wild type and Usp22 KO HSPC across gene bodies of all genes ( A ), of non-expressed genes ( B , top panels), of genes highly expressed in HSPC ( B , bottom panels), of all immune cell ISG ( C , top panels), and of the immune cell core ISG ( C , bottom panels). ( D ) Average ATAC sequencing signal around the TSS of genes upregulated in Usp22 KO HSPC (top panels), and of genes downregulated in Usp22 KO HSPC (bottom panels) comparing wild type and Usp22 KO HSPC. ( E ) Correlation between changes in TSS accessibility (measured by ATAC sequencing) and changes in mRNA expression comparing Usp22 KO and wild type HSPC. Each dot represents an individual gene, left panel shows all genes, and right panel only genes differentially expressed between wild type and Usp22 KO HSPC. ( F ) Average ATAC sequencing signal around the TSS of differentially expressed and ubiquitinated genes (top panel), and differentially expressed (DE) and differentially ubiquitinated (DU) ISG (bottom panel) in wild type and Usp22 KO HSPC. TSS = Transcription start site; TES = Transcription end site.

    Techniques Used: ChIP-sequencing, Sequencing, Expressing

    ( A ) Average H2Bub1 ChIP sequencing signal across gene bodies of IFN- α target genes (top panels) and IFN- γ target genes (bottom panels) in wild type and Usp22 KO HSPC. ( B ) Correlation between change in gene body monoubiquitination and change in mRNA expression comparing Usp22 KO against wild type HSPC for individual genes (dots), shown for all genes (left panel), or selectively for differentially expressed genes (right panel). ( C ) Correlation between change in gene body monoubiquitination and change in mRNA expression upon loss of Usp22 in HSPC for genes differentially expressed and ubiquitinated between wild type and Usp22 KO HPSC. ISG are highlighted in red. ( D ) Fraction of ISG within all genes detected in RNA sequencing and H2Bub1 ChIP sequencing (black bar), and the set of genes differentially expressed and ubiquitinated in Usp22 KO HSPC (red bar). p-value was determined using Fisher’s exact test. TSS = Transcription start site; TES = Transcription end site.
    Figure Legend Snippet: ( A ) Average H2Bub1 ChIP sequencing signal across gene bodies of IFN- α target genes (top panels) and IFN- γ target genes (bottom panels) in wild type and Usp22 KO HSPC. ( B ) Correlation between change in gene body monoubiquitination and change in mRNA expression comparing Usp22 KO against wild type HSPC for individual genes (dots), shown for all genes (left panel), or selectively for differentially expressed genes (right panel). ( C ) Correlation between change in gene body monoubiquitination and change in mRNA expression upon loss of Usp22 in HSPC for genes differentially expressed and ubiquitinated between wild type and Usp22 KO HPSC. ISG are highlighted in red. ( D ) Fraction of ISG within all genes detected in RNA sequencing and H2Bub1 ChIP sequencing (black bar), and the set of genes differentially expressed and ubiquitinated in Usp22 KO HSPC (red bar). p-value was determined using Fisher’s exact test. TSS = Transcription start site; TES = Transcription end site.

    Techniques Used: ChIP-sequencing, Expressing, RNA Sequencing Assay

    anti h2bub1  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc anti h2bub1
    ( A ) Transgenic Vav1-Cre mice were crossed with mice carrying a floxed Usp22 allele to obtain mice with pan-hematopoietic Usp22 deficiency ( Usp22 KO). ( B ) Usp22 genotype of individual cells within the indicated cell populations isolated from Usp22 KO mice determined by single-cell PCR (n=67-142 cells) (delta/delta = KO). ( C ) Quantitative reverse transcription (qRT-)PCR analysis for Usp22 mRNA expression in long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), multipotent progenitors (MPP) and common myeloid progenitors (CMP) from bone marrow, and B cells, CD4 + T cells, CD8a + T cells and granulocytes from the spleens of wild type and Usp22 KO mice (n=3-6). ( D ) Western Blot analysis for H2B and <t>H2Bub1</t> in bone marrow HSPC, and splenic B (CD19 + B220 + ), T (both CD4 + and CD8a + cells) and myeloid cells (CD11b + ) from wild type and Usp22 KO mice (n=8 mice per genotype). ( E ) GO term analysis of DEG upregulated in Usp22 KO versus wild type HSPC. ( F ) Heatmap showing the expression of genes differentially expressed between wild type and Usp22 KO HSPC within selected GO terms identified in ( E ). ( G ) Interferon serum levels in wild type and Usp22 KO mice (n=9-12). Geometric mean ± geometric s.d. ( C ), mean ± s.d. ( G ). * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; n.s., not significant by unpaired, two-tailed Student’s t-test.
    Anti H2bub1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti h2bub1/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti h2bub1 - by Bioz Stars, 2023-09
    95/100 stars

    Images

    1) Product Images from "Loss of Usp22 enhances histone H2B monoubiquitination and stimulates intracellular and systemic interferon immunity"

    Article Title: Loss of Usp22 enhances histone H2B monoubiquitination and stimulates intracellular and systemic interferon immunity

    Journal: bioRxiv

    doi: 10.1101/2021.04.09.439190

    ( A ) Transgenic Vav1-Cre mice were crossed with mice carrying a floxed Usp22 allele to obtain mice with pan-hematopoietic Usp22 deficiency ( Usp22 KO). ( B ) Usp22 genotype of individual cells within the indicated cell populations isolated from Usp22 KO mice determined by single-cell PCR (n=67-142 cells) (delta/delta = KO). ( C ) Quantitative reverse transcription (qRT-)PCR analysis for Usp22 mRNA expression in long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), multipotent progenitors (MPP) and common myeloid progenitors (CMP) from bone marrow, and B cells, CD4 + T cells, CD8a + T cells and granulocytes from the spleens of wild type and Usp22 KO mice (n=3-6). ( D ) Western Blot analysis for H2B and H2Bub1 in bone marrow HSPC, and splenic B (CD19 + B220 + ), T (both CD4 + and CD8a + cells) and myeloid cells (CD11b + ) from wild type and Usp22 KO mice (n=8 mice per genotype). ( E ) GO term analysis of DEG upregulated in Usp22 KO versus wild type HSPC. ( F ) Heatmap showing the expression of genes differentially expressed between wild type and Usp22 KO HSPC within selected GO terms identified in ( E ). ( G ) Interferon serum levels in wild type and Usp22 KO mice (n=9-12). Geometric mean ± geometric s.d. ( C ), mean ± s.d. ( G ). * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; n.s., not significant by unpaired, two-tailed Student’s t-test.
    Figure Legend Snippet: ( A ) Transgenic Vav1-Cre mice were crossed with mice carrying a floxed Usp22 allele to obtain mice with pan-hematopoietic Usp22 deficiency ( Usp22 KO). ( B ) Usp22 genotype of individual cells within the indicated cell populations isolated from Usp22 KO mice determined by single-cell PCR (n=67-142 cells) (delta/delta = KO). ( C ) Quantitative reverse transcription (qRT-)PCR analysis for Usp22 mRNA expression in long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), multipotent progenitors (MPP) and common myeloid progenitors (CMP) from bone marrow, and B cells, CD4 + T cells, CD8a + T cells and granulocytes from the spleens of wild type and Usp22 KO mice (n=3-6). ( D ) Western Blot analysis for H2B and H2Bub1 in bone marrow HSPC, and splenic B (CD19 + B220 + ), T (both CD4 + and CD8a + cells) and myeloid cells (CD11b + ) from wild type and Usp22 KO mice (n=8 mice per genotype). ( E ) GO term analysis of DEG upregulated in Usp22 KO versus wild type HSPC. ( F ) Heatmap showing the expression of genes differentially expressed between wild type and Usp22 KO HSPC within selected GO terms identified in ( E ). ( G ) Interferon serum levels in wild type and Usp22 KO mice (n=9-12). Geometric mean ± geometric s.d. ( C ), mean ± s.d. ( G ). * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; n.s., not significant by unpaired, two-tailed Student’s t-test.

    Techniques Used: Transgenic Assay, Isolation, Quantitative RT-PCR, Expressing, Western Blot, Two Tailed Test

    ( A ) Western Blot for H2B and H2Bub1 at day 4 post transduction of wild type and Usp22 KO HSPC with empty pMSCV-IRES-GFP (pMIG) or pMIG- Usp22 . ( B ) CD11b and B220 expression in cells differentiated in vitro for 1.5 weeks from Usp22 KO HSPC transduced with the indicated vectors. Transduced cells can be identified based on the expression of GFP. ( C ) Relative proportions of B220 + B cells and CD11b + myeloid cells (gated as in B ) within cells differentiated for 1.5 weeks in vitro from wild type and Usp22 KO HSPC transduced with the indicated gamma-retroviral expression vectors (n=3). The partial myeloid cell production in these experiments also from wild type progenitors presumably results from the cellular pre-expansion conditions required for viral transduction prior to the differentiation assay. Mean ± s.d.
    Figure Legend Snippet: ( A ) Western Blot for H2B and H2Bub1 at day 4 post transduction of wild type and Usp22 KO HSPC with empty pMSCV-IRES-GFP (pMIG) or pMIG- Usp22 . ( B ) CD11b and B220 expression in cells differentiated in vitro for 1.5 weeks from Usp22 KO HSPC transduced with the indicated vectors. Transduced cells can be identified based on the expression of GFP. ( C ) Relative proportions of B220 + B cells and CD11b + myeloid cells (gated as in B ) within cells differentiated for 1.5 weeks in vitro from wild type and Usp22 KO HSPC transduced with the indicated gamma-retroviral expression vectors (n=3). The partial myeloid cell production in these experiments also from wild type progenitors presumably results from the cellular pre-expansion conditions required for viral transduction prior to the differentiation assay. Mean ± s.d.

    Techniques Used: Western Blot, Transduction, Expressing, In Vitro, Differentiation Assay

    ( A - C ) Average H2Bub1 ChIP sequencing signal in wild type and Usp22 KO HSPC across gene bodies of all genes ( A ), of non-expressed genes ( B , top panels), of genes highly expressed in HSPC ( B , bottom panels), of all immune cell ISG ( C , top panels), and of the immune cell core ISG ( C , bottom panels). ( D ) Average ATAC sequencing signal around the TSS of genes upregulated in Usp22 KO HSPC (top panels), and of genes downregulated in Usp22 KO HSPC (bottom panels) comparing wild type and Usp22 KO HSPC. ( E ) Correlation between changes in TSS accessibility (measured by ATAC sequencing) and changes in mRNA expression comparing Usp22 KO and wild type HSPC. Each dot represents an individual gene, left panel shows all genes, and right panel only genes differentially expressed between wild type and Usp22 KO HSPC. ( F ) Average ATAC sequencing signal around the TSS of differentially expressed and ubiquitinated genes (top panel), and differentially expressed (DE) and differentially ubiquitinated (DU) ISG (bottom panel) in wild type and Usp22 KO HSPC. TSS = Transcription start site; TES = Transcription end site.
    Figure Legend Snippet: ( A - C ) Average H2Bub1 ChIP sequencing signal in wild type and Usp22 KO HSPC across gene bodies of all genes ( A ), of non-expressed genes ( B , top panels), of genes highly expressed in HSPC ( B , bottom panels), of all immune cell ISG ( C , top panels), and of the immune cell core ISG ( C , bottom panels). ( D ) Average ATAC sequencing signal around the TSS of genes upregulated in Usp22 KO HSPC (top panels), and of genes downregulated in Usp22 KO HSPC (bottom panels) comparing wild type and Usp22 KO HSPC. ( E ) Correlation between changes in TSS accessibility (measured by ATAC sequencing) and changes in mRNA expression comparing Usp22 KO and wild type HSPC. Each dot represents an individual gene, left panel shows all genes, and right panel only genes differentially expressed between wild type and Usp22 KO HSPC. ( F ) Average ATAC sequencing signal around the TSS of differentially expressed and ubiquitinated genes (top panel), and differentially expressed (DE) and differentially ubiquitinated (DU) ISG (bottom panel) in wild type and Usp22 KO HSPC. TSS = Transcription start site; TES = Transcription end site.

    Techniques Used: ChIP-sequencing, Sequencing, Expressing

    ( A ) Average H2Bub1 ChIP sequencing signal across gene bodies of IFN- α target genes (top panels) and IFN- γ target genes (bottom panels) in wild type and Usp22 KO HSPC. ( B ) Correlation between change in gene body monoubiquitination and change in mRNA expression comparing Usp22 KO against wild type HSPC for individual genes (dots), shown for all genes (left panel), or selectively for differentially expressed genes (right panel). ( C ) Correlation between change in gene body monoubiquitination and change in mRNA expression upon loss of Usp22 in HSPC for genes differentially expressed and ubiquitinated between wild type and Usp22 KO HPSC. ISG are highlighted in red. ( D ) Fraction of ISG within all genes detected in RNA sequencing and H2Bub1 ChIP sequencing (black bar), and the set of genes differentially expressed and ubiquitinated in Usp22 KO HSPC (red bar). p-value was determined using Fisher’s exact test. TSS = Transcription start site; TES = Transcription end site.
    Figure Legend Snippet: ( A ) Average H2Bub1 ChIP sequencing signal across gene bodies of IFN- α target genes (top panels) and IFN- γ target genes (bottom panels) in wild type and Usp22 KO HSPC. ( B ) Correlation between change in gene body monoubiquitination and change in mRNA expression comparing Usp22 KO against wild type HSPC for individual genes (dots), shown for all genes (left panel), or selectively for differentially expressed genes (right panel). ( C ) Correlation between change in gene body monoubiquitination and change in mRNA expression upon loss of Usp22 in HSPC for genes differentially expressed and ubiquitinated between wild type and Usp22 KO HPSC. ISG are highlighted in red. ( D ) Fraction of ISG within all genes detected in RNA sequencing and H2Bub1 ChIP sequencing (black bar), and the set of genes differentially expressed and ubiquitinated in Usp22 KO HSPC (red bar). p-value was determined using Fisher’s exact test. TSS = Transcription start site; TES = Transcription end site.

    Techniques Used: ChIP-sequencing, Expressing, RNA Sequencing Assay

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    • anti h2bub1 antibody  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc anti h2bub1 antibody
    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) <t>H2Bub1</t> and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).
    Anti H2bub1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti h2bub1  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc anti h2bub1
    (A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of H2B, <t>H2Bub1</t> and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.
    Anti H2bub1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    h2bub1  (Cell Signaling Technology Inc)
    95
    Cell Signaling Technology Inc h2bub1
    Downregulated expression of target genes by OsUBR7-mediated reduction of <t>H2Bub1</t> results in suppression of cell-cycle progression. (A) Diagrammatic representation of ChIP-seq maps (using an anti-H2Bub1 antibody; n = 2 biological repeats) for four gene loci at which the H2Bub1 level was completely suppressed or decreased in osubr7 seedlings . (B) ChIP–qPCR analysis to determine H2Bub1 levels in candidate target genes, including the two pleiotropic genes and two cell-cycle-related genes shown in (A) . P1 to P2 represent regions covered by the primers used to assess the level of H2Bub1 by qPCR following ChIP. Data were normalized to the input chromatin, OsNRAMP2 was used as the reference, and the values of H2Bub1 levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test). (C) qRT–PCR analysis of five pleiotropic genes selected from the ChIP-seq and transcriptome data in 14-day-old WT and osubr7 seedlings. Actin1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 (Student’s t -test). (D) Relative expression of six cell-cycle marker genes in 14-day-old WT and osubr7 seedlings. UFC1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t- test). NS, not significant. (E) Cell numbers during cell-cycle phases with 2C and 4C nuclei in WT and osubr7 seedlings (7 days old) measured by flow cytometry. (F) Percentage of cells in different phases of the cell cycle in WT and osubr7 seedlings. Data are means ± SD ( n = 4 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test).
    H2bub1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    h2bub1 antibody  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc h2bub1 antibody
    a–d Neonatal pups were treated with CASAAV-RNF20/40 (RNF Cas-KO) at a dose that transduced most CMs. Western blotting of ventricular apexes ( a ) was performed to measure protein levels of RNF20, RNF40, <t>H2Bub1,</t> and NPPA, with GAPDH internal control. RNF20, RNF40, and H2Bub1 were depleted, whereas NPPA was upregulated. n = 4 mice per group. Quantification of H2Bub1 ( b ) showed significant 74% reduction ( P = 0.0007), consistent with reduced RNF20/40 ubiquitin ligase activity. Reduction in tissue samples with multiple cell types underestimates changes in cardiomyocytes. Survival curve ( c ) demonstrated death of juvenile RNF Cas-KO mice in the majority of CMs. At P7, before the onset of lethality, echocardiography ( d ) showed that a subset of mice (arrowheads) exhibited cardiac dysfunction (reduced fractional shortening percentage and increased LV internal diameter at end systole). e Sections of RNF Cas-KO hearts ( n = 3 mice) at P7 showed dramatic upregulation of Myh7 YFP compared with controls ( n = 3 mice). Scale bars = 200 μm. f – n In order to avoid non-cell-autonomous secondary effects of heart failure, CASAAV-RNF20/40 was administered to newborn R26 Cas9-GFP/+ ; Myh7 YFP/+ pups at a mosaic dose. Analysis was performed at P28. f Representative flow cytometry analysis. Cells were gated on GFP (transduction marker) and then on YFP. g Quantification of YFP + transduced CMs. RNF Cas-KO markedly increased the fraction of transduced CMs that retained Myh7 YFP expression at P28 (92% of CASAAV-RNF20/40-transduced CMs, compared with 13.5% of AAV-Cre; P < 0.0001). h – n Analysis of CM maturation. RNF Cas-KO CMs were markedly smaller than controls ( h ; P < 0.0001), with a moderate increase in length-to-width ratio ( i; P = 0.0002). j These RNF20/40-depleted CMs had increased mononucleation ( P = 0.0207). k – l However, sarcomere organization appeared unperturbed. In total 89 YFP- and 93 YFP + CMs were randomly selected from three mice in equal proportions for quantification. m , n T-tubules were markedly disrupted, as determined by optical sectioning of freshly isolated, FM4–64-perfused hearts followed by quantitative analysis of T-tubule transverse element (TE) density ( P < 0.0001). In total 79 YFP- and 69 YFP + CMs were randomly selected from three mice in equal proportions for quantification. Collectively, these phenotypes observed in RNF Cas-KO CMs indicate that maturation is impaired. Source data are provided as a Source Data file. Scale bars in k and m = 5 μm. Violin plots: shape indicates data distribution; point, median; whiskers, starts at quartile and extends 1.5 times the interquartile distance. Bar plots: error bars denote standard error. Two-tailed t-test: * P < 0.05, ** P < 0.001.
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    Image Search Results


    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    doi: 10.1016/j.omtn.2023.02.014

    Figure Lengend Snippet: The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).

    Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

    Techniques: RNA Sequencing Assay, Infection, Expressing

    BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    doi: 10.1016/j.omtn.2023.02.014

    Figure Lengend Snippet: BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

    Techniques: Expressing, Infection, Staining, Immunohistochemistry

    H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    doi: 10.1016/j.omtn.2023.02.014

    Figure Lengend Snippet: H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

    Techniques: Expressing, ChIP-sequencing, Infection, Binding Assay

    TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    doi: 10.1016/j.omtn.2023.02.014

    Figure Lengend Snippet: TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

    Techniques: Expressing, Western Blot, Immunofluorescence, Staining

    Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    doi: 10.1016/j.omtn.2023.02.014

    Figure Lengend Snippet: Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.

    Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

    Techniques: Injection, Western Blot, Expressing, Fluorescence, Immunofluorescence, Staining, Micro-CT

    Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    doi: 10.1016/j.omtn.2023.02.014

    Figure Lengend Snippet: Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis

    Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

    Techniques:

    (A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of H2B, H2Bub1 and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.

    Journal: PLoS ONE

    Article Title: Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

    doi: 10.1371/journal.pone.0061447

    Figure Lengend Snippet: (A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of H2B, H2Bub1 and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.

    Article Snippet: ChIP analysis was performed with the ChIP Assay Kit (Millipore-Upstate, Schwalbach, Germany) as described by the manufacturer, isolating genomic DNA fragments generated by sonication, using antibodies anti-NKX2-1 (EP1584Y, Abgent), anti-H2B (53H3, Cell Signaling, Danvers, MA, USA), anti-H2Bub1 (D11, Cell Signaling), anti-H3K4me3 (mAbcam1012, Abcam, Cambridge, UK) and anti-H3K27me3 (mAbcam6002, Abcam).

    Techniques: Expressing, Amplification, Positive Control, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot

    The figure summarizes the regulations of the genes described in this study, highlighting a central position of NKX2-1. Chromosomal aberrations activate expression of MLL (11q23) and histones including H2B (6p22). MLL together with H2Bub1 generate an activatory chromatin structure at NKX2-1. This structure is reinforced by reduced expression of USP46 and E2F6 and elevated expression of RNF20/40, JMJD3 and HOPX, and mediates activation of NKX2-1. NKX2-1 in turn activates directly expression of HEY1 which performs inhibition of B-cell differentiation. Both, NKX2-1 and HEY1 contribute to the activatory chromatin structure by regulating RNF40 and E2F6, respectively. IL4/STAT3-signaling enhances expression of NKX2-1. TNFa, cGMP, cAMP and PRKCE support NFkB which activates both NKX2-1 and HEY1. Reduced expression of PDE6D and enhanced expression of NOS1 contribute to elevated cGMP. NOS1 and PRKCE are activated by NKX2-1. HEY1 mediates reduced expression of PDE4A resulting in elevated cAMP levels. Finally, NOTCH-signaling and TGFb-signaling (via SMAD) activate HEY1 expression. SMAD and NKX2-1 interact and coactivate HEY1 transcription.

    Journal: PLoS ONE

    Article Title: Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

    doi: 10.1371/journal.pone.0061447

    Figure Lengend Snippet: The figure summarizes the regulations of the genes described in this study, highlighting a central position of NKX2-1. Chromosomal aberrations activate expression of MLL (11q23) and histones including H2B (6p22). MLL together with H2Bub1 generate an activatory chromatin structure at NKX2-1. This structure is reinforced by reduced expression of USP46 and E2F6 and elevated expression of RNF20/40, JMJD3 and HOPX, and mediates activation of NKX2-1. NKX2-1 in turn activates directly expression of HEY1 which performs inhibition of B-cell differentiation. Both, NKX2-1 and HEY1 contribute to the activatory chromatin structure by regulating RNF40 and E2F6, respectively. IL4/STAT3-signaling enhances expression of NKX2-1. TNFa, cGMP, cAMP and PRKCE support NFkB which activates both NKX2-1 and HEY1. Reduced expression of PDE6D and enhanced expression of NOS1 contribute to elevated cGMP. NOS1 and PRKCE are activated by NKX2-1. HEY1 mediates reduced expression of PDE4A resulting in elevated cAMP levels. Finally, NOTCH-signaling and TGFb-signaling (via SMAD) activate HEY1 expression. SMAD and NKX2-1 interact and coactivate HEY1 transcription.

    Article Snippet: ChIP analysis was performed with the ChIP Assay Kit (Millipore-Upstate, Schwalbach, Germany) as described by the manufacturer, isolating genomic DNA fragments generated by sonication, using antibodies anti-NKX2-1 (EP1584Y, Abgent), anti-H2B (53H3, Cell Signaling, Danvers, MA, USA), anti-H2Bub1 (D11, Cell Signaling), anti-H3K4me3 (mAbcam1012, Abcam, Cambridge, UK) and anti-H3K27me3 (mAbcam6002, Abcam).

    Techniques: Expressing, Activation Assay, Inhibition, Cell Differentiation

    Downregulated expression of target genes by OsUBR7-mediated reduction of H2Bub1 results in suppression of cell-cycle progression. (A) Diagrammatic representation of ChIP-seq maps (using an anti-H2Bub1 antibody; n = 2 biological repeats) for four gene loci at which the H2Bub1 level was completely suppressed or decreased in osubr7 seedlings . (B) ChIP–qPCR analysis to determine H2Bub1 levels in candidate target genes, including the two pleiotropic genes and two cell-cycle-related genes shown in (A) . P1 to P2 represent regions covered by the primers used to assess the level of H2Bub1 by qPCR following ChIP. Data were normalized to the input chromatin, OsNRAMP2 was used as the reference, and the values of H2Bub1 levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test). (C) qRT–PCR analysis of five pleiotropic genes selected from the ChIP-seq and transcriptome data in 14-day-old WT and osubr7 seedlings. Actin1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 (Student’s t -test). (D) Relative expression of six cell-cycle marker genes in 14-day-old WT and osubr7 seedlings. UFC1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t- test). NS, not significant. (E) Cell numbers during cell-cycle phases with 2C and 4C nuclei in WT and osubr7 seedlings (7 days old) measured by flow cytometry. (F) Percentage of cells in different phases of the cell cycle in WT and osubr7 seedlings. Data are means ± SD ( n = 4 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test).

    Journal: Plant Communications

    Article Title: Rice OsUBR7 modulates plant height by regulating histone H2B monoubiquitination and cell proliferation

    doi: 10.1016/j.xplc.2022.100412

    Figure Lengend Snippet: Downregulated expression of target genes by OsUBR7-mediated reduction of H2Bub1 results in suppression of cell-cycle progression. (A) Diagrammatic representation of ChIP-seq maps (using an anti-H2Bub1 antibody; n = 2 biological repeats) for four gene loci at which the H2Bub1 level was completely suppressed or decreased in osubr7 seedlings . (B) ChIP–qPCR analysis to determine H2Bub1 levels in candidate target genes, including the two pleiotropic genes and two cell-cycle-related genes shown in (A) . P1 to P2 represent regions covered by the primers used to assess the level of H2Bub1 by qPCR following ChIP. Data were normalized to the input chromatin, OsNRAMP2 was used as the reference, and the values of H2Bub1 levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test). (C) qRT–PCR analysis of five pleiotropic genes selected from the ChIP-seq and transcriptome data in 14-day-old WT and osubr7 seedlings. Actin1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 (Student’s t -test). (D) Relative expression of six cell-cycle marker genes in 14-day-old WT and osubr7 seedlings. UFC1 was used as the reference, and the values of expression levels in the WT were set to 1. Values are means ± SD ( n = 3 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t- test). NS, not significant. (E) Cell numbers during cell-cycle phases with 2C and 4C nuclei in WT and osubr7 seedlings (7 days old) measured by flow cytometry. (F) Percentage of cells in different phases of the cell cycle in WT and osubr7 seedlings. Data are means ± SD ( n = 4 biological repeats). ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (Student’s t -test).

    Article Snippet: The reaction products were separated by SDS–PAGE, blotted, and analyzed via western blotting with antibodies against Ub (mouse, Abcam ab7254, 1:2000 dilution), MBP, H2Aub1 (rabbit, Cell Signaling Technology 8240T, 1:1000 dilution), and H2Bub1 (rabbit, Cell Signaling Technology 5546T, 1:2000 dilution).

    Techniques: Expressing, ChIP-sequencing, Quantitative RT-PCR, Marker, Flow Cytometry

    OsUBR7 monoubiquitinates H2B at K148, and OsUBC18 serves as its specific E2. (A) In vitro GST pull-down assay to examine the interaction between OsUBR7 and OsUBC18. OsUBR7 was fused with GST, OsUBC18 was fused with His, and GST served as a control. Precipitated His-OsUBC18 associated with immobilized GST-OsUBR7 was detected by western blotting with anti-His or anti-GST antibody. (B) BiFC assays in rice sheath protoplasts demonstrating that OsUBR7 interacted with OsUBC18. Scale bars, 10 μm. (C) Thioester assay for the activity of recombinant His-OsUBC18. Binding of OsUBC18 with Ub was detected with anti-Ub antibody. Thioester binding was disrupted in the presence of the reducing agent DTT. (D) In vitro ubiquitination assay using recombinant His-AtUBA2 as E1, His-OsUBC18 as E2, His-OsUBR7 as E3, and MBP-H2B as substrate. Ubiquitinated MBP-H2B was detected with anti-H2Bub1 or anti-MBP antibody. RT, reaction time (h). (E) Schematic of the C-terminal region of the MBP-tagged H2B (OsH2B.1) native construct (H2B_N) and its mutant forms, in which K136, K144, or K148 was replaced by R (H2B_K136R, H2B_K144R, and H2B_ K148R, respectively). (F) In vitro ubiquitination assay using the H2B native construct (H2B_N) or a mutant variant (H2B_K136R, H2B_K144R, or H2B_K148R) as a substrate. Ubiquitinated MBP-H2B was detected with the anti-H2Bub1 antibody. (G) Comparison of H2Bub1 levels between WT and osubr7 plants by western blotting. H3 protein was detected as a loading control. Values of relative band intensity (RBI) to the first lanes from the left are given below the bands. ∗∗ p < 0.01 (Student’s t -test). NS, not significant.

    Journal: Plant Communications

    Article Title: Rice OsUBR7 modulates plant height by regulating histone H2B monoubiquitination and cell proliferation

    doi: 10.1016/j.xplc.2022.100412

    Figure Lengend Snippet: OsUBR7 monoubiquitinates H2B at K148, and OsUBC18 serves as its specific E2. (A) In vitro GST pull-down assay to examine the interaction between OsUBR7 and OsUBC18. OsUBR7 was fused with GST, OsUBC18 was fused with His, and GST served as a control. Precipitated His-OsUBC18 associated with immobilized GST-OsUBR7 was detected by western blotting with anti-His or anti-GST antibody. (B) BiFC assays in rice sheath protoplasts demonstrating that OsUBR7 interacted with OsUBC18. Scale bars, 10 μm. (C) Thioester assay for the activity of recombinant His-OsUBC18. Binding of OsUBC18 with Ub was detected with anti-Ub antibody. Thioester binding was disrupted in the presence of the reducing agent DTT. (D) In vitro ubiquitination assay using recombinant His-AtUBA2 as E1, His-OsUBC18 as E2, His-OsUBR7 as E3, and MBP-H2B as substrate. Ubiquitinated MBP-H2B was detected with anti-H2Bub1 or anti-MBP antibody. RT, reaction time (h). (E) Schematic of the C-terminal region of the MBP-tagged H2B (OsH2B.1) native construct (H2B_N) and its mutant forms, in which K136, K144, or K148 was replaced by R (H2B_K136R, H2B_K144R, and H2B_ K148R, respectively). (F) In vitro ubiquitination assay using the H2B native construct (H2B_N) or a mutant variant (H2B_K136R, H2B_K144R, or H2B_K148R) as a substrate. Ubiquitinated MBP-H2B was detected with the anti-H2Bub1 antibody. (G) Comparison of H2Bub1 levels between WT and osubr7 plants by western blotting. H3 protein was detected as a loading control. Values of relative band intensity (RBI) to the first lanes from the left are given below the bands. ∗∗ p < 0.01 (Student’s t -test). NS, not significant.

    Article Snippet: The reaction products were separated by SDS–PAGE, blotted, and analyzed via western blotting with antibodies against Ub (mouse, Abcam ab7254, 1:2000 dilution), MBP, H2Aub1 (rabbit, Cell Signaling Technology 8240T, 1:1000 dilution), and H2Bub1 (rabbit, Cell Signaling Technology 5546T, 1:2000 dilution).

    Techniques: In Vitro, Pull Down Assay, Western Blot, Activity Assay, Recombinant, Binding Assay, Ubiquitin Assay, Construct, Mutagenesis, Variant Assay

    A working model for the molecular function of OsUBR7 in rice development. (A) In WT plants, OsUBR7 functions as an E3 ligase. It binds to an E2 conjugase (OsUBC18) and a substrate (H2B) in the target chromatin regions and transfers ubiquitin from OsUBC18 to K148 of H2B. This E1-OsUBC18-OsUBR7 H2Bub1 pathway, possibly together with the E1-E2-OsHUB1/2 H2Bub1 pathway, results in normal H2Bub1 levels at target gene loci (including cell-cycle-related and pleiotropic genes) for their normal transcription, ultimately properly controlling cell-cycle progression and normal organ development. (B) In osubr7 plants, the lack of OsUBR7 results in reduced H2Bub1 levels (basal H2Bub1 levels may depend on the OsHUB1/2-mediated pathway) and decreased expression of the target genes. The suppressed cell-cycle progression leads to semi-dwarfing and other trait variations of osubr7 plants.

    Journal: Plant Communications

    Article Title: Rice OsUBR7 modulates plant height by regulating histone H2B monoubiquitination and cell proliferation

    doi: 10.1016/j.xplc.2022.100412

    Figure Lengend Snippet: A working model for the molecular function of OsUBR7 in rice development. (A) In WT plants, OsUBR7 functions as an E3 ligase. It binds to an E2 conjugase (OsUBC18) and a substrate (H2B) in the target chromatin regions and transfers ubiquitin from OsUBC18 to K148 of H2B. This E1-OsUBC18-OsUBR7 H2Bub1 pathway, possibly together with the E1-E2-OsHUB1/2 H2Bub1 pathway, results in normal H2Bub1 levels at target gene loci (including cell-cycle-related and pleiotropic genes) for their normal transcription, ultimately properly controlling cell-cycle progression and normal organ development. (B) In osubr7 plants, the lack of OsUBR7 results in reduced H2Bub1 levels (basal H2Bub1 levels may depend on the OsHUB1/2-mediated pathway) and decreased expression of the target genes. The suppressed cell-cycle progression leads to semi-dwarfing and other trait variations of osubr7 plants.

    Article Snippet: The reaction products were separated by SDS–PAGE, blotted, and analyzed via western blotting with antibodies against Ub (mouse, Abcam ab7254, 1:2000 dilution), MBP, H2Aub1 (rabbit, Cell Signaling Technology 8240T, 1:1000 dilution), and H2Bub1 (rabbit, Cell Signaling Technology 5546T, 1:2000 dilution).

    Techniques: Expressing

    a–d Neonatal pups were treated with CASAAV-RNF20/40 (RNF Cas-KO) at a dose that transduced most CMs. Western blotting of ventricular apexes ( a ) was performed to measure protein levels of RNF20, RNF40, H2Bub1, and NPPA, with GAPDH internal control. RNF20, RNF40, and H2Bub1 were depleted, whereas NPPA was upregulated. n = 4 mice per group. Quantification of H2Bub1 ( b ) showed significant 74% reduction ( P = 0.0007), consistent with reduced RNF20/40 ubiquitin ligase activity. Reduction in tissue samples with multiple cell types underestimates changes in cardiomyocytes. Survival curve ( c ) demonstrated death of juvenile RNF Cas-KO mice in the majority of CMs. At P7, before the onset of lethality, echocardiography ( d ) showed that a subset of mice (arrowheads) exhibited cardiac dysfunction (reduced fractional shortening percentage and increased LV internal diameter at end systole). e Sections of RNF Cas-KO hearts ( n = 3 mice) at P7 showed dramatic upregulation of Myh7 YFP compared with controls ( n = 3 mice). Scale bars = 200 μm. f – n In order to avoid non-cell-autonomous secondary effects of heart failure, CASAAV-RNF20/40 was administered to newborn R26 Cas9-GFP/+ ; Myh7 YFP/+ pups at a mosaic dose. Analysis was performed at P28. f Representative flow cytometry analysis. Cells were gated on GFP (transduction marker) and then on YFP. g Quantification of YFP + transduced CMs. RNF Cas-KO markedly increased the fraction of transduced CMs that retained Myh7 YFP expression at P28 (92% of CASAAV-RNF20/40-transduced CMs, compared with 13.5% of AAV-Cre; P < 0.0001). h – n Analysis of CM maturation. RNF Cas-KO CMs were markedly smaller than controls ( h ; P < 0.0001), with a moderate increase in length-to-width ratio ( i; P = 0.0002). j These RNF20/40-depleted CMs had increased mononucleation ( P = 0.0207). k – l However, sarcomere organization appeared unperturbed. In total 89 YFP- and 93 YFP + CMs were randomly selected from three mice in equal proportions for quantification. m , n T-tubules were markedly disrupted, as determined by optical sectioning of freshly isolated, FM4–64-perfused hearts followed by quantitative analysis of T-tubule transverse element (TE) density ( P < 0.0001). In total 79 YFP- and 69 YFP + CMs were randomly selected from three mice in equal proportions for quantification. Collectively, these phenotypes observed in RNF Cas-KO CMs indicate that maturation is impaired. Source data are provided as a Source Data file. Scale bars in k and m = 5 μm. Violin plots: shape indicates data distribution; point, median; whiskers, starts at quartile and extends 1.5 times the interquartile distance. Bar plots: error bars denote standard error. Two-tailed t-test: * P < 0.05, ** P < 0.001.

    Journal: Nature Communications

    Article Title: Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

    doi: 10.1038/s41467-021-24743-z

    Figure Lengend Snippet: a–d Neonatal pups were treated with CASAAV-RNF20/40 (RNF Cas-KO) at a dose that transduced most CMs. Western blotting of ventricular apexes ( a ) was performed to measure protein levels of RNF20, RNF40, H2Bub1, and NPPA, with GAPDH internal control. RNF20, RNF40, and H2Bub1 were depleted, whereas NPPA was upregulated. n = 4 mice per group. Quantification of H2Bub1 ( b ) showed significant 74% reduction ( P = 0.0007), consistent with reduced RNF20/40 ubiquitin ligase activity. Reduction in tissue samples with multiple cell types underestimates changes in cardiomyocytes. Survival curve ( c ) demonstrated death of juvenile RNF Cas-KO mice in the majority of CMs. At P7, before the onset of lethality, echocardiography ( d ) showed that a subset of mice (arrowheads) exhibited cardiac dysfunction (reduced fractional shortening percentage and increased LV internal diameter at end systole). e Sections of RNF Cas-KO hearts ( n = 3 mice) at P7 showed dramatic upregulation of Myh7 YFP compared with controls ( n = 3 mice). Scale bars = 200 μm. f – n In order to avoid non-cell-autonomous secondary effects of heart failure, CASAAV-RNF20/40 was administered to newborn R26 Cas9-GFP/+ ; Myh7 YFP/+ pups at a mosaic dose. Analysis was performed at P28. f Representative flow cytometry analysis. Cells were gated on GFP (transduction marker) and then on YFP. g Quantification of YFP + transduced CMs. RNF Cas-KO markedly increased the fraction of transduced CMs that retained Myh7 YFP expression at P28 (92% of CASAAV-RNF20/40-transduced CMs, compared with 13.5% of AAV-Cre; P < 0.0001). h – n Analysis of CM maturation. RNF Cas-KO CMs were markedly smaller than controls ( h ; P < 0.0001), with a moderate increase in length-to-width ratio ( i; P = 0.0002). j These RNF20/40-depleted CMs had increased mononucleation ( P = 0.0207). k – l However, sarcomere organization appeared unperturbed. In total 89 YFP- and 93 YFP + CMs were randomly selected from three mice in equal proportions for quantification. m , n T-tubules were markedly disrupted, as determined by optical sectioning of freshly isolated, FM4–64-perfused hearts followed by quantitative analysis of T-tubule transverse element (TE) density ( P < 0.0001). In total 79 YFP- and 69 YFP + CMs were randomly selected from three mice in equal proportions for quantification. Collectively, these phenotypes observed in RNF Cas-KO CMs indicate that maturation is impaired. Source data are provided as a Source Data file. Scale bars in k and m = 5 μm. Violin plots: shape indicates data distribution; point, median; whiskers, starts at quartile and extends 1.5 times the interquartile distance. Bar plots: error bars denote standard error. Two-tailed t-test: * P < 0.05, ** P < 0.001.

    Article Snippet: In all, 5 µl of H2Bub1 antibody (Cell Signaling 5546), 3 µl of H3K4me3 antibody (Active Motif 39159), or 5 µl of H3K36me3 antibody (Cell Signaling 4909 S) was added to the remaining 750 µl of Dynabeads, along with 250 µl of fresh BSA solution, and incubated overnight at 4 °C.

    Techniques: Western Blot, Activity Assay, Flow Cytometry, Transduction, Marker, Expressing, Isolation, Two Tailed Test

    a Representative genome browser view of neonatal and adult H2Bub1 ChIP-seq replicates. Dashed red box highlights a gene with dynamic H2Bub1 occupancy. b Profile plot showing average H2Bub1 signal at gene bodies. Plot shows gene bodies and 50 kb upstream of the TSS or downstream of the TSE. c Genes with detectable H2Bub1 ChIP-seq signal at P1, P28, or both timepoints. d Distribution of changes in H2Bub1 signal intensity during maturation, for all genes marked by H2Bub1 in at least one timepoint. e H2Bub1 P1 ChIP signal (top) or the change of H2Bub1 ChIP signal during maturation (bottom) as a function of the gene’s maturational change in RNA expression. The change in RNA was binned to contain the indicated range of log2 fold-change values. The change in gene expression correlated with the change in H2Bub1 occupancy, but not with the absolute H2Bub1 occupancy. Numbers at the top of plot (n) indicate genes per bin. Center lines in box plots indicate the median, while boxes show 25th and 75th percentiles. Whiskers denote the maximum observation within the 75th percentile +1.5 times the interquartile range, or the minimum observation within the 25th percentile −1.5 times the interquartile range. P -value: Steel–Dwass vs. H2Bub1 P1 ChIP signal in the (−1,0] bin; *** P < 0.001. f Maturational change in expression versus maturational change in H2Bub1 signal plotted for genes differentially expressed in RNF20/40-depleted CMs. Color indicates the direction of differential expression in RNF20/40-depleted CMs. Genes with maturational gain in H2Bub1 and RNA expression were predominantly downregulated in RNF20/40 depletion (blue, upper-right quadrant), whereas genes with maturational loss in H2Bub1 and RNA expression were mostly upregulated in RNF20/40 depeletion (red, lower-left quadrant). g Ingenuity pathway analysis of functional terms enriched amongst the indicated genes. Left: Red-colored genes in the lower-left quadrant of panel f (i.e., genes that decrease H2Bub1 and gene expression during maturation and are upregulated in RNF20/40 depletion). Right: Blue-colored genes in the upper-right quadrant of panel f (i.e., genes that increase H2Bub1 and gene expression during maturation and are downregulated in RNF20/40 depletion). P -values were calculated by IPA Core Analysis. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

    doi: 10.1038/s41467-021-24743-z

    Figure Lengend Snippet: a Representative genome browser view of neonatal and adult H2Bub1 ChIP-seq replicates. Dashed red box highlights a gene with dynamic H2Bub1 occupancy. b Profile plot showing average H2Bub1 signal at gene bodies. Plot shows gene bodies and 50 kb upstream of the TSS or downstream of the TSE. c Genes with detectable H2Bub1 ChIP-seq signal at P1, P28, or both timepoints. d Distribution of changes in H2Bub1 signal intensity during maturation, for all genes marked by H2Bub1 in at least one timepoint. e H2Bub1 P1 ChIP signal (top) or the change of H2Bub1 ChIP signal during maturation (bottom) as a function of the gene’s maturational change in RNA expression. The change in RNA was binned to contain the indicated range of log2 fold-change values. The change in gene expression correlated with the change in H2Bub1 occupancy, but not with the absolute H2Bub1 occupancy. Numbers at the top of plot (n) indicate genes per bin. Center lines in box plots indicate the median, while boxes show 25th and 75th percentiles. Whiskers denote the maximum observation within the 75th percentile +1.5 times the interquartile range, or the minimum observation within the 25th percentile −1.5 times the interquartile range. P -value: Steel–Dwass vs. H2Bub1 P1 ChIP signal in the (−1,0] bin; *** P < 0.001. f Maturational change in expression versus maturational change in H2Bub1 signal plotted for genes differentially expressed in RNF20/40-depleted CMs. Color indicates the direction of differential expression in RNF20/40-depleted CMs. Genes with maturational gain in H2Bub1 and RNA expression were predominantly downregulated in RNF20/40 depletion (blue, upper-right quadrant), whereas genes with maturational loss in H2Bub1 and RNA expression were mostly upregulated in RNF20/40 depeletion (red, lower-left quadrant). g Ingenuity pathway analysis of functional terms enriched amongst the indicated genes. Left: Red-colored genes in the lower-left quadrant of panel f (i.e., genes that decrease H2Bub1 and gene expression during maturation and are upregulated in RNF20/40 depletion). Right: Blue-colored genes in the upper-right quadrant of panel f (i.e., genes that increase H2Bub1 and gene expression during maturation and are downregulated in RNF20/40 depletion). P -values were calculated by IPA Core Analysis. Source data are provided as a Source Data file.

    Article Snippet: In all, 5 µl of H2Bub1 antibody (Cell Signaling 5546), 3 µl of H3K4me3 antibody (Active Motif 39159), or 5 µl of H3K36me3 antibody (Cell Signaling 4909 S) was added to the remaining 750 µl of Dynabeads, along with 250 µl of fresh BSA solution, and incubated overnight at 4 °C.

    Techniques: ChIP-sequencing, RNA Expression, Expressing, Functional Assay