9425s rrid ab 2797700  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 9425s rrid ab 2797700

    9425s Rrid Ab 2797700, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/9425s rrid ab 2797700/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    9425s rrid ab 2797700 - by Bioz Stars, 2023-01
    94/100 stars

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    1) Product Images from "Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions"

    Article Title: Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

    Journal: eLife

    doi: 10.7554/eLife.73524


    Figure Legend Snippet:

    Techniques Used: Software

    9425s rrid ab 2797700  (Cell Signaling Technology Inc)


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  • 94

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    Cell Signaling Technology Inc 9425s rrid ab 2797700

    9425s Rrid Ab 2797700, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/9425s rrid ab 2797700/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    9425s rrid ab 2797700 - by Bioz Stars, 2023-01
    94/100 stars

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    1) Product Images from "Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions"

    Article Title: Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

    Journal: eLife

    doi: 10.7554/eLife.73524


    Figure Legend Snippet:

    Techniques Used: Software

    anti rnf20  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti rnf20

    Anti Rnf20, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rnf20/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rnf20 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions"

    Article Title: Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

    Journal: eLife

    doi: 10.7554/eLife.73524


    Figure Legend Snippet:

    Techniques Used: Software

    rnf20 antibodies  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rnf20 antibodies
    <t>RNF20</t> alters the expression of epithelial-mesenchymal transition (EMT) markers. (A) Expression of E-cadherin, Vimentin and RNF20 in the MDA-MB231 and BT549 cells was analyzed by Western blotting. Actin was served as a loading control. (B) Expression of RNF20 and E-cadherin was analyzed by qPCR in MDA-MB231 and BT549 cells infected with empty vector (Ctr) or RNF20-expressing vector. *P < 0.01 by Student’s t test. (C) Expression of E-cadherin, and RNF20 was analyzed by Western blotting in BT474 cells with stable empty vector or knockdown of RNF20 expression as well as shRNF20-expressing BT474 cells infected with plvx-neo-RNF20. (D) Expression of RNF20 and E-cadherin was analyzed by qPCR in cells from (C) . *P < 0.01 by Student’s t test.
    Rnf20 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnf20 antibodies/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rnf20 antibodies - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer"

    Article Title: RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2020.613470

    RNF20 alters the expression of epithelial-mesenchymal transition (EMT) markers. (A) Expression of E-cadherin, Vimentin and RNF20 in the MDA-MB231 and BT549 cells was analyzed by Western blotting. Actin was served as a loading control. (B) Expression of RNF20 and E-cadherin was analyzed by qPCR in MDA-MB231 and BT549 cells infected with empty vector (Ctr) or RNF20-expressing vector. *P < 0.01 by Student’s t test. (C) Expression of E-cadherin, and RNF20 was analyzed by Western blotting in BT474 cells with stable empty vector or knockdown of RNF20 expression as well as shRNF20-expressing BT474 cells infected with plvx-neo-RNF20. (D) Expression of RNF20 and E-cadherin was analyzed by qPCR in cells from (C) . *P < 0.01 by Student’s t test.
    Figure Legend Snippet: RNF20 alters the expression of epithelial-mesenchymal transition (EMT) markers. (A) Expression of E-cadherin, Vimentin and RNF20 in the MDA-MB231 and BT549 cells was analyzed by Western blotting. Actin was served as a loading control. (B) Expression of RNF20 and E-cadherin was analyzed by qPCR in MDA-MB231 and BT549 cells infected with empty vector (Ctr) or RNF20-expressing vector. *P < 0.01 by Student’s t test. (C) Expression of E-cadherin, and RNF20 was analyzed by Western blotting in BT474 cells with stable empty vector or knockdown of RNF20 expression as well as shRNF20-expressing BT474 cells infected with plvx-neo-RNF20. (D) Expression of RNF20 and E-cadherin was analyzed by qPCR in cells from (C) . *P < 0.01 by Student’s t test.

    Techniques Used: Expressing, Western Blot, Infection, Plasmid Preparation

    RNF20 enhances breast cancer cell migration and invasion in vitro . (A–C) Migratory (A, B) and invasive ability (C) of MDA-MB231 and BT549 cells with stable empty vector or RNF20 expression were analyzed. The percentage of migratory and invasive cells is shown in the bar graphs (mean ± SD in three separate experiments). *P < 0.01 by Student’s t test: 100 µm.
    Figure Legend Snippet: RNF20 enhances breast cancer cell migration and invasion in vitro . (A–C) Migratory (A, B) and invasive ability (C) of MDA-MB231 and BT549 cells with stable empty vector or RNF20 expression were analyzed. The percentage of migratory and invasive cells is shown in the bar graphs (mean ± SD in three separate experiments). *P < 0.01 by Student’s t test: 100 µm.

    Techniques Used: Migration, In Vitro, Plasmid Preparation, Expressing

    RNF20 interacts with Snail. (A) 293T cells were transiently coexpressed with Flag-tagged RNF20 and HA-tagged Snail. Cell extracts were immunoprecipitated separately with Flag or HA antibodies, and the associated RNF20 and Snail were examined by Western blotting, respectively. (B) Schematic diagram showing the structure of Snail and two deletion mutations. (C) Full-length and deletion mutants of Snail were coexpressed with RNF20 in 293T cells. After immunoprecipitation of RN20, associated Snail was analyzed by Western blotting.
    Figure Legend Snippet: RNF20 interacts with Snail. (A) 293T cells were transiently coexpressed with Flag-tagged RNF20 and HA-tagged Snail. Cell extracts were immunoprecipitated separately with Flag or HA antibodies, and the associated RNF20 and Snail were examined by Western blotting, respectively. (B) Schematic diagram showing the structure of Snail and two deletion mutations. (C) Full-length and deletion mutants of Snail were coexpressed with RNF20 in 293T cells. After immunoprecipitation of RN20, associated Snail was analyzed by Western blotting.

    Techniques Used: Immunoprecipitation, Western Blot

    RNF20 interacts with G9a. 293T cells were transiently coexpressed with Flag-tagged G9a, HA-tagged RNF20. Cell extract were immunoprecipitated separately with Flag or HA antibodies, and the associated RNF20 and G9a were examined by Western blotting respectively.
    Figure Legend Snippet: RNF20 interacts with G9a. 293T cells were transiently coexpressed with Flag-tagged G9a, HA-tagged RNF20. Cell extract were immunoprecipitated separately with Flag or HA antibodies, and the associated RNF20 and G9a were examined by Western blotting respectively.

    Techniques Used: Immunoprecipitation, Western Blot

    RNF20 is recruited to the E-cadherin promoter for epigenetic silencing of E-cadherin expression by Snail. (A, B) RNF20 was expressed in MDA-MB231 and BT549 cells, and RNF20, H3K9me2, and H3K9 acetylation at the E-cadherin promoter were analyzed by the ChIP assay. Quantitative real-time PCR was also performed to analyze ChIP samples in (A) (mean ± SD from three separate experiments). *P < 0.01 by Student’s t test.
    Figure Legend Snippet: RNF20 is recruited to the E-cadherin promoter for epigenetic silencing of E-cadherin expression by Snail. (A, B) RNF20 was expressed in MDA-MB231 and BT549 cells, and RNF20, H3K9me2, and H3K9 acetylation at the E-cadherin promoter were analyzed by the ChIP assay. Quantitative real-time PCR was also performed to analyze ChIP samples in (A) (mean ± SD from three separate experiments). *P < 0.01 by Student’s t test.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    RNF20 promotes tumorsphere and colony formation. (A) Tumorsphere formation of MDA-MB231 and BT549 cells with stable empty vector (Ctr) or overexpression of RNF20 was measured. (B) The formation of colonies was measured from MDA-MB231 and BT549 cells with stable empty vector (Ctr) or overexpression of RNF20, as well as BT474 cells with stable empty vector (Ctr) or knockdown of RNF20 expression. Data are presented as a percentage of empty vector cells lines (mean ± SD in three separate experiments). *P < 0.01 by Student’s t test (C) Kaplan-Meier survival analysis for overall survival (OS) of patients in a breast cancer dataset according to RNF20 protein expression status. The p value was determined using the log-rank test.
    Figure Legend Snippet: RNF20 promotes tumorsphere and colony formation. (A) Tumorsphere formation of MDA-MB231 and BT549 cells with stable empty vector (Ctr) or overexpression of RNF20 was measured. (B) The formation of colonies was measured from MDA-MB231 and BT549 cells with stable empty vector (Ctr) or overexpression of RNF20, as well as BT474 cells with stable empty vector (Ctr) or knockdown of RNF20 expression. Data are presented as a percentage of empty vector cells lines (mean ± SD in three separate experiments). *P < 0.01 by Student’s t test (C) Kaplan-Meier survival analysis for overall survival (OS) of patients in a breast cancer dataset according to RNF20 protein expression status. The p value was determined using the log-rank test.

    Techniques Used: Plasmid Preparation, Over Expression, Expressing

    anti stat5  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti stat5
    Anti Stat5, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti stat5/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti stat5 - by Bioz Stars, 2023-01
    94/100 stars

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    rabbit anti rnf20 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti rnf20 antibody
    A. Images of tumorspheres from 3 lung adenocarcinoma tissues (Samples 1, 2, and 3). B. Western Blot analysis of CD133 (as a surface biomarker of CICs), CD44 (as another surface biomarker of CICs), USP22, <t>RNF20,</t> and H2Bub1, shows enriched USP22 protein in tumorspheres compared to original cancer tissues. C. Flow cytometry analysis of CD133 expression demonstrates a strong CD133 expression in about 30% of tumorsphere cells. D. Western Blot analysis demonstrates USP22 is markedly elevated, while H2Bub1 were significantly deceased in CD133+ cancer cells compared to CD133− cells sorted from tumorspheres. E. Flow cytometry analysis of CD44 expression demonstrates a strong CD44 expression in about 12% to 44% of tumorsphere cells. F. Western Blot analysis demonstrates USP22 and CD133 are markedly elevated, while H2Bub1 and RNF20 were markedly or moderately deceased in CD44+ cancer cells compared to CD44− cells sorted from tumorspheres. G. Mouse xenografts generated by both CD133− and CD133+ cancer cells isolated from tumorspheres, indicating CD133+ cells may represent CICs with stem cell characteristics in tumorspheres.
    Rabbit Anti Rnf20 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti rnf20 antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti rnf20 antibody - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Targeting USP22 Suppresses Tumorigenicity and Enhances Cisplatin Sensitivity Through ALDH1A3 Downregulation in Cancer-initiating Cells from Lung Adenocarcinoma"

    Article Title: Targeting USP22 Suppresses Tumorigenicity and Enhances Cisplatin Sensitivity Through ALDH1A3 Downregulation in Cancer-initiating Cells from Lung Adenocarcinoma

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-18-0042

    A. Images of tumorspheres from 3 lung adenocarcinoma tissues (Samples 1, 2, and 3). B. Western Blot analysis of CD133 (as a surface biomarker of CICs), CD44 (as another surface biomarker of CICs), USP22, RNF20, and H2Bub1, shows enriched USP22 protein in tumorspheres compared to original cancer tissues. C. Flow cytometry analysis of CD133 expression demonstrates a strong CD133 expression in about 30% of tumorsphere cells. D. Western Blot analysis demonstrates USP22 is markedly elevated, while H2Bub1 were significantly deceased in CD133+ cancer cells compared to CD133− cells sorted from tumorspheres. E. Flow cytometry analysis of CD44 expression demonstrates a strong CD44 expression in about 12% to 44% of tumorsphere cells. F. Western Blot analysis demonstrates USP22 and CD133 are markedly elevated, while H2Bub1 and RNF20 were markedly or moderately deceased in CD44+ cancer cells compared to CD44− cells sorted from tumorspheres. G. Mouse xenografts generated by both CD133− and CD133+ cancer cells isolated from tumorspheres, indicating CD133+ cells may represent CICs with stem cell characteristics in tumorspheres.
    Figure Legend Snippet: A. Images of tumorspheres from 3 lung adenocarcinoma tissues (Samples 1, 2, and 3). B. Western Blot analysis of CD133 (as a surface biomarker of CICs), CD44 (as another surface biomarker of CICs), USP22, RNF20, and H2Bub1, shows enriched USP22 protein in tumorspheres compared to original cancer tissues. C. Flow cytometry analysis of CD133 expression demonstrates a strong CD133 expression in about 30% of tumorsphere cells. D. Western Blot analysis demonstrates USP22 is markedly elevated, while H2Bub1 were significantly deceased in CD133+ cancer cells compared to CD133− cells sorted from tumorspheres. E. Flow cytometry analysis of CD44 expression demonstrates a strong CD44 expression in about 12% to 44% of tumorsphere cells. F. Western Blot analysis demonstrates USP22 and CD133 are markedly elevated, while H2Bub1 and RNF20 were markedly or moderately deceased in CD44+ cancer cells compared to CD44− cells sorted from tumorspheres. G. Mouse xenografts generated by both CD133− and CD133+ cancer cells isolated from tumorspheres, indicating CD133+ cells may represent CICs with stem cell characteristics in tumorspheres.

    Techniques Used: Western Blot, Biomarker Assay, Flow Cytometry, Expressing, Generated, Isolation

    A. Morphology changes of tumorspheres in differentiating culture medium (left panel); Western Blot analysis of USP22, CD133, RNF20 and H2Bub1 in tumorspheres in differentiating conditions (right panel). B. Western Blot analysis of USP22, CD133 and H2Bub1 in GFP-CTRL and USP22-KD cells (left panel); Quantitative analysis of tumorsphere formation upon USP22 knockdown, GFP-CTRL for cells transfected with control shRNA, USP22-KD for specific shRNA targeting USP22 (**P < 0.01, compared with Control, middle panel) and Tumorspheres (right panel). C. In vivo tumorigenesis of GFP-CTRL and USP22-KD tumorsphere cells, demonstrating USP22 knockdown suppressed both tumorsphere formation and tumorigenesis of lung adenocarcinoma CICs.
    Figure Legend Snippet: A. Morphology changes of tumorspheres in differentiating culture medium (left panel); Western Blot analysis of USP22, CD133, RNF20 and H2Bub1 in tumorspheres in differentiating conditions (right panel). B. Western Blot analysis of USP22, CD133 and H2Bub1 in GFP-CTRL and USP22-KD cells (left panel); Quantitative analysis of tumorsphere formation upon USP22 knockdown, GFP-CTRL for cells transfected with control shRNA, USP22-KD for specific shRNA targeting USP22 (**P < 0.01, compared with Control, middle panel) and Tumorspheres (right panel). C. In vivo tumorigenesis of GFP-CTRL and USP22-KD tumorsphere cells, demonstrating USP22 knockdown suppressed both tumorsphere formation and tumorigenesis of lung adenocarcinoma CICs.

    Techniques Used: Western Blot, Transfection, shRNA, In Vivo

    rabbit anti rnf20 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti rnf20 antibody
    A. Images of tumorspheres from 3 lung adenocarcinoma tissues (Samples 1, 2, and 3). B. Western Blot analysis of CD133 (as a surface biomarker of CICs), CD44 (as another surface biomarker of CICs), USP22, <t>RNF20,</t> and H2Bub1, shows enriched USP22 protein in tumorspheres compared to original cancer tissues. C. Flow cytometry analysis of CD133 expression demonstrates a strong CD133 expression in about 30% of tumorsphere cells. D. Western Blot analysis demonstrates USP22 is markedly elevated, while H2Bub1 were significantly deceased in CD133+ cancer cells compared to CD133− cells sorted from tumorspheres. E. Flow cytometry analysis of CD44 expression demonstrates a strong CD44 expression in about 12% to 44% of tumorsphere cells. F. Western Blot analysis demonstrates USP22 and CD133 are markedly elevated, while H2Bub1 and RNF20 were markedly or moderately deceased in CD44+ cancer cells compared to CD44− cells sorted from tumorspheres. G. Mouse xenografts generated by both CD133− and CD133+ cancer cells isolated from tumorspheres, indicating CD133+ cells may represent CICs with stem cell characteristics in tumorspheres.
    Rabbit Anti Rnf20 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti rnf20 antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti rnf20 antibody - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Targeting USP22 Suppresses Tumorigenicity and Enhances Cisplatin Sensitivity Through ALDH1A3 Downregulation in Cancer-initiating Cells from Lung Adenocarcinoma"

    Article Title: Targeting USP22 Suppresses Tumorigenicity and Enhances Cisplatin Sensitivity Through ALDH1A3 Downregulation in Cancer-initiating Cells from Lung Adenocarcinoma

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-18-0042

    A. Images of tumorspheres from 3 lung adenocarcinoma tissues (Samples 1, 2, and 3). B. Western Blot analysis of CD133 (as a surface biomarker of CICs), CD44 (as another surface biomarker of CICs), USP22, RNF20, and H2Bub1, shows enriched USP22 protein in tumorspheres compared to original cancer tissues. C. Flow cytometry analysis of CD133 expression demonstrates a strong CD133 expression in about 30% of tumorsphere cells. D. Western Blot analysis demonstrates USP22 is markedly elevated, while H2Bub1 were significantly deceased in CD133+ cancer cells compared to CD133− cells sorted from tumorspheres. E. Flow cytometry analysis of CD44 expression demonstrates a strong CD44 expression in about 12% to 44% of tumorsphere cells. F. Western Blot analysis demonstrates USP22 and CD133 are markedly elevated, while H2Bub1 and RNF20 were markedly or moderately deceased in CD44+ cancer cells compared to CD44− cells sorted from tumorspheres. G. Mouse xenografts generated by both CD133− and CD133+ cancer cells isolated from tumorspheres, indicating CD133+ cells may represent CICs with stem cell characteristics in tumorspheres.
    Figure Legend Snippet: A. Images of tumorspheres from 3 lung adenocarcinoma tissues (Samples 1, 2, and 3). B. Western Blot analysis of CD133 (as a surface biomarker of CICs), CD44 (as another surface biomarker of CICs), USP22, RNF20, and H2Bub1, shows enriched USP22 protein in tumorspheres compared to original cancer tissues. C. Flow cytometry analysis of CD133 expression demonstrates a strong CD133 expression in about 30% of tumorsphere cells. D. Western Blot analysis demonstrates USP22 is markedly elevated, while H2Bub1 were significantly deceased in CD133+ cancer cells compared to CD133− cells sorted from tumorspheres. E. Flow cytometry analysis of CD44 expression demonstrates a strong CD44 expression in about 12% to 44% of tumorsphere cells. F. Western Blot analysis demonstrates USP22 and CD133 are markedly elevated, while H2Bub1 and RNF20 were markedly or moderately deceased in CD44+ cancer cells compared to CD44− cells sorted from tumorspheres. G. Mouse xenografts generated by both CD133− and CD133+ cancer cells isolated from tumorspheres, indicating CD133+ cells may represent CICs with stem cell characteristics in tumorspheres.

    Techniques Used: Western Blot, Biomarker Assay, Flow Cytometry, Expressing, Generated, Isolation

    A. Morphology changes of tumorspheres in differentiating culture medium (left panel); Western Blot analysis of USP22, CD133, RNF20 and H2Bub1 in tumorspheres in differentiating conditions (right panel). B. Western Blot analysis of USP22, CD133 and H2Bub1 in GFP-CTRL and USP22-KD cells (left panel); Quantitative analysis of tumorsphere formation upon USP22 knockdown, GFP-CTRL for cells transfected with control shRNA, USP22-KD for specific shRNA targeting USP22 (**P < 0.01, compared with Control, middle panel) and Tumorspheres (right panel). C. In vivo tumorigenesis of GFP-CTRL and USP22-KD tumorsphere cells, demonstrating USP22 knockdown suppressed both tumorsphere formation and tumorigenesis of lung adenocarcinoma CICs.
    Figure Legend Snippet: A. Morphology changes of tumorspheres in differentiating culture medium (left panel); Western Blot analysis of USP22, CD133, RNF20 and H2Bub1 in tumorspheres in differentiating conditions (right panel). B. Western Blot analysis of USP22, CD133 and H2Bub1 in GFP-CTRL and USP22-KD cells (left panel); Quantitative analysis of tumorsphere formation upon USP22 knockdown, GFP-CTRL for cells transfected with control shRNA, USP22-KD for specific shRNA targeting USP22 (**P < 0.01, compared with Control, middle panel) and Tumorspheres (right panel). C. In vivo tumorigenesis of GFP-CTRL and USP22-KD tumorsphere cells, demonstrating USP22 knockdown suppressed both tumorsphere formation and tumorigenesis of lung adenocarcinoma CICs.

    Techniques Used: Western Blot, Transfection, shRNA, In Vivo

    rnf20  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rnf20
    Downregulation of <t>RNF20</t> mRNA in lung adenocarcinoma tissues and its association with histone modification in lung epithelial cell lines. (a) Box-and-Whisker plot of RNF20 mRNA level in lung adenocarcinoma (LUAD). Box represents first and third quartiles, thick band is median value, and bars extend to ± the interquartile range divided by the square root of the number of samples were applied to describe RNF20 gene expression values. Compared to normal lung tissues (Gr2, n=59), RNA20 mRNA was significantly decreased in LUAD (Gr1, n = 517) (fold change/FC = 0.86, and P= 9.12E-12). (b) Western blot analysis of RNF20, H2Bub1, H3K4/79-me3 and USP22 in lung epithelial cells at 72h post-transfection of control or RNF20 siRNA. Beta-actin served as the loading control.
    Rnf20, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnf20/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rnf20 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma"

    Article Title: Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma

    Journal: International journal of cancer

    doi: 10.1002/ijc.30769

    Downregulation of RNF20 mRNA in lung adenocarcinoma tissues and its association with histone modification in lung epithelial cell lines. (a) Box-and-Whisker plot of RNF20 mRNA level in lung adenocarcinoma (LUAD). Box represents first and third quartiles, thick band is median value, and bars extend to ± the interquartile range divided by the square root of the number of samples were applied to describe RNF20 gene expression values. Compared to normal lung tissues (Gr2, n=59), RNA20 mRNA was significantly decreased in LUAD (Gr1, n = 517) (fold change/FC = 0.86, and P= 9.12E-12). (b) Western blot analysis of RNF20, H2Bub1, H3K4/79-me3 and USP22 in lung epithelial cells at 72h post-transfection of control or RNF20 siRNA. Beta-actin served as the loading control.
    Figure Legend Snippet: Downregulation of RNF20 mRNA in lung adenocarcinoma tissues and its association with histone modification in lung epithelial cell lines. (a) Box-and-Whisker plot of RNF20 mRNA level in lung adenocarcinoma (LUAD). Box represents first and third quartiles, thick band is median value, and bars extend to ± the interquartile range divided by the square root of the number of samples were applied to describe RNF20 gene expression values. Compared to normal lung tissues (Gr2, n=59), RNA20 mRNA was significantly decreased in LUAD (Gr1, n = 517) (fold change/FC = 0.86, and P= 9.12E-12). (b) Western blot analysis of RNF20, H2Bub1, H3K4/79-me3 and USP22 in lung epithelial cells at 72h post-transfection of control or RNF20 siRNA. Beta-actin served as the loading control.

    Techniques Used: Modification, Whisker Assay, Expressing, Western Blot, Transfection

    Changes of gene expression profile and gene set in A549, H1299, H460 cells upon downregulation of H2Bub1. (a) Unsupervised hierarchical clustering analysis of global expression profiles in lung epithelial cells upon RNF20 knockdown. The two top rows represent two independent siRNA repeats, RNF is for RNF20 siRNA, and C is for control siRNA. (b) qRT–PCR and (c) Western blot analysis for selected genes. The level of each gene in cancer cell transfected with RNF20 siRNA is the average ratio of triplicate samples, and is presented as the ratio to control sample transfected with scramble siRNA (* P < 0.05, compared with control). (d). Selected gene sets that were enriched upon RNF20 knockdown in these lung cancer cells.
    Figure Legend Snippet: Changes of gene expression profile and gene set in A549, H1299, H460 cells upon downregulation of H2Bub1. (a) Unsupervised hierarchical clustering analysis of global expression profiles in lung epithelial cells upon RNF20 knockdown. The two top rows represent two independent siRNA repeats, RNF is for RNF20 siRNA, and C is for control siRNA. (b) qRT–PCR and (c) Western blot analysis for selected genes. The level of each gene in cancer cell transfected with RNF20 siRNA is the average ratio of triplicate samples, and is presented as the ratio to control sample transfected with scramble siRNA (* P < 0.05, compared with control). (d). Selected gene sets that were enriched upon RNF20 knockdown in these lung cancer cells.

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Transfection

    Knockdown of RNF20 enhances proliferation, migration and inhibits retinoic acid-induced squamous differentiation of BEAS-2B cells. (a) Enriched gene sets in BEAS-2B cells with silenced RNF20 and decreased H2Bub1 (P < 0.05, FDR < 5%). (b) Western blot for RNF20 and H2Bub1, p53, Myc, and ALDH1A1 proteins. (c) Enhanced cellular proliferation and (d) migration upon RNF20 knockdown in BEAS-2B cells. After 72 h of siRNA transfection, cell proliferation was measured. At 48 h post-transfection, 5 x 104 cells transfected with either control or RNF20 siRNA were further subjected to transwell migration assay for 12 h, and the number of migrated cells was counted (*P < 0.05, compared to control siRNA). (e) qRT–PCR analysis of IVL, a squamous differentiation marker, mRNA in BEAS-2B cells transfected with control or RNF20 siRNA and cultured in the differentiating medium BEDM, data were presented as ratio to non-differentiating medium control (*P < 0.05, ** P < 0.01, compared with control). (f) Micrographs show morphology changes of BEAS-2B cells after transfection with control siRNA (left panel) or RNF20 siRNA (right panel) and then subcultured in BEDM. Arrows point to “fried egg” morphology of squamous cells in control siRNA-transfected BEAS-2B cells.
    Figure Legend Snippet: Knockdown of RNF20 enhances proliferation, migration and inhibits retinoic acid-induced squamous differentiation of BEAS-2B cells. (a) Enriched gene sets in BEAS-2B cells with silenced RNF20 and decreased H2Bub1 (P < 0.05, FDR < 5%). (b) Western blot for RNF20 and H2Bub1, p53, Myc, and ALDH1A1 proteins. (c) Enhanced cellular proliferation and (d) migration upon RNF20 knockdown in BEAS-2B cells. After 72 h of siRNA transfection, cell proliferation was measured. At 48 h post-transfection, 5 x 104 cells transfected with either control or RNF20 siRNA were further subjected to transwell migration assay for 12 h, and the number of migrated cells was counted (*P < 0.05, compared to control siRNA). (e) qRT–PCR analysis of IVL, a squamous differentiation marker, mRNA in BEAS-2B cells transfected with control or RNF20 siRNA and cultured in the differentiating medium BEDM, data were presented as ratio to non-differentiating medium control (*P < 0.05, ** P < 0.01, compared with control). (f) Micrographs show morphology changes of BEAS-2B cells after transfection with control siRNA (left panel) or RNF20 siRNA (right panel) and then subcultured in BEDM. Arrows point to “fried egg” morphology of squamous cells in control siRNA-transfected BEAS-2B cells.

    Techniques Used: Migration, Western Blot, Transfection, Transwell Migration Assay, Quantitative RT-PCR, Marker, Cell Culture

    Impact of RNF20 knockdown on in vitro proliferation, migration, and invasion of lung cancer cell lines. (a) Western blot analysis of cell lysates of A549 and H460 cells transfected with either control or RNF20 siRNA and harvested at 72 h post-transfection. Actin served as loading control, total and phosphorylated Erk/Akt (T/P-Erk/Akt), p53, p21 E-Cadherin, and Vimentin were detected. The p53 blots were from separately developed blots for each cell, and the below Actin is its loading control. (b) Histogram of proliferation shows RNF20 knockdown increased the in vitro proliferation of both A549 and H460 cells over 72h (*P < 0.05, compared to control siRNA). (c) Representative photomicrographs of the migration chamber at the times indicated in control or RNF20 siRNA-transfected cells is shown on the left, with quantitative analysis of migrated cells shown on the right (N = 3 replicates per cell type). Error bars show SD (*P < 0.05, compared with control siRNA). (d) Lower surface of Matrigel transwell membranes seeded with cancer cells previously transfected with indicated siRNAs, 24 h after incubation (left), and corresponding quantitative analysis of invading cells (right). Data are shown as mean values graphed for indicated cells on the right (N = 3 replicates per cell type). Error bars show SD (*P < 0.05, **P < 0.01, compared with control siRNA).
    Figure Legend Snippet: Impact of RNF20 knockdown on in vitro proliferation, migration, and invasion of lung cancer cell lines. (a) Western blot analysis of cell lysates of A549 and H460 cells transfected with either control or RNF20 siRNA and harvested at 72 h post-transfection. Actin served as loading control, total and phosphorylated Erk/Akt (T/P-Erk/Akt), p53, p21 E-Cadherin, and Vimentin were detected. The p53 blots were from separately developed blots for each cell, and the below Actin is its loading control. (b) Histogram of proliferation shows RNF20 knockdown increased the in vitro proliferation of both A549 and H460 cells over 72h (*P < 0.05, compared to control siRNA). (c) Representative photomicrographs of the migration chamber at the times indicated in control or RNF20 siRNA-transfected cells is shown on the left, with quantitative analysis of migrated cells shown on the right (N = 3 replicates per cell type). Error bars show SD (*P < 0.05, compared with control siRNA). (d) Lower surface of Matrigel transwell membranes seeded with cancer cells previously transfected with indicated siRNAs, 24 h after incubation (left), and corresponding quantitative analysis of invading cells (right). Data are shown as mean values graphed for indicated cells on the right (N = 3 replicates per cell type). Error bars show SD (*P < 0.05, **P < 0.01, compared with control siRNA).

    Techniques Used: In Vitro, Migration, Western Blot, Transfection, Incubation

    Downregulation of H2Bub1 enhanced resistance to cisplatin in lung cancer cells. The levels of RNF20, H2Bub1, p21, p53, and apoptotic markers caspase-3/PARP cleaved products (T/C- Caspase-3/PARP for total and cleaved proteins) in (a) A549 and (b). H460 cells treated with cisplatin were analyzed by Western blot. Beta-actin was used as loading control. (c) Representative flow cytometry profile of A549 and H460 cells transfected with either control (left panel) or RNF20 siRNA (right panel). After cells were treated for 72 h with 20 uM Cisplatin, apoptosis was measured by flow cytometry analysis of Annexin-V (labeled with Alexa Fluor 488) staining in X axis and propidium iodide staining in Y axis. (d) Quantitative analysis of the experiments shows that apoptotic cells were significantly reduced in both A549 and H460 cells upon downregulation of H2Bub1. The experiment was repeated three times and data represent the average of the early apoptotic and late apoptotic cells (* P < 0.05; ** P < 0.01).
    Figure Legend Snippet: Downregulation of H2Bub1 enhanced resistance to cisplatin in lung cancer cells. The levels of RNF20, H2Bub1, p21, p53, and apoptotic markers caspase-3/PARP cleaved products (T/C- Caspase-3/PARP for total and cleaved proteins) in (a) A549 and (b). H460 cells treated with cisplatin were analyzed by Western blot. Beta-actin was used as loading control. (c) Representative flow cytometry profile of A549 and H460 cells transfected with either control (left panel) or RNF20 siRNA (right panel). After cells were treated for 72 h with 20 uM Cisplatin, apoptosis was measured by flow cytometry analysis of Annexin-V (labeled with Alexa Fluor 488) staining in X axis and propidium iodide staining in Y axis. (d) Quantitative analysis of the experiments shows that apoptotic cells were significantly reduced in both A549 and H460 cells upon downregulation of H2Bub1. The experiment was repeated three times and data represent the average of the early apoptotic and late apoptotic cells (* P < 0.05; ** P < 0.01).

    Techniques Used: Western Blot, Flow Cytometry, Transfection, Labeling, Staining

    anti bre1a  (Cell Signaling Technology Inc)


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    anti bre1a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti bre1a
    Anti Bre1a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti bre1a igg  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti bre1a igg
    PCR primers for RT-PCR
    Rabbit Anti Bre1a Igg, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Bre1a, a Histone H2B Ubiquitin Ligase, Regulates the Cell Cycle and Differentiation of Neural Precursor Cells"

    Article Title: Bre1a, a Histone H2B Ubiquitin Ligase, Regulates the Cell Cycle and Differentiation of Neural Precursor Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3832-13.2014

    PCR primers for RT-PCR
    Figure Legend Snippet: PCR primers for RT-PCR

    Techniques Used: Sequencing

    Bre1a expression in the developing brain. A–L, The expression of Bre1a was determined in coronal cryosections of forebrains from E10.5 (A–D), E12.5 (E–I), E14.5 (J), and E16.5 (K) embryos, and P0 pups (L) by immunostaining or by in situ hybridization (J, top). B, Coronal cryosections of E10.5 dorsal forebrain were immunostained for Bre1a and H2Bub1. Boxed areas in the top panels are shown in the bottom panels with higher magnification. Nonmitotic cells that weakly express Bre1a and H2Bub1 are indicated by arrowheads, and some mitotic cells are indicated by dotted circles and arrows. C, Double immunostaining for Bre1a and phospho-histone 3 (PH3) shows Bre1aweak/PH3− cells (arrowheads). D, Colocalization of Bre1a and βIII-tubulin in cells nearby the basal surface is indicated by arrowheads. E–H, Coronal cryosections of E12.5 brain were double-immunostained for Bre1a and H2Bub1 (E), and boxed areas in the cortex (F), dorsoventral boundary (G), and medial ganglionic eminence (H) are shown with higher magnification. Nonmitotic cells that weakly express Bre1a and H2Bub1 are indicated by arrowheads, and some mitotic cells are indicated by dotted circles and arrows. I, Coronal cryosections of E12.5 brain were immunostained for PH3, and then, double immunostained for Bre1a and H2Bub1. Alexa Fluor-633 signal for PH3 is pseudo-colored and shown in blue. LV, Lateral ventricle. Scale bars: A, E, J–L, 200 μm; B–D, F–I, 50 μm.
    Figure Legend Snippet: Bre1a expression in the developing brain. A–L, The expression of Bre1a was determined in coronal cryosections of forebrains from E10.5 (A–D), E12.5 (E–I), E14.5 (J), and E16.5 (K) embryos, and P0 pups (L) by immunostaining or by in situ hybridization (J, top). B, Coronal cryosections of E10.5 dorsal forebrain were immunostained for Bre1a and H2Bub1. Boxed areas in the top panels are shown in the bottom panels with higher magnification. Nonmitotic cells that weakly express Bre1a and H2Bub1 are indicated by arrowheads, and some mitotic cells are indicated by dotted circles and arrows. C, Double immunostaining for Bre1a and phospho-histone 3 (PH3) shows Bre1aweak/PH3− cells (arrowheads). D, Colocalization of Bre1a and βIII-tubulin in cells nearby the basal surface is indicated by arrowheads. E–H, Coronal cryosections of E12.5 brain were double-immunostained for Bre1a and H2Bub1 (E), and boxed areas in the cortex (F), dorsoventral boundary (G), and medial ganglionic eminence (H) are shown with higher magnification. Nonmitotic cells that weakly express Bre1a and H2Bub1 are indicated by arrowheads, and some mitotic cells are indicated by dotted circles and arrows. I, Coronal cryosections of E12.5 brain were immunostained for PH3, and then, double immunostained for Bre1a and H2Bub1. Alexa Fluor-633 signal for PH3 is pseudo-colored and shown in blue. LV, Lateral ventricle. Scale bars: A, E, J–L, 200 μm; B–D, F–I, 50 μm.

    Techniques Used: Expressing, Immunostaining, In Situ Hybridization, Double Immunostaining

    Bre1a knockdown impairs the proliferation of neural precursor cells. A–C, F, H, Primary neurospheres derived from the E13.5 cortex were infected with lentivirus expressing control or Bre1a shRNAs and GFP, and resultant GFP+ secondary neurospheres were subjected for analyses. A, Quantitative RT-PCR for Bre1a expression. B, GFP+ secondary neurospheres were plated and immunostained for GFP and Bre1a. Arrowheads and arrows indicate Bre1a shRNA-expressing and nonexpressing cells, respectively. C, Protein extracts from neurospheres expressing Bre1a shRNAs or control shRNA were subjected to Western blotting using the indicated antibodies. D, E, G, Primary neuroepithelial cells from E10.5 forebrains were transfected with Bre1a shRNA or control shRNA (D), together with GFP expression plasmid only, GFP and Bre1a expression plasmids (E), or GFP and miRNA expression plasmids (G). We used two miRNAs for p57Kip2 (1 and 2) or control miRNA. The cells were cultured in proliferation conditions for 3 d and were incubated with 20 μm EdU for 2 h before fixation. D, Cells were immunostained for GFP, followed by visualization for EdU. Arrowheads indicate GFP+/EdU+ cells and arrows indicate GFP+/EdU− cells. E, G, Quantification of GFP+/EdU+ cells in total GFP+ cells. F, H, Quantitative RT-PCR for p21Cip1, p27Kip1, and p57Kip2 (F) and for Cdk2 (H). Scale bars, 50 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (A, E, F, H) or Tukey-Kramer's post hoc comparison (G).
    Figure Legend Snippet: Bre1a knockdown impairs the proliferation of neural precursor cells. A–C, F, H, Primary neurospheres derived from the E13.5 cortex were infected with lentivirus expressing control or Bre1a shRNAs and GFP, and resultant GFP+ secondary neurospheres were subjected for analyses. A, Quantitative RT-PCR for Bre1a expression. B, GFP+ secondary neurospheres were plated and immunostained for GFP and Bre1a. Arrowheads and arrows indicate Bre1a shRNA-expressing and nonexpressing cells, respectively. C, Protein extracts from neurospheres expressing Bre1a shRNAs or control shRNA were subjected to Western blotting using the indicated antibodies. D, E, G, Primary neuroepithelial cells from E10.5 forebrains were transfected with Bre1a shRNA or control shRNA (D), together with GFP expression plasmid only, GFP and Bre1a expression plasmids (E), or GFP and miRNA expression plasmids (G). We used two miRNAs for p57Kip2 (1 and 2) or control miRNA. The cells were cultured in proliferation conditions for 3 d and were incubated with 20 μm EdU for 2 h before fixation. D, Cells were immunostained for GFP, followed by visualization for EdU. Arrowheads indicate GFP+/EdU+ cells and arrows indicate GFP+/EdU− cells. E, G, Quantification of GFP+/EdU+ cells in total GFP+ cells. F, H, Quantitative RT-PCR for p21Cip1, p27Kip1, and p57Kip2 (F) and for Cdk2 (H). Scale bars, 50 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (A, E, F, H) or Tukey-Kramer's post hoc comparison (G).

    Techniques Used: Derivative Assay, Infection, Expressing, Quantitative RT-PCR, shRNA, Western Blot, Transfection, Plasmid Preparation, Cell Culture, Incubation

    Bre1a knockdown in neural precursor cells reduces the proportion of mitotic cells. A–D, Bre1a shRNA or control shRNA and GFP expression plasmids were microinjected into the lateral ventricle (LV) of E13.5 embryos and electroporated into the dorsal forebrain in utero. The embryos were fixed 24 h after electroporation, and the brains were analyzed. A, GFP+ Bre1a shRNA-expressing cells (arrowheads) are less immunopositive for Bre1a than surrounding GFP− cells (arrows). B, Cryosections were immunostained for GFP and c-Cas3, followed by Hoechst nuclear staining. C, D, EdU was injected intraperitoneally into dams 2 h before fixation. Coronal cryosections were immunostained for GFP, followed by EdU visualization (C), and GFP +/EdU+ cells were counted (D). Bre1a expression plasmids were coelectroporated with Bre1a shRNA 3 to rescue the effects of Bre1a knockdown. E–H, Bre1a or control shRNA plasmids together with GFP and H2B-mCherry expression plasmids were coelectroporated into the dorsal forebrain of E13.5 embryos in utero. Twenty-four hours after electroporation, coronal slices were made and cultured on a hydrophilic transparent membrane in serum-free media for 30 h. E, Time-lapse images of GFP+/mCherry+ cells in the VZ/SVZ. Arrowheads indicate cells undergoing cell divisions, which were precisely recognized by the observation of H2B-mCherry expression (red). F–H, The percentage of GFP+ cells that underwent mitosis during the 30 h culture (F), the duration of the G2/M phase of GFP+ cells (G), and the migration speed of GFP+ cells in the G2 phase (H) in the VZ/SVZ were quantified. Scale bars: A, 20 μm; B, 100 μm; C, E, 30 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (D) or Student's t test (F–H).
    Figure Legend Snippet: Bre1a knockdown in neural precursor cells reduces the proportion of mitotic cells. A–D, Bre1a shRNA or control shRNA and GFP expression plasmids were microinjected into the lateral ventricle (LV) of E13.5 embryos and electroporated into the dorsal forebrain in utero. The embryos were fixed 24 h after electroporation, and the brains were analyzed. A, GFP+ Bre1a shRNA-expressing cells (arrowheads) are less immunopositive for Bre1a than surrounding GFP− cells (arrows). B, Cryosections were immunostained for GFP and c-Cas3, followed by Hoechst nuclear staining. C, D, EdU was injected intraperitoneally into dams 2 h before fixation. Coronal cryosections were immunostained for GFP, followed by EdU visualization (C), and GFP +/EdU+ cells were counted (D). Bre1a expression plasmids were coelectroporated with Bre1a shRNA 3 to rescue the effects of Bre1a knockdown. E–H, Bre1a or control shRNA plasmids together with GFP and H2B-mCherry expression plasmids were coelectroporated into the dorsal forebrain of E13.5 embryos in utero. Twenty-four hours after electroporation, coronal slices were made and cultured on a hydrophilic transparent membrane in serum-free media for 30 h. E, Time-lapse images of GFP+/mCherry+ cells in the VZ/SVZ. Arrowheads indicate cells undergoing cell divisions, which were precisely recognized by the observation of H2B-mCherry expression (red). F–H, The percentage of GFP+ cells that underwent mitosis during the 30 h culture (F), the duration of the G2/M phase of GFP+ cells (G), and the migration speed of GFP+ cells in the G2 phase (H) in the VZ/SVZ were quantified. Scale bars: A, 20 μm; B, 100 μm; C, E, 30 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (D) or Student's t test (F–H).

    Techniques Used: shRNA, Expressing, In Utero, Electroporation, Staining, Injection, Cell Culture, Migration

    Bre1a knockdown impairs the migration of neural precursor cells. Bre1a or control shRNA and GFP expression plasmids were microinjected into the lateral ventricle of E13.5 embryos and electroporated into the dorsal forebrain in utero. A, Three days after the electroporation, the embryos were fixed and coronal cryosections of the brains were immunostained for GFP. B, GFP+ cells in the VZ/SVZ, IMZ, or CP were counted. C, D, Twenty-four hours after the electroporation, coronal slices were made and cultured in serum-free media for 30 h. C, Time-lapse images of GFP+ cells in the CP. Arrowheads indicate migrating cells. D, Migration speed of GFP+ cells in the CP was quantified. Scale bars: A, 100 μm; C, 20 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (B) or by Student's t test (D).
    Figure Legend Snippet: Bre1a knockdown impairs the migration of neural precursor cells. Bre1a or control shRNA and GFP expression plasmids were microinjected into the lateral ventricle of E13.5 embryos and electroporated into the dorsal forebrain in utero. A, Three days after the electroporation, the embryos were fixed and coronal cryosections of the brains were immunostained for GFP. B, GFP+ cells in the VZ/SVZ, IMZ, or CP were counted. C, D, Twenty-four hours after the electroporation, coronal slices were made and cultured in serum-free media for 30 h. C, Time-lapse images of GFP+ cells in the CP. Arrowheads indicate migrating cells. D, Migration speed of GFP+ cells in the CP was quantified. Scale bars: A, 100 μm; C, 20 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (B) or by Student's t test (D).

    Techniques Used: Migration, shRNA, Expressing, In Utero, Electroporation, Cell Culture

    Bre1a or Bre1aΔC overexpression does not inhibit cell migration. A, Schematic structures of full-length Bre1a and Bre1a that lacks the ring finger domain at the C terminus (Bre1aΔC) are shown. B–E, Expression plasmids for full-length Bre1a or Bre1aΔC were coelectroporated with GFP expression plasmid only (B, C) or with Bre1a shRNA and GFP expression plasmids (D, E) into the dorsal forebrain of E13.5 embryos in utero. Three days after the electroporation, the embryos were fixed and coronal cryosections of the brains were immunostained for GFP (B, D). C, E, GFP-positive cells in the VZ/SVZ, IMZ, or CP were counted. Scale bar, 100 μm. Error bars indicate SEM, and n values are shown in columns.
    Figure Legend Snippet: Bre1a or Bre1aΔC overexpression does not inhibit cell migration. A, Schematic structures of full-length Bre1a and Bre1a that lacks the ring finger domain at the C terminus (Bre1aΔC) are shown. B–E, Expression plasmids for full-length Bre1a or Bre1aΔC were coelectroporated with GFP expression plasmid only (B, C) or with Bre1a shRNA and GFP expression plasmids (D, E) into the dorsal forebrain of E13.5 embryos in utero. Three days after the electroporation, the embryos were fixed and coronal cryosections of the brains were immunostained for GFP (B, D). C, E, GFP-positive cells in the VZ/SVZ, IMZ, or CP were counted. Scale bar, 100 μm. Error bars indicate SEM, and n values are shown in columns.

    Techniques Used: Over Expression, Migration, Expressing, Plasmid Preparation, shRNA, In Utero, Electroporation

    Bre1a knockdown affects the cortical layer formation in the postnatal brain. A, B, Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E13.5 embryos in utero, and the pups were perfused on P0. A, Coronal cryosections of the brains were immunostained for GFP (right), followed by Hoechst nuclear staining (left). The CP was divided into four layers based on the density of nuclei: the mantle zone (MZ); and upper (u), middle (m), and lower (l) CP. The borders between layers are indicated by dotted lines. B, Quantification of GFP+ cells distributed in the upper, middle, and lower CP in the P0 brain. C–F, Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E14.5 embryos in utero, and the pups were perfused on P14. For rescue experiments, Bre1a expression plasmids were coelectroporated with Bre1a shRNA 2. C–E, Coronal cryosections were immunostained for GFP (middle), followed by Hoechst nuclear staining (left), or were subjected to in situ hybridization with a probe for Rorb, a marker of cortical layer IV (right). F, Quantitative analysis of GFP+ cells in cortical layers II/III and IV. Scale bars, 100 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by Student's or Welch's t test.
    Figure Legend Snippet: Bre1a knockdown affects the cortical layer formation in the postnatal brain. A, B, Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E13.5 embryos in utero, and the pups were perfused on P0. A, Coronal cryosections of the brains were immunostained for GFP (right), followed by Hoechst nuclear staining (left). The CP was divided into four layers based on the density of nuclei: the mantle zone (MZ); and upper (u), middle (m), and lower (l) CP. The borders between layers are indicated by dotted lines. B, Quantification of GFP+ cells distributed in the upper, middle, and lower CP in the P0 brain. C–F, Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E14.5 embryos in utero, and the pups were perfused on P14. For rescue experiments, Bre1a expression plasmids were coelectroporated with Bre1a shRNA 2. C–E, Coronal cryosections were immunostained for GFP (middle), followed by Hoechst nuclear staining (left), or were subjected to in situ hybridization with a probe for Rorb, a marker of cortical layer IV (right). F, Quantitative analysis of GFP+ cells in cortical layers II/III and IV. Scale bars, 100 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by Student's or Welch's t test.

    Techniques Used: shRNA, Expressing, In Utero, Staining, In Situ Hybridization, Marker

    Bre1a knockdown delays the birth date of differentiating neuroblasts. Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E14.5 embryos in utero, and the pups were perfused on P14. For rescue experiments, Bre1a expression plasmids were coelectroporated with Bre1a shRNA 2. A, B, D–F, Dams received an intraperitoneal injection of EdU 24 h (A, B) or 48 h (D–F) after the electroporation. Coronal cryosections were immunostained for GFP (left), followed by EdU visualization (middle), and the merged images are shown (right). Arrowheads indicate GFP and EdU double-positive cells. C, G, Quantification of GFP+/EdU+ cells in cortical layers II/III and IV. Scale bars, 50 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by Student's or Welch's t test.
    Figure Legend Snippet: Bre1a knockdown delays the birth date of differentiating neuroblasts. Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E14.5 embryos in utero, and the pups were perfused on P14. For rescue experiments, Bre1a expression plasmids were coelectroporated with Bre1a shRNA 2. A, B, D–F, Dams received an intraperitoneal injection of EdU 24 h (A, B) or 48 h (D–F) after the electroporation. Coronal cryosections were immunostained for GFP (left), followed by EdU visualization (middle), and the merged images are shown (right). Arrowheads indicate GFP and EdU double-positive cells. C, G, Quantification of GFP+/EdU+ cells in cortical layers II/III and IV. Scale bars, 50 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by Student's or Welch's t test.

    Techniques Used: shRNA, Expressing, In Utero, Injection, Electroporation

    Bre1a facilitates the differentiation of neural precursor cells. A, B, Primary neuroepithelial cells from E10.5 forebrains were transfected with Bre1a or control shRNAs and GFP expression plasmids, and cultured in differentiation condition for 3 d. Bre1a or control expression plasmids were cotransfected with Bre1a-shRNA 2 for rescue experiments. A, Cells were immunostained for GFP and βIII tubulin. Arrowheads and arrows indicate GFP+/βIII tubulin+ and GFP+/βIII tubulin− cells, respectively. B, Quantification of GFP+/βIII tubulin+ cells. C–J, Bre1a or control shRNA, pCX-Bre1a, or control expression plasmids were microinjected into the lateral ventricle of E13.5 embryos and electroporated into the dorsal forebrain in utero. The embryos were fixed 24 h after electroporation, and the brains were analyzed. C–J, Cryosections were immunostained for GFP and Tbr2 (C, G) or Pax6 (E, I). GFP+/Tbr2+ (D, H) or GFP+/Pax6+ (F, J) cells were counted. Scale bars: A, 50 μm; C, E, G, I, 100 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (B, D, F) or by Student's t test (H, J).
    Figure Legend Snippet: Bre1a facilitates the differentiation of neural precursor cells. A, B, Primary neuroepithelial cells from E10.5 forebrains were transfected with Bre1a or control shRNAs and GFP expression plasmids, and cultured in differentiation condition for 3 d. Bre1a or control expression plasmids were cotransfected with Bre1a-shRNA 2 for rescue experiments. A, Cells were immunostained for GFP and βIII tubulin. Arrowheads and arrows indicate GFP+/βIII tubulin+ and GFP+/βIII tubulin− cells, respectively. B, Quantification of GFP+/βIII tubulin+ cells. C–J, Bre1a or control shRNA, pCX-Bre1a, or control expression plasmids were microinjected into the lateral ventricle of E13.5 embryos and electroporated into the dorsal forebrain in utero. The embryos were fixed 24 h after electroporation, and the brains were analyzed. C–J, Cryosections were immunostained for GFP and Tbr2 (C, G) or Pax6 (E, I). GFP+/Tbr2+ (D, H) or GFP+/Pax6+ (F, J) cells were counted. Scale bars: A, 50 μm; C, E, G, I, 100 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (B, D, F) or by Student's t test (H, J).

    Techniques Used: Transfection, Expressing, Cell Culture, shRNA, In Utero, Electroporation

    Bre1a knockdown upregulates Hes5 expression in neural precursor cells. A, B, Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E13.5 embryos in utero, and brains were analyzed 3 d after the electroporation. Coronal cryosections were immunostained for GFP (left) or subjected to in situ hybridization with an RNA probe for Hes5 (right). Arrowheads indicate ectopic Hes5+ cells outside the VZ. The boxed area is magnified in the inset. Scale bar, 50 μm. C, D, G, H, Primary neurosphere cells derived from E13.5 cortex were infected with lentiviruses expressing Bre1a or control shRNAs and were cultured for 4–7 d. Lentivirus-infected GFP+ neurospheres were used for quantitative RT-PCR analysis of Hes5 (C), Hes1 (D), Fezf1 (G), or Fezf2 (H). For rescue experiments, Bre1a-expressing retrovirus was coinfected with Bre1a shRNA-expressing lentivirus. E, F, Firefly luciferase reporter assays in Neuro2a cells using the following plasmids: a plasmid containing the promoter region of the Hes5 (E) or Hes1 (F) gene; the Renilla luciferase reporter plasmid as an internal control; pCX-NICD, and the indicated plasmid for expression of Bre1a, Bre1aΔC, Fezf1, Fezf2, and Fezf1, and Fezf2 miRNAs. The relative promoter activities were compared with activity in the absence of pCX-NICD. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison or Student's t test.
    Figure Legend Snippet: Bre1a knockdown upregulates Hes5 expression in neural precursor cells. A, B, Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E13.5 embryos in utero, and brains were analyzed 3 d after the electroporation. Coronal cryosections were immunostained for GFP (left) or subjected to in situ hybridization with an RNA probe for Hes5 (right). Arrowheads indicate ectopic Hes5+ cells outside the VZ. The boxed area is magnified in the inset. Scale bar, 50 μm. C, D, G, H, Primary neurosphere cells derived from E13.5 cortex were infected with lentiviruses expressing Bre1a or control shRNAs and were cultured for 4–7 d. Lentivirus-infected GFP+ neurospheres were used for quantitative RT-PCR analysis of Hes5 (C), Hes1 (D), Fezf1 (G), or Fezf2 (H). For rescue experiments, Bre1a-expressing retrovirus was coinfected with Bre1a shRNA-expressing lentivirus. E, F, Firefly luciferase reporter assays in Neuro2a cells using the following plasmids: a plasmid containing the promoter region of the Hes5 (E) or Hes1 (F) gene; the Renilla luciferase reporter plasmid as an internal control; pCX-NICD, and the indicated plasmid for expression of Bre1a, Bre1aΔC, Fezf1, Fezf2, and Fezf1, and Fezf2 miRNAs. The relative promoter activities were compared with activity in the absence of pCX-NICD. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison or Student's t test.

    Techniques Used: Expressing, shRNA, In Utero, Electroporation, In Situ Hybridization, Derivative Assay, Infection, Cell Culture, Quantitative RT-PCR, Luciferase, Plasmid Preparation, Activity Assay

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    Cell Signaling Technology Inc 9425s rrid ab 2797700

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    <t>RNF20</t> alters the expression of epithelial-mesenchymal transition (EMT) markers. (A) Expression of E-cadherin, Vimentin and RNF20 in the MDA-MB231 and BT549 cells was analyzed by Western blotting. Actin was served as a loading control. (B) Expression of RNF20 and E-cadherin was analyzed by qPCR in MDA-MB231 and BT549 cells infected with empty vector (Ctr) or RNF20-expressing vector. *P < 0.01 by Student’s t test. (C) Expression of E-cadherin, and RNF20 was analyzed by Western blotting in BT474 cells with stable empty vector or knockdown of RNF20 expression as well as shRNF20-expressing BT474 cells infected with plvx-neo-RNF20. (D) Expression of RNF20 and E-cadherin was analyzed by qPCR in cells from (C) . *P < 0.01 by Student’s t test.
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    <t>RNF20</t> alters the expression of epithelial-mesenchymal transition (EMT) markers. (A) Expression of E-cadherin, Vimentin and RNF20 in the MDA-MB231 and BT549 cells was analyzed by Western blotting. Actin was served as a loading control. (B) Expression of RNF20 and E-cadherin was analyzed by qPCR in MDA-MB231 and BT549 cells infected with empty vector (Ctr) or RNF20-expressing vector. *P < 0.01 by Student’s t test. (C) Expression of E-cadherin, and RNF20 was analyzed by Western blotting in BT474 cells with stable empty vector or knockdown of RNF20 expression as well as shRNF20-expressing BT474 cells infected with plvx-neo-RNF20. (D) Expression of RNF20 and E-cadherin was analyzed by qPCR in cells from (C) . *P < 0.01 by Student’s t test.
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    A. Images of tumorspheres from 3 lung adenocarcinoma tissues (Samples 1, 2, and 3). B. Western Blot analysis of CD133 (as a surface biomarker of CICs), CD44 (as another surface biomarker of CICs), USP22, <t>RNF20,</t> and H2Bub1, shows enriched USP22 protein in tumorspheres compared to original cancer tissues. C. Flow cytometry analysis of CD133 expression demonstrates a strong CD133 expression in about 30% of tumorsphere cells. D. Western Blot analysis demonstrates USP22 is markedly elevated, while H2Bub1 were significantly deceased in CD133+ cancer cells compared to CD133− cells sorted from tumorspheres. E. Flow cytometry analysis of CD44 expression demonstrates a strong CD44 expression in about 12% to 44% of tumorsphere cells. F. Western Blot analysis demonstrates USP22 and CD133 are markedly elevated, while H2Bub1 and RNF20 were markedly or moderately deceased in CD44+ cancer cells compared to CD44− cells sorted from tumorspheres. G. Mouse xenografts generated by both CD133− and CD133+ cancer cells isolated from tumorspheres, indicating CD133+ cells may represent CICs with stem cell characteristics in tumorspheres.
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    Downregulation of <t>RNF20</t> mRNA in lung adenocarcinoma tissues and its association with histone modification in lung epithelial cell lines. (a) Box-and-Whisker plot of RNF20 mRNA level in lung adenocarcinoma (LUAD). Box represents first and third quartiles, thick band is median value, and bars extend to ± the interquartile range divided by the square root of the number of samples were applied to describe RNF20 gene expression values. Compared to normal lung tissues (Gr2, n=59), RNA20 mRNA was significantly decreased in LUAD (Gr1, n = 517) (fold change/FC = 0.86, and P= 9.12E-12). (b) Western blot analysis of RNF20, H2Bub1, H3K4/79-me3 and USP22 in lung epithelial cells at 72h post-transfection of control or RNF20 siRNA. Beta-actin served as the loading control.
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    Downregulation of <t>RNF20</t> mRNA in lung adenocarcinoma tissues and its association with histone modification in lung epithelial cell lines. (a) Box-and-Whisker plot of RNF20 mRNA level in lung adenocarcinoma (LUAD). Box represents first and third quartiles, thick band is median value, and bars extend to ± the interquartile range divided by the square root of the number of samples were applied to describe RNF20 gene expression values. Compared to normal lung tissues (Gr2, n=59), RNA20 mRNA was significantly decreased in LUAD (Gr1, n = 517) (fold change/FC = 0.86, and P= 9.12E-12). (b) Western blot analysis of RNF20, H2Bub1, H3K4/79-me3 and USP22 in lung epithelial cells at 72h post-transfection of control or RNF20 siRNA. Beta-actin served as the loading control.
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    Image Search Results


    Journal: eLife

    Article Title: Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

    doi: 10.7554/eLife.73524

    Figure Lengend Snippet:

    Article Snippet: Antibody , Anti-RNF20 (rabbit polyclonal) , Cell Signaling Technology , Cat# 9425S;RRID: AB_2797700 , WB (1:1000).

    Techniques: Software

    Journal: eLife

    Article Title: Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

    doi: 10.7554/eLife.73524

    Figure Lengend Snippet:

    Article Snippet: Antibody , Anti-RNF20 (rabbit polyclonal) , Cell Signaling Technology , Cat# 9425S;RRID: AB_2797700 , WB (1:1000).

    Techniques: Software

    RNF20 alters the expression of epithelial-mesenchymal transition (EMT) markers. (A) Expression of E-cadherin, Vimentin and RNF20 in the MDA-MB231 and BT549 cells was analyzed by Western blotting. Actin was served as a loading control. (B) Expression of RNF20 and E-cadherin was analyzed by qPCR in MDA-MB231 and BT549 cells infected with empty vector (Ctr) or RNF20-expressing vector. *P < 0.01 by Student’s t test. (C) Expression of E-cadherin, and RNF20 was analyzed by Western blotting in BT474 cells with stable empty vector or knockdown of RNF20 expression as well as shRNF20-expressing BT474 cells infected with plvx-neo-RNF20. (D) Expression of RNF20 and E-cadherin was analyzed by qPCR in cells from (C) . *P < 0.01 by Student’s t test.

    Journal: Frontiers in Oncology

    Article Title: RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer

    doi: 10.3389/fonc.2020.613470

    Figure Lengend Snippet: RNF20 alters the expression of epithelial-mesenchymal transition (EMT) markers. (A) Expression of E-cadherin, Vimentin and RNF20 in the MDA-MB231 and BT549 cells was analyzed by Western blotting. Actin was served as a loading control. (B) Expression of RNF20 and E-cadherin was analyzed by qPCR in MDA-MB231 and BT549 cells infected with empty vector (Ctr) or RNF20-expressing vector. *P < 0.01 by Student’s t test. (C) Expression of E-cadherin, and RNF20 was analyzed by Western blotting in BT474 cells with stable empty vector or knockdown of RNF20 expression as well as shRNF20-expressing BT474 cells infected with plvx-neo-RNF20. (D) Expression of RNF20 and E-cadherin was analyzed by qPCR in cells from (C) . *P < 0.01 by Student’s t test.

    Article Snippet: RNF20 antibodies and Snail antibodies were from Abcam and Cell Signaling Technology respectively.

    Techniques: Expressing, Western Blot, Infection, Plasmid Preparation

    RNF20 enhances breast cancer cell migration and invasion in vitro . (A–C) Migratory (A, B) and invasive ability (C) of MDA-MB231 and BT549 cells with stable empty vector or RNF20 expression were analyzed. The percentage of migratory and invasive cells is shown in the bar graphs (mean ± SD in three separate experiments). *P < 0.01 by Student’s t test: 100 µm.

    Journal: Frontiers in Oncology

    Article Title: RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer

    doi: 10.3389/fonc.2020.613470

    Figure Lengend Snippet: RNF20 enhances breast cancer cell migration and invasion in vitro . (A–C) Migratory (A, B) and invasive ability (C) of MDA-MB231 and BT549 cells with stable empty vector or RNF20 expression were analyzed. The percentage of migratory and invasive cells is shown in the bar graphs (mean ± SD in three separate experiments). *P < 0.01 by Student’s t test: 100 µm.

    Article Snippet: RNF20 antibodies and Snail antibodies were from Abcam and Cell Signaling Technology respectively.

    Techniques: Migration, In Vitro, Plasmid Preparation, Expressing

    RNF20 interacts with Snail. (A) 293T cells were transiently coexpressed with Flag-tagged RNF20 and HA-tagged Snail. Cell extracts were immunoprecipitated separately with Flag or HA antibodies, and the associated RNF20 and Snail were examined by Western blotting, respectively. (B) Schematic diagram showing the structure of Snail and two deletion mutations. (C) Full-length and deletion mutants of Snail were coexpressed with RNF20 in 293T cells. After immunoprecipitation of RN20, associated Snail was analyzed by Western blotting.

    Journal: Frontiers in Oncology

    Article Title: RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer

    doi: 10.3389/fonc.2020.613470

    Figure Lengend Snippet: RNF20 interacts with Snail. (A) 293T cells were transiently coexpressed with Flag-tagged RNF20 and HA-tagged Snail. Cell extracts were immunoprecipitated separately with Flag or HA antibodies, and the associated RNF20 and Snail were examined by Western blotting, respectively. (B) Schematic diagram showing the structure of Snail and two deletion mutations. (C) Full-length and deletion mutants of Snail were coexpressed with RNF20 in 293T cells. After immunoprecipitation of RN20, associated Snail was analyzed by Western blotting.

    Article Snippet: RNF20 antibodies and Snail antibodies were from Abcam and Cell Signaling Technology respectively.

    Techniques: Immunoprecipitation, Western Blot

    RNF20 interacts with G9a. 293T cells were transiently coexpressed with Flag-tagged G9a, HA-tagged RNF20. Cell extract were immunoprecipitated separately with Flag or HA antibodies, and the associated RNF20 and G9a were examined by Western blotting respectively.

    Journal: Frontiers in Oncology

    Article Title: RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer

    doi: 10.3389/fonc.2020.613470

    Figure Lengend Snippet: RNF20 interacts with G9a. 293T cells were transiently coexpressed with Flag-tagged G9a, HA-tagged RNF20. Cell extract were immunoprecipitated separately with Flag or HA antibodies, and the associated RNF20 and G9a were examined by Western blotting respectively.

    Article Snippet: RNF20 antibodies and Snail antibodies were from Abcam and Cell Signaling Technology respectively.

    Techniques: Immunoprecipitation, Western Blot

    RNF20 is recruited to the E-cadherin promoter for epigenetic silencing of E-cadherin expression by Snail. (A, B) RNF20 was expressed in MDA-MB231 and BT549 cells, and RNF20, H3K9me2, and H3K9 acetylation at the E-cadherin promoter were analyzed by the ChIP assay. Quantitative real-time PCR was also performed to analyze ChIP samples in (A) (mean ± SD from three separate experiments). *P < 0.01 by Student’s t test.

    Journal: Frontiers in Oncology

    Article Title: RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer

    doi: 10.3389/fonc.2020.613470

    Figure Lengend Snippet: RNF20 is recruited to the E-cadherin promoter for epigenetic silencing of E-cadherin expression by Snail. (A, B) RNF20 was expressed in MDA-MB231 and BT549 cells, and RNF20, H3K9me2, and H3K9 acetylation at the E-cadherin promoter were analyzed by the ChIP assay. Quantitative real-time PCR was also performed to analyze ChIP samples in (A) (mean ± SD from three separate experiments). *P < 0.01 by Student’s t test.

    Article Snippet: RNF20 antibodies and Snail antibodies were from Abcam and Cell Signaling Technology respectively.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    RNF20 promotes tumorsphere and colony formation. (A) Tumorsphere formation of MDA-MB231 and BT549 cells with stable empty vector (Ctr) or overexpression of RNF20 was measured. (B) The formation of colonies was measured from MDA-MB231 and BT549 cells with stable empty vector (Ctr) or overexpression of RNF20, as well as BT474 cells with stable empty vector (Ctr) or knockdown of RNF20 expression. Data are presented as a percentage of empty vector cells lines (mean ± SD in three separate experiments). *P < 0.01 by Student’s t test (C) Kaplan-Meier survival analysis for overall survival (OS) of patients in a breast cancer dataset according to RNF20 protein expression status. The p value was determined using the log-rank test.

    Journal: Frontiers in Oncology

    Article Title: RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer

    doi: 10.3389/fonc.2020.613470

    Figure Lengend Snippet: RNF20 promotes tumorsphere and colony formation. (A) Tumorsphere formation of MDA-MB231 and BT549 cells with stable empty vector (Ctr) or overexpression of RNF20 was measured. (B) The formation of colonies was measured from MDA-MB231 and BT549 cells with stable empty vector (Ctr) or overexpression of RNF20, as well as BT474 cells with stable empty vector (Ctr) or knockdown of RNF20 expression. Data are presented as a percentage of empty vector cells lines (mean ± SD in three separate experiments). *P < 0.01 by Student’s t test (C) Kaplan-Meier survival analysis for overall survival (OS) of patients in a breast cancer dataset according to RNF20 protein expression status. The p value was determined using the log-rank test.

    Article Snippet: RNF20 antibodies and Snail antibodies were from Abcam and Cell Signaling Technology respectively.

    Techniques: Plasmid Preparation, Over Expression, Expressing

    A. Images of tumorspheres from 3 lung adenocarcinoma tissues (Samples 1, 2, and 3). B. Western Blot analysis of CD133 (as a surface biomarker of CICs), CD44 (as another surface biomarker of CICs), USP22, RNF20, and H2Bub1, shows enriched USP22 protein in tumorspheres compared to original cancer tissues. C. Flow cytometry analysis of CD133 expression demonstrates a strong CD133 expression in about 30% of tumorsphere cells. D. Western Blot analysis demonstrates USP22 is markedly elevated, while H2Bub1 were significantly deceased in CD133+ cancer cells compared to CD133− cells sorted from tumorspheres. E. Flow cytometry analysis of CD44 expression demonstrates a strong CD44 expression in about 12% to 44% of tumorsphere cells. F. Western Blot analysis demonstrates USP22 and CD133 are markedly elevated, while H2Bub1 and RNF20 were markedly or moderately deceased in CD44+ cancer cells compared to CD44− cells sorted from tumorspheres. G. Mouse xenografts generated by both CD133− and CD133+ cancer cells isolated from tumorspheres, indicating CD133+ cells may represent CICs with stem cell characteristics in tumorspheres.

    Journal: Molecular cancer research : MCR

    Article Title: Targeting USP22 Suppresses Tumorigenicity and Enhances Cisplatin Sensitivity Through ALDH1A3 Downregulation in Cancer-initiating Cells from Lung Adenocarcinoma

    doi: 10.1158/1541-7786.MCR-18-0042

    Figure Lengend Snippet: A. Images of tumorspheres from 3 lung adenocarcinoma tissues (Samples 1, 2, and 3). B. Western Blot analysis of CD133 (as a surface biomarker of CICs), CD44 (as another surface biomarker of CICs), USP22, RNF20, and H2Bub1, shows enriched USP22 protein in tumorspheres compared to original cancer tissues. C. Flow cytometry analysis of CD133 expression demonstrates a strong CD133 expression in about 30% of tumorsphere cells. D. Western Blot analysis demonstrates USP22 is markedly elevated, while H2Bub1 were significantly deceased in CD133+ cancer cells compared to CD133− cells sorted from tumorspheres. E. Flow cytometry analysis of CD44 expression demonstrates a strong CD44 expression in about 12% to 44% of tumorsphere cells. F. Western Blot analysis demonstrates USP22 and CD133 are markedly elevated, while H2Bub1 and RNF20 were markedly or moderately deceased in CD44+ cancer cells compared to CD44− cells sorted from tumorspheres. G. Mouse xenografts generated by both CD133− and CD133+ cancer cells isolated from tumorspheres, indicating CD133+ cells may represent CICs with stem cell characteristics in tumorspheres.

    Article Snippet: Rabbit anti-RNF20 antibody (#9425) was purchased from Cell Signaling Technology (Beverly, CA, USA).

    Techniques: Western Blot, Biomarker Assay, Flow Cytometry, Expressing, Generated, Isolation

    A. Morphology changes of tumorspheres in differentiating culture medium (left panel); Western Blot analysis of USP22, CD133, RNF20 and H2Bub1 in tumorspheres in differentiating conditions (right panel). B. Western Blot analysis of USP22, CD133 and H2Bub1 in GFP-CTRL and USP22-KD cells (left panel); Quantitative analysis of tumorsphere formation upon USP22 knockdown, GFP-CTRL for cells transfected with control shRNA, USP22-KD for specific shRNA targeting USP22 (**P < 0.01, compared with Control, middle panel) and Tumorspheres (right panel). C. In vivo tumorigenesis of GFP-CTRL and USP22-KD tumorsphere cells, demonstrating USP22 knockdown suppressed both tumorsphere formation and tumorigenesis of lung adenocarcinoma CICs.

    Journal: Molecular cancer research : MCR

    Article Title: Targeting USP22 Suppresses Tumorigenicity and Enhances Cisplatin Sensitivity Through ALDH1A3 Downregulation in Cancer-initiating Cells from Lung Adenocarcinoma

    doi: 10.1158/1541-7786.MCR-18-0042

    Figure Lengend Snippet: A. Morphology changes of tumorspheres in differentiating culture medium (left panel); Western Blot analysis of USP22, CD133, RNF20 and H2Bub1 in tumorspheres in differentiating conditions (right panel). B. Western Blot analysis of USP22, CD133 and H2Bub1 in GFP-CTRL and USP22-KD cells (left panel); Quantitative analysis of tumorsphere formation upon USP22 knockdown, GFP-CTRL for cells transfected with control shRNA, USP22-KD for specific shRNA targeting USP22 (**P < 0.01, compared with Control, middle panel) and Tumorspheres (right panel). C. In vivo tumorigenesis of GFP-CTRL and USP22-KD tumorsphere cells, demonstrating USP22 knockdown suppressed both tumorsphere formation and tumorigenesis of lung adenocarcinoma CICs.

    Article Snippet: Rabbit anti-RNF20 antibody (#9425) was purchased from Cell Signaling Technology (Beverly, CA, USA).

    Techniques: Western Blot, Transfection, shRNA, In Vivo

    Downregulation of RNF20 mRNA in lung adenocarcinoma tissues and its association with histone modification in lung epithelial cell lines. (a) Box-and-Whisker plot of RNF20 mRNA level in lung adenocarcinoma (LUAD). Box represents first and third quartiles, thick band is median value, and bars extend to ± the interquartile range divided by the square root of the number of samples were applied to describe RNF20 gene expression values. Compared to normal lung tissues (Gr2, n=59), RNA20 mRNA was significantly decreased in LUAD (Gr1, n = 517) (fold change/FC = 0.86, and P= 9.12E-12). (b) Western blot analysis of RNF20, H2Bub1, H3K4/79-me3 and USP22 in lung epithelial cells at 72h post-transfection of control or RNF20 siRNA. Beta-actin served as the loading control.

    Journal: International journal of cancer

    Article Title: Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma

    doi: 10.1002/ijc.30769

    Figure Lengend Snippet: Downregulation of RNF20 mRNA in lung adenocarcinoma tissues and its association with histone modification in lung epithelial cell lines. (a) Box-and-Whisker plot of RNF20 mRNA level in lung adenocarcinoma (LUAD). Box represents first and third quartiles, thick band is median value, and bars extend to ± the interquartile range divided by the square root of the number of samples were applied to describe RNF20 gene expression values. Compared to normal lung tissues (Gr2, n=59), RNA20 mRNA was significantly decreased in LUAD (Gr1, n = 517) (fold change/FC = 0.86, and P= 9.12E-12). (b) Western blot analysis of RNF20, H2Bub1, H3K4/79-me3 and USP22 in lung epithelial cells at 72h post-transfection of control or RNF20 siRNA. Beta-actin served as the loading control.

    Article Snippet: The mouse monoclonal antibodies against Met, Myc, BMF, E2F2, p21, p53, ALDH1A1, total and cleaved caspase-3/PARP (Asp214), RNF20, Cyclin D3, H2B, trimethylated H3K4/K79 were purchased from Cell Signaling Technology (Beverly, CA USA) and Abcam (Cambridge, MA).

    Techniques: Modification, Whisker Assay, Expressing, Western Blot, Transfection

    Changes of gene expression profile and gene set in A549, H1299, H460 cells upon downregulation of H2Bub1. (a) Unsupervised hierarchical clustering analysis of global expression profiles in lung epithelial cells upon RNF20 knockdown. The two top rows represent two independent siRNA repeats, RNF is for RNF20 siRNA, and C is for control siRNA. (b) qRT–PCR and (c) Western blot analysis for selected genes. The level of each gene in cancer cell transfected with RNF20 siRNA is the average ratio of triplicate samples, and is presented as the ratio to control sample transfected with scramble siRNA (* P < 0.05, compared with control). (d). Selected gene sets that were enriched upon RNF20 knockdown in these lung cancer cells.

    Journal: International journal of cancer

    Article Title: Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma

    doi: 10.1002/ijc.30769

    Figure Lengend Snippet: Changes of gene expression profile and gene set in A549, H1299, H460 cells upon downregulation of H2Bub1. (a) Unsupervised hierarchical clustering analysis of global expression profiles in lung epithelial cells upon RNF20 knockdown. The two top rows represent two independent siRNA repeats, RNF is for RNF20 siRNA, and C is for control siRNA. (b) qRT–PCR and (c) Western blot analysis for selected genes. The level of each gene in cancer cell transfected with RNF20 siRNA is the average ratio of triplicate samples, and is presented as the ratio to control sample transfected with scramble siRNA (* P < 0.05, compared with control). (d). Selected gene sets that were enriched upon RNF20 knockdown in these lung cancer cells.

    Article Snippet: The mouse monoclonal antibodies against Met, Myc, BMF, E2F2, p21, p53, ALDH1A1, total and cleaved caspase-3/PARP (Asp214), RNF20, Cyclin D3, H2B, trimethylated H3K4/K79 were purchased from Cell Signaling Technology (Beverly, CA USA) and Abcam (Cambridge, MA).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection

    Knockdown of RNF20 enhances proliferation, migration and inhibits retinoic acid-induced squamous differentiation of BEAS-2B cells. (a) Enriched gene sets in BEAS-2B cells with silenced RNF20 and decreased H2Bub1 (P < 0.05, FDR < 5%). (b) Western blot for RNF20 and H2Bub1, p53, Myc, and ALDH1A1 proteins. (c) Enhanced cellular proliferation and (d) migration upon RNF20 knockdown in BEAS-2B cells. After 72 h of siRNA transfection, cell proliferation was measured. At 48 h post-transfection, 5 x 104 cells transfected with either control or RNF20 siRNA were further subjected to transwell migration assay for 12 h, and the number of migrated cells was counted (*P < 0.05, compared to control siRNA). (e) qRT–PCR analysis of IVL, a squamous differentiation marker, mRNA in BEAS-2B cells transfected with control or RNF20 siRNA and cultured in the differentiating medium BEDM, data were presented as ratio to non-differentiating medium control (*P < 0.05, ** P < 0.01, compared with control). (f) Micrographs show morphology changes of BEAS-2B cells after transfection with control siRNA (left panel) or RNF20 siRNA (right panel) and then subcultured in BEDM. Arrows point to “fried egg” morphology of squamous cells in control siRNA-transfected BEAS-2B cells.

    Journal: International journal of cancer

    Article Title: Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma

    doi: 10.1002/ijc.30769

    Figure Lengend Snippet: Knockdown of RNF20 enhances proliferation, migration and inhibits retinoic acid-induced squamous differentiation of BEAS-2B cells. (a) Enriched gene sets in BEAS-2B cells with silenced RNF20 and decreased H2Bub1 (P < 0.05, FDR < 5%). (b) Western blot for RNF20 and H2Bub1, p53, Myc, and ALDH1A1 proteins. (c) Enhanced cellular proliferation and (d) migration upon RNF20 knockdown in BEAS-2B cells. After 72 h of siRNA transfection, cell proliferation was measured. At 48 h post-transfection, 5 x 104 cells transfected with either control or RNF20 siRNA were further subjected to transwell migration assay for 12 h, and the number of migrated cells was counted (*P < 0.05, compared to control siRNA). (e) qRT–PCR analysis of IVL, a squamous differentiation marker, mRNA in BEAS-2B cells transfected with control or RNF20 siRNA and cultured in the differentiating medium BEDM, data were presented as ratio to non-differentiating medium control (*P < 0.05, ** P < 0.01, compared with control). (f) Micrographs show morphology changes of BEAS-2B cells after transfection with control siRNA (left panel) or RNF20 siRNA (right panel) and then subcultured in BEDM. Arrows point to “fried egg” morphology of squamous cells in control siRNA-transfected BEAS-2B cells.

    Article Snippet: The mouse monoclonal antibodies against Met, Myc, BMF, E2F2, p21, p53, ALDH1A1, total and cleaved caspase-3/PARP (Asp214), RNF20, Cyclin D3, H2B, trimethylated H3K4/K79 were purchased from Cell Signaling Technology (Beverly, CA USA) and Abcam (Cambridge, MA).

    Techniques: Migration, Western Blot, Transfection, Transwell Migration Assay, Quantitative RT-PCR, Marker, Cell Culture

    Impact of RNF20 knockdown on in vitro proliferation, migration, and invasion of lung cancer cell lines. (a) Western blot analysis of cell lysates of A549 and H460 cells transfected with either control or RNF20 siRNA and harvested at 72 h post-transfection. Actin served as loading control, total and phosphorylated Erk/Akt (T/P-Erk/Akt), p53, p21 E-Cadherin, and Vimentin were detected. The p53 blots were from separately developed blots for each cell, and the below Actin is its loading control. (b) Histogram of proliferation shows RNF20 knockdown increased the in vitro proliferation of both A549 and H460 cells over 72h (*P < 0.05, compared to control siRNA). (c) Representative photomicrographs of the migration chamber at the times indicated in control or RNF20 siRNA-transfected cells is shown on the left, with quantitative analysis of migrated cells shown on the right (N = 3 replicates per cell type). Error bars show SD (*P < 0.05, compared with control siRNA). (d) Lower surface of Matrigel transwell membranes seeded with cancer cells previously transfected with indicated siRNAs, 24 h after incubation (left), and corresponding quantitative analysis of invading cells (right). Data are shown as mean values graphed for indicated cells on the right (N = 3 replicates per cell type). Error bars show SD (*P < 0.05, **P < 0.01, compared with control siRNA).

    Journal: International journal of cancer

    Article Title: Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma

    doi: 10.1002/ijc.30769

    Figure Lengend Snippet: Impact of RNF20 knockdown on in vitro proliferation, migration, and invasion of lung cancer cell lines. (a) Western blot analysis of cell lysates of A549 and H460 cells transfected with either control or RNF20 siRNA and harvested at 72 h post-transfection. Actin served as loading control, total and phosphorylated Erk/Akt (T/P-Erk/Akt), p53, p21 E-Cadherin, and Vimentin were detected. The p53 blots were from separately developed blots for each cell, and the below Actin is its loading control. (b) Histogram of proliferation shows RNF20 knockdown increased the in vitro proliferation of both A549 and H460 cells over 72h (*P < 0.05, compared to control siRNA). (c) Representative photomicrographs of the migration chamber at the times indicated in control or RNF20 siRNA-transfected cells is shown on the left, with quantitative analysis of migrated cells shown on the right (N = 3 replicates per cell type). Error bars show SD (*P < 0.05, compared with control siRNA). (d) Lower surface of Matrigel transwell membranes seeded with cancer cells previously transfected with indicated siRNAs, 24 h after incubation (left), and corresponding quantitative analysis of invading cells (right). Data are shown as mean values graphed for indicated cells on the right (N = 3 replicates per cell type). Error bars show SD (*P < 0.05, **P < 0.01, compared with control siRNA).

    Article Snippet: The mouse monoclonal antibodies against Met, Myc, BMF, E2F2, p21, p53, ALDH1A1, total and cleaved caspase-3/PARP (Asp214), RNF20, Cyclin D3, H2B, trimethylated H3K4/K79 were purchased from Cell Signaling Technology (Beverly, CA USA) and Abcam (Cambridge, MA).

    Techniques: In Vitro, Migration, Western Blot, Transfection, Incubation

    Downregulation of H2Bub1 enhanced resistance to cisplatin in lung cancer cells. The levels of RNF20, H2Bub1, p21, p53, and apoptotic markers caspase-3/PARP cleaved products (T/C- Caspase-3/PARP for total and cleaved proteins) in (a) A549 and (b). H460 cells treated with cisplatin were analyzed by Western blot. Beta-actin was used as loading control. (c) Representative flow cytometry profile of A549 and H460 cells transfected with either control (left panel) or RNF20 siRNA (right panel). After cells were treated for 72 h with 20 uM Cisplatin, apoptosis was measured by flow cytometry analysis of Annexin-V (labeled with Alexa Fluor 488) staining in X axis and propidium iodide staining in Y axis. (d) Quantitative analysis of the experiments shows that apoptotic cells were significantly reduced in both A549 and H460 cells upon downregulation of H2Bub1. The experiment was repeated three times and data represent the average of the early apoptotic and late apoptotic cells (* P < 0.05; ** P < 0.01).

    Journal: International journal of cancer

    Article Title: Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma

    doi: 10.1002/ijc.30769

    Figure Lengend Snippet: Downregulation of H2Bub1 enhanced resistance to cisplatin in lung cancer cells. The levels of RNF20, H2Bub1, p21, p53, and apoptotic markers caspase-3/PARP cleaved products (T/C- Caspase-3/PARP for total and cleaved proteins) in (a) A549 and (b). H460 cells treated with cisplatin were analyzed by Western blot. Beta-actin was used as loading control. (c) Representative flow cytometry profile of A549 and H460 cells transfected with either control (left panel) or RNF20 siRNA (right panel). After cells were treated for 72 h with 20 uM Cisplatin, apoptosis was measured by flow cytometry analysis of Annexin-V (labeled with Alexa Fluor 488) staining in X axis and propidium iodide staining in Y axis. (d) Quantitative analysis of the experiments shows that apoptotic cells were significantly reduced in both A549 and H460 cells upon downregulation of H2Bub1. The experiment was repeated three times and data represent the average of the early apoptotic and late apoptotic cells (* P < 0.05; ** P < 0.01).

    Article Snippet: The mouse monoclonal antibodies against Met, Myc, BMF, E2F2, p21, p53, ALDH1A1, total and cleaved caspase-3/PARP (Asp214), RNF20, Cyclin D3, H2B, trimethylated H3K4/K79 were purchased from Cell Signaling Technology (Beverly, CA USA) and Abcam (Cambridge, MA).

    Techniques: Western Blot, Flow Cytometry, Transfection, Labeling, Staining

    PCR primers for RT-PCR

    Journal: The Journal of Neuroscience

    Article Title: Bre1a, a Histone H2B Ubiquitin Ligase, Regulates the Cell Cycle and Differentiation of Neural Precursor Cells

    doi: 10.1523/JNEUROSCI.3832-13.2014

    Figure Lengend Snippet: PCR primers for RT-PCR

    Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-Bre1a IgG (ab32629, Abcam); rabbit anti-Bre1a IgG (catalog #9425, Cell Signaling Technology); rabbit anti-H2B IgG (Millipore); mouse anti-ubiquitylated H2B IgG (Millipore); mouse anti-phospho-histone H3 IgG (Cell Signaling Technology); mouse anti-GAPDH IgG (Santa Cruz Biotechnology); mouse anti-proliferating cell nuclear antigen (PCNA) IgG (DAKO); rabbit anti-green fluorescent protein (GFP) IgG (Invitrogen); rat anti-GFP IgG (Nacalai Tesque); rabbit anti-cleaved caspase 3 (c-Cas3) IgG (Cell Signaling Technology); rabbit anti-paired box gene 6 (Pax6) IgG (Millipore); and rabbit anti-eomesodermin homolog (Eomes/Tbr2) IgG (Abcam).

    Techniques: Sequencing

    Bre1a expression in the developing brain. A–L, The expression of Bre1a was determined in coronal cryosections of forebrains from E10.5 (A–D), E12.5 (E–I), E14.5 (J), and E16.5 (K) embryos, and P0 pups (L) by immunostaining or by in situ hybridization (J, top). B, Coronal cryosections of E10.5 dorsal forebrain were immunostained for Bre1a and H2Bub1. Boxed areas in the top panels are shown in the bottom panels with higher magnification. Nonmitotic cells that weakly express Bre1a and H2Bub1 are indicated by arrowheads, and some mitotic cells are indicated by dotted circles and arrows. C, Double immunostaining for Bre1a and phospho-histone 3 (PH3) shows Bre1aweak/PH3− cells (arrowheads). D, Colocalization of Bre1a and βIII-tubulin in cells nearby the basal surface is indicated by arrowheads. E–H, Coronal cryosections of E12.5 brain were double-immunostained for Bre1a and H2Bub1 (E), and boxed areas in the cortex (F), dorsoventral boundary (G), and medial ganglionic eminence (H) are shown with higher magnification. Nonmitotic cells that weakly express Bre1a and H2Bub1 are indicated by arrowheads, and some mitotic cells are indicated by dotted circles and arrows. I, Coronal cryosections of E12.5 brain were immunostained for PH3, and then, double immunostained for Bre1a and H2Bub1. Alexa Fluor-633 signal for PH3 is pseudo-colored and shown in blue. LV, Lateral ventricle. Scale bars: A, E, J–L, 200 μm; B–D, F–I, 50 μm.

    Journal: The Journal of Neuroscience

    Article Title: Bre1a, a Histone H2B Ubiquitin Ligase, Regulates the Cell Cycle and Differentiation of Neural Precursor Cells

    doi: 10.1523/JNEUROSCI.3832-13.2014

    Figure Lengend Snippet: Bre1a expression in the developing brain. A–L, The expression of Bre1a was determined in coronal cryosections of forebrains from E10.5 (A–D), E12.5 (E–I), E14.5 (J), and E16.5 (K) embryos, and P0 pups (L) by immunostaining or by in situ hybridization (J, top). B, Coronal cryosections of E10.5 dorsal forebrain were immunostained for Bre1a and H2Bub1. Boxed areas in the top panels are shown in the bottom panels with higher magnification. Nonmitotic cells that weakly express Bre1a and H2Bub1 are indicated by arrowheads, and some mitotic cells are indicated by dotted circles and arrows. C, Double immunostaining for Bre1a and phospho-histone 3 (PH3) shows Bre1aweak/PH3− cells (arrowheads). D, Colocalization of Bre1a and βIII-tubulin in cells nearby the basal surface is indicated by arrowheads. E–H, Coronal cryosections of E12.5 brain were double-immunostained for Bre1a and H2Bub1 (E), and boxed areas in the cortex (F), dorsoventral boundary (G), and medial ganglionic eminence (H) are shown with higher magnification. Nonmitotic cells that weakly express Bre1a and H2Bub1 are indicated by arrowheads, and some mitotic cells are indicated by dotted circles and arrows. I, Coronal cryosections of E12.5 brain were immunostained for PH3, and then, double immunostained for Bre1a and H2Bub1. Alexa Fluor-633 signal for PH3 is pseudo-colored and shown in blue. LV, Lateral ventricle. Scale bars: A, E, J–L, 200 μm; B–D, F–I, 50 μm.

    Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-Bre1a IgG (ab32629, Abcam); rabbit anti-Bre1a IgG (catalog #9425, Cell Signaling Technology); rabbit anti-H2B IgG (Millipore); mouse anti-ubiquitylated H2B IgG (Millipore); mouse anti-phospho-histone H3 IgG (Cell Signaling Technology); mouse anti-GAPDH IgG (Santa Cruz Biotechnology); mouse anti-proliferating cell nuclear antigen (PCNA) IgG (DAKO); rabbit anti-green fluorescent protein (GFP) IgG (Invitrogen); rat anti-GFP IgG (Nacalai Tesque); rabbit anti-cleaved caspase 3 (c-Cas3) IgG (Cell Signaling Technology); rabbit anti-paired box gene 6 (Pax6) IgG (Millipore); and rabbit anti-eomesodermin homolog (Eomes/Tbr2) IgG (Abcam).

    Techniques: Expressing, Immunostaining, In Situ Hybridization, Double Immunostaining

    Bre1a knockdown impairs the proliferation of neural precursor cells. A–C, F, H, Primary neurospheres derived from the E13.5 cortex were infected with lentivirus expressing control or Bre1a shRNAs and GFP, and resultant GFP+ secondary neurospheres were subjected for analyses. A, Quantitative RT-PCR for Bre1a expression. B, GFP+ secondary neurospheres were plated and immunostained for GFP and Bre1a. Arrowheads and arrows indicate Bre1a shRNA-expressing and nonexpressing cells, respectively. C, Protein extracts from neurospheres expressing Bre1a shRNAs or control shRNA were subjected to Western blotting using the indicated antibodies. D, E, G, Primary neuroepithelial cells from E10.5 forebrains were transfected with Bre1a shRNA or control shRNA (D), together with GFP expression plasmid only, GFP and Bre1a expression plasmids (E), or GFP and miRNA expression plasmids (G). We used two miRNAs for p57Kip2 (1 and 2) or control miRNA. The cells were cultured in proliferation conditions for 3 d and were incubated with 20 μm EdU for 2 h before fixation. D, Cells were immunostained for GFP, followed by visualization for EdU. Arrowheads indicate GFP+/EdU+ cells and arrows indicate GFP+/EdU− cells. E, G, Quantification of GFP+/EdU+ cells in total GFP+ cells. F, H, Quantitative RT-PCR for p21Cip1, p27Kip1, and p57Kip2 (F) and for Cdk2 (H). Scale bars, 50 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (A, E, F, H) or Tukey-Kramer's post hoc comparison (G).

    Journal: The Journal of Neuroscience

    Article Title: Bre1a, a Histone H2B Ubiquitin Ligase, Regulates the Cell Cycle and Differentiation of Neural Precursor Cells

    doi: 10.1523/JNEUROSCI.3832-13.2014

    Figure Lengend Snippet: Bre1a knockdown impairs the proliferation of neural precursor cells. A–C, F, H, Primary neurospheres derived from the E13.5 cortex were infected with lentivirus expressing control or Bre1a shRNAs and GFP, and resultant GFP+ secondary neurospheres were subjected for analyses. A, Quantitative RT-PCR for Bre1a expression. B, GFP+ secondary neurospheres were plated and immunostained for GFP and Bre1a. Arrowheads and arrows indicate Bre1a shRNA-expressing and nonexpressing cells, respectively. C, Protein extracts from neurospheres expressing Bre1a shRNAs or control shRNA were subjected to Western blotting using the indicated antibodies. D, E, G, Primary neuroepithelial cells from E10.5 forebrains were transfected with Bre1a shRNA or control shRNA (D), together with GFP expression plasmid only, GFP and Bre1a expression plasmids (E), or GFP and miRNA expression plasmids (G). We used two miRNAs for p57Kip2 (1 and 2) or control miRNA. The cells were cultured in proliferation conditions for 3 d and were incubated with 20 μm EdU for 2 h before fixation. D, Cells were immunostained for GFP, followed by visualization for EdU. Arrowheads indicate GFP+/EdU+ cells and arrows indicate GFP+/EdU− cells. E, G, Quantification of GFP+/EdU+ cells in total GFP+ cells. F, H, Quantitative RT-PCR for p21Cip1, p27Kip1, and p57Kip2 (F) and for Cdk2 (H). Scale bars, 50 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (A, E, F, H) or Tukey-Kramer's post hoc comparison (G).

    Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-Bre1a IgG (ab32629, Abcam); rabbit anti-Bre1a IgG (catalog #9425, Cell Signaling Technology); rabbit anti-H2B IgG (Millipore); mouse anti-ubiquitylated H2B IgG (Millipore); mouse anti-phospho-histone H3 IgG (Cell Signaling Technology); mouse anti-GAPDH IgG (Santa Cruz Biotechnology); mouse anti-proliferating cell nuclear antigen (PCNA) IgG (DAKO); rabbit anti-green fluorescent protein (GFP) IgG (Invitrogen); rat anti-GFP IgG (Nacalai Tesque); rabbit anti-cleaved caspase 3 (c-Cas3) IgG (Cell Signaling Technology); rabbit anti-paired box gene 6 (Pax6) IgG (Millipore); and rabbit anti-eomesodermin homolog (Eomes/Tbr2) IgG (Abcam).

    Techniques: Derivative Assay, Infection, Expressing, Quantitative RT-PCR, shRNA, Western Blot, Transfection, Plasmid Preparation, Cell Culture, Incubation

    Bre1a knockdown in neural precursor cells reduces the proportion of mitotic cells. A–D, Bre1a shRNA or control shRNA and GFP expression plasmids were microinjected into the lateral ventricle (LV) of E13.5 embryos and electroporated into the dorsal forebrain in utero. The embryos were fixed 24 h after electroporation, and the brains were analyzed. A, GFP+ Bre1a shRNA-expressing cells (arrowheads) are less immunopositive for Bre1a than surrounding GFP− cells (arrows). B, Cryosections were immunostained for GFP and c-Cas3, followed by Hoechst nuclear staining. C, D, EdU was injected intraperitoneally into dams 2 h before fixation. Coronal cryosections were immunostained for GFP, followed by EdU visualization (C), and GFP +/EdU+ cells were counted (D). Bre1a expression plasmids were coelectroporated with Bre1a shRNA 3 to rescue the effects of Bre1a knockdown. E–H, Bre1a or control shRNA plasmids together with GFP and H2B-mCherry expression plasmids were coelectroporated into the dorsal forebrain of E13.5 embryos in utero. Twenty-four hours after electroporation, coronal slices were made and cultured on a hydrophilic transparent membrane in serum-free media for 30 h. E, Time-lapse images of GFP+/mCherry+ cells in the VZ/SVZ. Arrowheads indicate cells undergoing cell divisions, which were precisely recognized by the observation of H2B-mCherry expression (red). F–H, The percentage of GFP+ cells that underwent mitosis during the 30 h culture (F), the duration of the G2/M phase of GFP+ cells (G), and the migration speed of GFP+ cells in the G2 phase (H) in the VZ/SVZ were quantified. Scale bars: A, 20 μm; B, 100 μm; C, E, 30 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (D) or Student's t test (F–H).

    Journal: The Journal of Neuroscience

    Article Title: Bre1a, a Histone H2B Ubiquitin Ligase, Regulates the Cell Cycle and Differentiation of Neural Precursor Cells

    doi: 10.1523/JNEUROSCI.3832-13.2014

    Figure Lengend Snippet: Bre1a knockdown in neural precursor cells reduces the proportion of mitotic cells. A–D, Bre1a shRNA or control shRNA and GFP expression plasmids were microinjected into the lateral ventricle (LV) of E13.5 embryos and electroporated into the dorsal forebrain in utero. The embryos were fixed 24 h after electroporation, and the brains were analyzed. A, GFP+ Bre1a shRNA-expressing cells (arrowheads) are less immunopositive for Bre1a than surrounding GFP− cells (arrows). B, Cryosections were immunostained for GFP and c-Cas3, followed by Hoechst nuclear staining. C, D, EdU was injected intraperitoneally into dams 2 h before fixation. Coronal cryosections were immunostained for GFP, followed by EdU visualization (C), and GFP +/EdU+ cells were counted (D). Bre1a expression plasmids were coelectroporated with Bre1a shRNA 3 to rescue the effects of Bre1a knockdown. E–H, Bre1a or control shRNA plasmids together with GFP and H2B-mCherry expression plasmids were coelectroporated into the dorsal forebrain of E13.5 embryos in utero. Twenty-four hours after electroporation, coronal slices were made and cultured on a hydrophilic transparent membrane in serum-free media for 30 h. E, Time-lapse images of GFP+/mCherry+ cells in the VZ/SVZ. Arrowheads indicate cells undergoing cell divisions, which were precisely recognized by the observation of H2B-mCherry expression (red). F–H, The percentage of GFP+ cells that underwent mitosis during the 30 h culture (F), the duration of the G2/M phase of GFP+ cells (G), and the migration speed of GFP+ cells in the G2 phase (H) in the VZ/SVZ were quantified. Scale bars: A, 20 μm; B, 100 μm; C, E, 30 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (D) or Student's t test (F–H).

    Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-Bre1a IgG (ab32629, Abcam); rabbit anti-Bre1a IgG (catalog #9425, Cell Signaling Technology); rabbit anti-H2B IgG (Millipore); mouse anti-ubiquitylated H2B IgG (Millipore); mouse anti-phospho-histone H3 IgG (Cell Signaling Technology); mouse anti-GAPDH IgG (Santa Cruz Biotechnology); mouse anti-proliferating cell nuclear antigen (PCNA) IgG (DAKO); rabbit anti-green fluorescent protein (GFP) IgG (Invitrogen); rat anti-GFP IgG (Nacalai Tesque); rabbit anti-cleaved caspase 3 (c-Cas3) IgG (Cell Signaling Technology); rabbit anti-paired box gene 6 (Pax6) IgG (Millipore); and rabbit anti-eomesodermin homolog (Eomes/Tbr2) IgG (Abcam).

    Techniques: shRNA, Expressing, In Utero, Electroporation, Staining, Injection, Cell Culture, Migration

    Bre1a knockdown impairs the migration of neural precursor cells. Bre1a or control shRNA and GFP expression plasmids were microinjected into the lateral ventricle of E13.5 embryos and electroporated into the dorsal forebrain in utero. A, Three days after the electroporation, the embryos were fixed and coronal cryosections of the brains were immunostained for GFP. B, GFP+ cells in the VZ/SVZ, IMZ, or CP were counted. C, D, Twenty-four hours after the electroporation, coronal slices were made and cultured in serum-free media for 30 h. C, Time-lapse images of GFP+ cells in the CP. Arrowheads indicate migrating cells. D, Migration speed of GFP+ cells in the CP was quantified. Scale bars: A, 100 μm; C, 20 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (B) or by Student's t test (D).

    Journal: The Journal of Neuroscience

    Article Title: Bre1a, a Histone H2B Ubiquitin Ligase, Regulates the Cell Cycle and Differentiation of Neural Precursor Cells

    doi: 10.1523/JNEUROSCI.3832-13.2014

    Figure Lengend Snippet: Bre1a knockdown impairs the migration of neural precursor cells. Bre1a or control shRNA and GFP expression plasmids were microinjected into the lateral ventricle of E13.5 embryos and electroporated into the dorsal forebrain in utero. A, Three days after the electroporation, the embryos were fixed and coronal cryosections of the brains were immunostained for GFP. B, GFP+ cells in the VZ/SVZ, IMZ, or CP were counted. C, D, Twenty-four hours after the electroporation, coronal slices were made and cultured in serum-free media for 30 h. C, Time-lapse images of GFP+ cells in the CP. Arrowheads indicate migrating cells. D, Migration speed of GFP+ cells in the CP was quantified. Scale bars: A, 100 μm; C, 20 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (B) or by Student's t test (D).

    Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-Bre1a IgG (ab32629, Abcam); rabbit anti-Bre1a IgG (catalog #9425, Cell Signaling Technology); rabbit anti-H2B IgG (Millipore); mouse anti-ubiquitylated H2B IgG (Millipore); mouse anti-phospho-histone H3 IgG (Cell Signaling Technology); mouse anti-GAPDH IgG (Santa Cruz Biotechnology); mouse anti-proliferating cell nuclear antigen (PCNA) IgG (DAKO); rabbit anti-green fluorescent protein (GFP) IgG (Invitrogen); rat anti-GFP IgG (Nacalai Tesque); rabbit anti-cleaved caspase 3 (c-Cas3) IgG (Cell Signaling Technology); rabbit anti-paired box gene 6 (Pax6) IgG (Millipore); and rabbit anti-eomesodermin homolog (Eomes/Tbr2) IgG (Abcam).

    Techniques: Migration, shRNA, Expressing, In Utero, Electroporation, Cell Culture

    Bre1a or Bre1aΔC overexpression does not inhibit cell migration. A, Schematic structures of full-length Bre1a and Bre1a that lacks the ring finger domain at the C terminus (Bre1aΔC) are shown. B–E, Expression plasmids for full-length Bre1a or Bre1aΔC were coelectroporated with GFP expression plasmid only (B, C) or with Bre1a shRNA and GFP expression plasmids (D, E) into the dorsal forebrain of E13.5 embryos in utero. Three days after the electroporation, the embryos were fixed and coronal cryosections of the brains were immunostained for GFP (B, D). C, E, GFP-positive cells in the VZ/SVZ, IMZ, or CP were counted. Scale bar, 100 μm. Error bars indicate SEM, and n values are shown in columns.

    Journal: The Journal of Neuroscience

    Article Title: Bre1a, a Histone H2B Ubiquitin Ligase, Regulates the Cell Cycle and Differentiation of Neural Precursor Cells

    doi: 10.1523/JNEUROSCI.3832-13.2014

    Figure Lengend Snippet: Bre1a or Bre1aΔC overexpression does not inhibit cell migration. A, Schematic structures of full-length Bre1a and Bre1a that lacks the ring finger domain at the C terminus (Bre1aΔC) are shown. B–E, Expression plasmids for full-length Bre1a or Bre1aΔC were coelectroporated with GFP expression plasmid only (B, C) or with Bre1a shRNA and GFP expression plasmids (D, E) into the dorsal forebrain of E13.5 embryos in utero. Three days after the electroporation, the embryos were fixed and coronal cryosections of the brains were immunostained for GFP (B, D). C, E, GFP-positive cells in the VZ/SVZ, IMZ, or CP were counted. Scale bar, 100 μm. Error bars indicate SEM, and n values are shown in columns.

    Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-Bre1a IgG (ab32629, Abcam); rabbit anti-Bre1a IgG (catalog #9425, Cell Signaling Technology); rabbit anti-H2B IgG (Millipore); mouse anti-ubiquitylated H2B IgG (Millipore); mouse anti-phospho-histone H3 IgG (Cell Signaling Technology); mouse anti-GAPDH IgG (Santa Cruz Biotechnology); mouse anti-proliferating cell nuclear antigen (PCNA) IgG (DAKO); rabbit anti-green fluorescent protein (GFP) IgG (Invitrogen); rat anti-GFP IgG (Nacalai Tesque); rabbit anti-cleaved caspase 3 (c-Cas3) IgG (Cell Signaling Technology); rabbit anti-paired box gene 6 (Pax6) IgG (Millipore); and rabbit anti-eomesodermin homolog (Eomes/Tbr2) IgG (Abcam).

    Techniques: Over Expression, Migration, Expressing, Plasmid Preparation, shRNA, In Utero, Electroporation

    Bre1a knockdown affects the cortical layer formation in the postnatal brain. A, B, Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E13.5 embryos in utero, and the pups were perfused on P0. A, Coronal cryosections of the brains were immunostained for GFP (right), followed by Hoechst nuclear staining (left). The CP was divided into four layers based on the density of nuclei: the mantle zone (MZ); and upper (u), middle (m), and lower (l) CP. The borders between layers are indicated by dotted lines. B, Quantification of GFP+ cells distributed in the upper, middle, and lower CP in the P0 brain. C–F, Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E14.5 embryos in utero, and the pups were perfused on P14. For rescue experiments, Bre1a expression plasmids were coelectroporated with Bre1a shRNA 2. C–E, Coronal cryosections were immunostained for GFP (middle), followed by Hoechst nuclear staining (left), or were subjected to in situ hybridization with a probe for Rorb, a marker of cortical layer IV (right). F, Quantitative analysis of GFP+ cells in cortical layers II/III and IV. Scale bars, 100 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by Student's or Welch's t test.

    Journal: The Journal of Neuroscience

    Article Title: Bre1a, a Histone H2B Ubiquitin Ligase, Regulates the Cell Cycle and Differentiation of Neural Precursor Cells

    doi: 10.1523/JNEUROSCI.3832-13.2014

    Figure Lengend Snippet: Bre1a knockdown affects the cortical layer formation in the postnatal brain. A, B, Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E13.5 embryos in utero, and the pups were perfused on P0. A, Coronal cryosections of the brains were immunostained for GFP (right), followed by Hoechst nuclear staining (left). The CP was divided into four layers based on the density of nuclei: the mantle zone (MZ); and upper (u), middle (m), and lower (l) CP. The borders between layers are indicated by dotted lines. B, Quantification of GFP+ cells distributed in the upper, middle, and lower CP in the P0 brain. C–F, Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E14.5 embryos in utero, and the pups were perfused on P14. For rescue experiments, Bre1a expression plasmids were coelectroporated with Bre1a shRNA 2. C–E, Coronal cryosections were immunostained for GFP (middle), followed by Hoechst nuclear staining (left), or were subjected to in situ hybridization with a probe for Rorb, a marker of cortical layer IV (right). F, Quantitative analysis of GFP+ cells in cortical layers II/III and IV. Scale bars, 100 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by Student's or Welch's t test.

    Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-Bre1a IgG (ab32629, Abcam); rabbit anti-Bre1a IgG (catalog #9425, Cell Signaling Technology); rabbit anti-H2B IgG (Millipore); mouse anti-ubiquitylated H2B IgG (Millipore); mouse anti-phospho-histone H3 IgG (Cell Signaling Technology); mouse anti-GAPDH IgG (Santa Cruz Biotechnology); mouse anti-proliferating cell nuclear antigen (PCNA) IgG (DAKO); rabbit anti-green fluorescent protein (GFP) IgG (Invitrogen); rat anti-GFP IgG (Nacalai Tesque); rabbit anti-cleaved caspase 3 (c-Cas3) IgG (Cell Signaling Technology); rabbit anti-paired box gene 6 (Pax6) IgG (Millipore); and rabbit anti-eomesodermin homolog (Eomes/Tbr2) IgG (Abcam).

    Techniques: shRNA, Expressing, In Utero, Staining, In Situ Hybridization, Marker

    Bre1a knockdown delays the birth date of differentiating neuroblasts. Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E14.5 embryos in utero, and the pups were perfused on P14. For rescue experiments, Bre1a expression plasmids were coelectroporated with Bre1a shRNA 2. A, B, D–F, Dams received an intraperitoneal injection of EdU 24 h (A, B) or 48 h (D–F) after the electroporation. Coronal cryosections were immunostained for GFP (left), followed by EdU visualization (middle), and the merged images are shown (right). Arrowheads indicate GFP and EdU double-positive cells. C, G, Quantification of GFP+/EdU+ cells in cortical layers II/III and IV. Scale bars, 50 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by Student's or Welch's t test.

    Journal: The Journal of Neuroscience

    Article Title: Bre1a, a Histone H2B Ubiquitin Ligase, Regulates the Cell Cycle and Differentiation of Neural Precursor Cells

    doi: 10.1523/JNEUROSCI.3832-13.2014

    Figure Lengend Snippet: Bre1a knockdown delays the birth date of differentiating neuroblasts. Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E14.5 embryos in utero, and the pups were perfused on P14. For rescue experiments, Bre1a expression plasmids were coelectroporated with Bre1a shRNA 2. A, B, D–F, Dams received an intraperitoneal injection of EdU 24 h (A, B) or 48 h (D–F) after the electroporation. Coronal cryosections were immunostained for GFP (left), followed by EdU visualization (middle), and the merged images are shown (right). Arrowheads indicate GFP and EdU double-positive cells. C, G, Quantification of GFP+/EdU+ cells in cortical layers II/III and IV. Scale bars, 50 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by Student's or Welch's t test.

    Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-Bre1a IgG (ab32629, Abcam); rabbit anti-Bre1a IgG (catalog #9425, Cell Signaling Technology); rabbit anti-H2B IgG (Millipore); mouse anti-ubiquitylated H2B IgG (Millipore); mouse anti-phospho-histone H3 IgG (Cell Signaling Technology); mouse anti-GAPDH IgG (Santa Cruz Biotechnology); mouse anti-proliferating cell nuclear antigen (PCNA) IgG (DAKO); rabbit anti-green fluorescent protein (GFP) IgG (Invitrogen); rat anti-GFP IgG (Nacalai Tesque); rabbit anti-cleaved caspase 3 (c-Cas3) IgG (Cell Signaling Technology); rabbit anti-paired box gene 6 (Pax6) IgG (Millipore); and rabbit anti-eomesodermin homolog (Eomes/Tbr2) IgG (Abcam).

    Techniques: shRNA, Expressing, In Utero, Injection, Electroporation

    Bre1a facilitates the differentiation of neural precursor cells. A, B, Primary neuroepithelial cells from E10.5 forebrains were transfected with Bre1a or control shRNAs and GFP expression plasmids, and cultured in differentiation condition for 3 d. Bre1a or control expression plasmids were cotransfected with Bre1a-shRNA 2 for rescue experiments. A, Cells were immunostained for GFP and βIII tubulin. Arrowheads and arrows indicate GFP+/βIII tubulin+ and GFP+/βIII tubulin− cells, respectively. B, Quantification of GFP+/βIII tubulin+ cells. C–J, Bre1a or control shRNA, pCX-Bre1a, or control expression plasmids were microinjected into the lateral ventricle of E13.5 embryos and electroporated into the dorsal forebrain in utero. The embryos were fixed 24 h after electroporation, and the brains were analyzed. C–J, Cryosections were immunostained for GFP and Tbr2 (C, G) or Pax6 (E, I). GFP+/Tbr2+ (D, H) or GFP+/Pax6+ (F, J) cells were counted. Scale bars: A, 50 μm; C, E, G, I, 100 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (B, D, F) or by Student's t test (H, J).

    Journal: The Journal of Neuroscience

    Article Title: Bre1a, a Histone H2B Ubiquitin Ligase, Regulates the Cell Cycle and Differentiation of Neural Precursor Cells

    doi: 10.1523/JNEUROSCI.3832-13.2014

    Figure Lengend Snippet: Bre1a facilitates the differentiation of neural precursor cells. A, B, Primary neuroepithelial cells from E10.5 forebrains were transfected with Bre1a or control shRNAs and GFP expression plasmids, and cultured in differentiation condition for 3 d. Bre1a or control expression plasmids were cotransfected with Bre1a-shRNA 2 for rescue experiments. A, Cells were immunostained for GFP and βIII tubulin. Arrowheads and arrows indicate GFP+/βIII tubulin+ and GFP+/βIII tubulin− cells, respectively. B, Quantification of GFP+/βIII tubulin+ cells. C–J, Bre1a or control shRNA, pCX-Bre1a, or control expression plasmids were microinjected into the lateral ventricle of E13.5 embryos and electroporated into the dorsal forebrain in utero. The embryos were fixed 24 h after electroporation, and the brains were analyzed. C–J, Cryosections were immunostained for GFP and Tbr2 (C, G) or Pax6 (E, I). GFP+/Tbr2+ (D, H) or GFP+/Pax6+ (F, J) cells were counted. Scale bars: A, 50 μm; C, E, G, I, 100 μm. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison (B, D, F) or by Student's t test (H, J).

    Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-Bre1a IgG (ab32629, Abcam); rabbit anti-Bre1a IgG (catalog #9425, Cell Signaling Technology); rabbit anti-H2B IgG (Millipore); mouse anti-ubiquitylated H2B IgG (Millipore); mouse anti-phospho-histone H3 IgG (Cell Signaling Technology); mouse anti-GAPDH IgG (Santa Cruz Biotechnology); mouse anti-proliferating cell nuclear antigen (PCNA) IgG (DAKO); rabbit anti-green fluorescent protein (GFP) IgG (Invitrogen); rat anti-GFP IgG (Nacalai Tesque); rabbit anti-cleaved caspase 3 (c-Cas3) IgG (Cell Signaling Technology); rabbit anti-paired box gene 6 (Pax6) IgG (Millipore); and rabbit anti-eomesodermin homolog (Eomes/Tbr2) IgG (Abcam).

    Techniques: Transfection, Expressing, Cell Culture, shRNA, In Utero, Electroporation

    Bre1a knockdown upregulates Hes5 expression in neural precursor cells. A, B, Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E13.5 embryos in utero, and brains were analyzed 3 d after the electroporation. Coronal cryosections were immunostained for GFP (left) or subjected to in situ hybridization with an RNA probe for Hes5 (right). Arrowheads indicate ectopic Hes5+ cells outside the VZ. The boxed area is magnified in the inset. Scale bar, 50 μm. C, D, G, H, Primary neurosphere cells derived from E13.5 cortex were infected with lentiviruses expressing Bre1a or control shRNAs and were cultured for 4–7 d. Lentivirus-infected GFP+ neurospheres were used for quantitative RT-PCR analysis of Hes5 (C), Hes1 (D), Fezf1 (G), or Fezf2 (H). For rescue experiments, Bre1a-expressing retrovirus was coinfected with Bre1a shRNA-expressing lentivirus. E, F, Firefly luciferase reporter assays in Neuro2a cells using the following plasmids: a plasmid containing the promoter region of the Hes5 (E) or Hes1 (F) gene; the Renilla luciferase reporter plasmid as an internal control; pCX-NICD, and the indicated plasmid for expression of Bre1a, Bre1aΔC, Fezf1, Fezf2, and Fezf1, and Fezf2 miRNAs. The relative promoter activities were compared with activity in the absence of pCX-NICD. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison or Student's t test.

    Journal: The Journal of Neuroscience

    Article Title: Bre1a, a Histone H2B Ubiquitin Ligase, Regulates the Cell Cycle and Differentiation of Neural Precursor Cells

    doi: 10.1523/JNEUROSCI.3832-13.2014

    Figure Lengend Snippet: Bre1a knockdown upregulates Hes5 expression in neural precursor cells. A, B, Bre1a or control shRNA and GFP expression plasmids were electroporated into the dorsal forebrain of E13.5 embryos in utero, and brains were analyzed 3 d after the electroporation. Coronal cryosections were immunostained for GFP (left) or subjected to in situ hybridization with an RNA probe for Hes5 (right). Arrowheads indicate ectopic Hes5+ cells outside the VZ. The boxed area is magnified in the inset. Scale bar, 50 μm. C, D, G, H, Primary neurosphere cells derived from E13.5 cortex were infected with lentiviruses expressing Bre1a or control shRNAs and were cultured for 4–7 d. Lentivirus-infected GFP+ neurospheres were used for quantitative RT-PCR analysis of Hes5 (C), Hes1 (D), Fezf1 (G), or Fezf2 (H). For rescue experiments, Bre1a-expressing retrovirus was coinfected with Bre1a shRNA-expressing lentivirus. E, F, Firefly luciferase reporter assays in Neuro2a cells using the following plasmids: a plasmid containing the promoter region of the Hes5 (E) or Hes1 (F) gene; the Renilla luciferase reporter plasmid as an internal control; pCX-NICD, and the indicated plasmid for expression of Bre1a, Bre1aΔC, Fezf1, Fezf2, and Fezf1, and Fezf2 miRNAs. The relative promoter activities were compared with activity in the absence of pCX-NICD. Error bars indicate SEM, and n values are shown in columns. *p < 0.05 by one-way ANOVA followed by Dunnett's post hoc comparison or Student's t test.

    Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-Bre1a IgG (ab32629, Abcam); rabbit anti-Bre1a IgG (catalog #9425, Cell Signaling Technology); rabbit anti-H2B IgG (Millipore); mouse anti-ubiquitylated H2B IgG (Millipore); mouse anti-phospho-histone H3 IgG (Cell Signaling Technology); mouse anti-GAPDH IgG (Santa Cruz Biotechnology); mouse anti-proliferating cell nuclear antigen (PCNA) IgG (DAKO); rabbit anti-green fluorescent protein (GFP) IgG (Invitrogen); rat anti-GFP IgG (Nacalai Tesque); rabbit anti-cleaved caspase 3 (c-Cas3) IgG (Cell Signaling Technology); rabbit anti-paired box gene 6 (Pax6) IgG (Millipore); and rabbit anti-eomesodermin homolog (Eomes/Tbr2) IgG (Abcam).

    Techniques: Expressing, shRNA, In Utero, Electroporation, In Situ Hybridization, Derivative Assay, Infection, Cell Culture, Quantitative RT-PCR, Luciferase, Plasmid Preparation, Activity Assay