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  • 99
    Thermo Fisher lipofectamine 2000
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 466214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gfp
    Localization of <t>Aub</t> and Ago3 in the egg-chamber and embryos depends on Vasa A) Box plot showing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > <t>GFP-Vas</t> WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Aub positive pole plasm, as determined by immunohistochemical detection of Aub. Experiments were performed in 3 independent replicates. B) Box plot representing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Ago3 positive pole plasm, as determined by immunohistochemical detection of Ago3 protein. Experiments were performed in 3 independent replicates. C) Mass spectormetry analysis of GFP-Vas WT (left panel) and GFP-Vas DQAD (right panel) co-IPs. Comparison of the fold-change in abundance of proteins based on spectral count ratio (enrichment > 2 fold), and statistical significance (p
    Gfp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7375 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gfp
    Localization of <t>Aub</t> and Ago3 in the egg-chamber and embryos depends on Vasa A) Box plot showing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > <t>GFP-Vas</t> WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Aub positive pole plasm, as determined by immunohistochemical detection of Aub. Experiments were performed in 3 independent replicates. B) Box plot representing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Ago3 positive pole plasm, as determined by immunohistochemical detection of Ago3 protein. Experiments were performed in 3 independent replicates. C) Mass spectormetry analysis of GFP-Vas WT (left panel) and GFP-Vas DQAD (right panel) co-IPs. Comparison of the fold-change in abundance of proteins based on spectral count ratio (enrichment > 2 fold), and statistical significance (p
    Gfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor
    Localization of <t>Aub</t> and Ago3 in the egg-chamber and embryos depends on Vasa A) Box plot showing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > <t>GFP-Vas</t> WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Aub positive pole plasm, as determined by immunohistochemical detection of Aub. Experiments were performed in 3 independent replicates. B) Box plot representing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Ago3 positive pole plasm, as determined by immunohistochemical detection of Ago3 protein. Experiments were performed in 3 independent replicates. C) Mass spectormetry analysis of GFP-Vas WT (left panel) and GFP-Vas DQAD (right panel) co-IPs. Comparison of the fold-change in abundance of proteins based on spectral count ratio (enrichment > 2 fold), and statistical significance (p
    Alexa Fluor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gfp tag polyclonal antibody
    Localization of <t>Aub</t> and Ago3 in the egg-chamber and embryos depends on Vasa A) Box plot showing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > <t>GFP-Vas</t> WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Aub positive pole plasm, as determined by immunohistochemical detection of Aub. Experiments were performed in 3 independent replicates. B) Box plot representing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Ago3 positive pole plasm, as determined by immunohistochemical detection of Ago3 protein. Experiments were performed in 3 independent replicates. C) Mass spectormetry analysis of GFP-Vas WT (left panel) and GFP-Vas DQAD (right panel) co-IPs. Comparison of the fold-change in abundance of proteins based on spectral count ratio (enrichment > 2 fold), and statistical significance (p
    Gfp Tag Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 8757 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gfp  (Abcam)
    99
    Abcam gfp
    Proximity ligation assay shows distinct proximity between histones (old versus new) and lagging strand-enriched DNA replication machinery components in GSCs. ( a ) A representative GSC showing PLA signals between lagging-strand-specific ligase-HA and new <t>H3-mKO</t> (top), and a representative GSC showing PLA signals between ligase-HA and old <t>H3-GFP</t> (bottom). ( b ) Quantification of the number of PLA puncta per nucleus between ligase and histones (old versus new) in GSCs and SGs. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. In GSCs, PLA puncta between ligase and new H3-mKO: 26.5; ( n =35); between ligase and old H3-GFP: 18.5; ( n =53). In SGs, PLA puncta between ligase and new H3-mKO: 16.7; ( n =24); between ligase and old H3-GFP: 21.9; ( n =21) **: P
    Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 9437 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fbs
    Proximity ligation assay shows distinct proximity between histones (old versus new) and lagging strand-enriched DNA replication machinery components in GSCs. ( a ) A representative GSC showing PLA signals between lagging-strand-specific ligase-HA and new <t>H3-mKO</t> (top), and a representative GSC showing PLA signals between ligase-HA and old <t>H3-GFP</t> (bottom). ( b ) Quantification of the number of PLA puncta per nucleus between ligase and histones (old versus new) in GSCs and SGs. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. In GSCs, PLA puncta between ligase and new H3-mKO: 26.5; ( n =35); between ligase and old H3-GFP: 18.5; ( n =53). In SGs, PLA puncta between ligase and new H3-mKO: 16.7; ( n =24); between ligase and old H3-GFP: 21.9; ( n =21) **: P
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    Becton Dickinson flow cytometry
    Proximity ligation assay shows distinct proximity between histones (old versus new) and lagging strand-enriched DNA replication machinery components in GSCs. ( a ) A representative GSC showing PLA signals between lagging-strand-specific ligase-HA and new <t>H3-mKO</t> (top), and a representative GSC showing PLA signals between ligase-HA and old <t>H3-GFP</t> (bottom). ( b ) Quantification of the number of PLA puncta per nucleus between ligase and histones (old versus new) in GSCs and SGs. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. In GSCs, PLA puncta between ligase and new H3-mKO: 26.5; ( n =35); between ligase and old H3-GFP: 18.5; ( n =53). In SGs, PLA puncta between ligase and new H3-mKO: 16.7; ( n =24); between ligase and old H3-GFP: 21.9; ( n =21) **: P
    Flow Cytometry, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 188544 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti flag m2 antibody
    Proximity ligation assay shows distinct proximity between histones (old versus new) and lagging strand-enriched DNA replication machinery components in GSCs. ( a ) A representative GSC showing PLA signals between lagging-strand-specific ligase-HA and new <t>H3-mKO</t> (top), and a representative GSC showing PLA signals between ligase-HA and old <t>H3-GFP</t> (bottom). ( b ) Quantification of the number of PLA puncta per nucleus between ligase and histones (old versus new) in GSCs and SGs. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. In GSCs, PLA puncta between ligase and new H3-mKO: 26.5; ( n =35); between ligase and old H3-GFP: 18.5; ( n =53). In SGs, PLA puncta between ligase and new H3-mKO: 16.7; ( n =24); between ligase and old H3-GFP: 21.9; ( n =21) **: P
    Monoclonal Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 37942 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore polybrene
    Proximity ligation assay shows distinct proximity between histones (old versus new) and lagging strand-enriched DNA replication machinery components in GSCs. ( a ) A representative GSC showing PLA signals between lagging-strand-specific ligase-HA and new <t>H3-mKO</t> (top), and a representative GSC showing PLA signals between ligase-HA and old <t>H3-GFP</t> (bottom). ( b ) Quantification of the number of PLA puncta per nucleus between ligase and histones (old versus new) in GSCs and SGs. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. In GSCs, PLA puncta between ligase and new H3-mKO: 26.5; ( n =35); between ligase and old H3-GFP: 18.5; ( n =53). In SGs, PLA puncta between ligase and new H3-mKO: 16.7; ( n =24); between ligase and old H3-GFP: 21.9; ( n =21) **: P
    Polybrene, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 71712 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti gfp
    Interactome of <t>GFP-MKRN1</t> RINGmut reveals putative ubiquitylation substrates. Experiments were performed using SILAC-based MS. Asymmetrical z-scores of combined SILAC ratios (n = 3 replicates) are shown. Proteins are detected in at least two out of three replicates. ( A ) Protein interactome of GFP-MKRN1 RINGmut in HEK293T cells analysed by quantitative mass spectrometry. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,097 protein groups were quantified in at least two out of three replicates ( Supplemental Table S1 ). MKRN1 and interesting candidate ubiquitylation targets are highlighted. ( B ) Quantitative comparison of the interactome of GFP-MKRN1 wt and GFP-MKRN1 RINGmut shows that potential ubiquitylation candidates identified in ( A ) are enriched in GFP-MKRN1 RINGmut over GFP-MKRN1 wt . Comparison reveals 137 proteins to be significantly enriched (MKRN1 RINGmut over MKRN1 wt with FDR
    Anti Gfp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 5342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dmem
    Interactome of <t>GFP-MKRN1</t> RINGmut reveals putative ubiquitylation substrates. Experiments were performed using SILAC-based MS. Asymmetrical z-scores of combined SILAC ratios (n = 3 replicates) are shown. Proteins are detected in at least two out of three replicates. ( A ) Protein interactome of GFP-MKRN1 RINGmut in HEK293T cells analysed by quantitative mass spectrometry. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,097 protein groups were quantified in at least two out of three replicates ( Supplemental Table S1 ). MKRN1 and interesting candidate ubiquitylation targets are highlighted. ( B ) Quantitative comparison of the interactome of GFP-MKRN1 wt and GFP-MKRN1 RINGmut shows that potential ubiquitylation candidates identified in ( A ) are enriched in GFP-MKRN1 RINGmut over GFP-MKRN1 wt . Comparison reveals 137 proteins to be significantly enriched (MKRN1 RINGmut over MKRN1 wt with FDR
    Dmem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 147955 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dapi
    Interactome of <t>GFP-MKRN1</t> RINGmut reveals putative ubiquitylation substrates. Experiments were performed using SILAC-based MS. Asymmetrical z-scores of combined SILAC ratios (n = 3 replicates) are shown. Proteins are detected in at least two out of three replicates. ( A ) Protein interactome of GFP-MKRN1 RINGmut in HEK293T cells analysed by quantitative mass spectrometry. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,097 protein groups were quantified in at least two out of three replicates ( Supplemental Table S1 ). MKRN1 and interesting candidate ubiquitylation targets are highlighted. ( B ) Quantitative comparison of the interactome of GFP-MKRN1 wt and GFP-MKRN1 RINGmut shows that potential ubiquitylation candidates identified in ( A ) are enriched in GFP-MKRN1 RINGmut over GFP-MKRN1 wt . Comparison reveals 137 proteins to be significantly enriched (MKRN1 RINGmut over MKRN1 wt with FDR
    Dapi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 155801 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore protease inhibitor cocktail
    Interactome of <t>GFP-MKRN1</t> RINGmut reveals putative ubiquitylation substrates. Experiments were performed using SILAC-based MS. Asymmetrical z-scores of combined SILAC ratios (n = 3 replicates) are shown. Proteins are detected in at least two out of three replicates. ( A ) Protein interactome of GFP-MKRN1 RINGmut in HEK293T cells analysed by quantitative mass spectrometry. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,097 protein groups were quantified in at least two out of three replicates ( Supplemental Table S1 ). MKRN1 and interesting candidate ubiquitylation targets are highlighted. ( B ) Quantitative comparison of the interactome of GFP-MKRN1 wt and GFP-MKRN1 RINGmut shows that potential ubiquitylation candidates identified in ( A ) are enriched in GFP-MKRN1 RINGmut over GFP-MKRN1 wt . Comparison reveals 137 proteins to be significantly enriched (MKRN1 RINGmut over MKRN1 wt with FDR
    Protease Inhibitor Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 135165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher penicillin streptomycin
    Interactome of <t>GFP-MKRN1</t> RINGmut reveals putative ubiquitylation substrates. Experiments were performed using SILAC-based MS. Asymmetrical z-scores of combined SILAC ratios (n = 3 replicates) are shown. Proteins are detected in at least two out of three replicates. ( A ) Protein interactome of GFP-MKRN1 RINGmut in HEK293T cells analysed by quantitative mass spectrometry. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,097 protein groups were quantified in at least two out of three replicates ( Supplemental Table S1 ). MKRN1 and interesting candidate ubiquitylation targets are highlighted. ( B ) Quantitative comparison of the interactome of GFP-MKRN1 wt and GFP-MKRN1 RINGmut shows that potential ubiquitylation candidates identified in ( A ) are enriched in GFP-MKRN1 RINGmut over GFP-MKRN1 wt . Comparison reveals 137 proteins to be significantly enriched (MKRN1 RINGmut over MKRN1 wt with FDR
    Penicillin Streptomycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 156248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pbs
    Interactome of <t>GFP-MKRN1</t> RINGmut reveals putative ubiquitylation substrates. Experiments were performed using SILAC-based MS. Asymmetrical z-scores of combined SILAC ratios (n = 3 replicates) are shown. Proteins are detected in at least two out of three replicates. ( A ) Protein interactome of GFP-MKRN1 RINGmut in HEK293T cells analysed by quantitative mass spectrometry. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,097 protein groups were quantified in at least two out of three replicates ( Supplemental Table S1 ). MKRN1 and interesting candidate ubiquitylation targets are highlighted. ( B ) Quantitative comparison of the interactome of GFP-MKRN1 wt and GFP-MKRN1 RINGmut shows that potential ubiquitylation candidates identified in ( A ) are enriched in GFP-MKRN1 RINGmut over GFP-MKRN1 wt . Comparison reveals 137 proteins to be significantly enriched (MKRN1 RINGmut over MKRN1 wt with FDR
    Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 45702 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 488
    Interactome of <t>GFP-MKRN1</t> RINGmut reveals putative ubiquitylation substrates. Experiments were performed using SILAC-based MS. Asymmetrical z-scores of combined SILAC ratios (n = 3 replicates) are shown. Proteins are detected in at least two out of three replicates. ( A ) Protein interactome of GFP-MKRN1 RINGmut in HEK293T cells analysed by quantitative mass spectrometry. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,097 protein groups were quantified in at least two out of three replicates ( Supplemental Table S1 ). MKRN1 and interesting candidate ubiquitylation targets are highlighted. ( B ) Quantitative comparison of the interactome of GFP-MKRN1 wt and GFP-MKRN1 RINGmut shows that potential ubiquitylation candidates identified in ( A ) are enriched in GFP-MKRN1 RINGmut over GFP-MKRN1 wt . Comparison reveals 137 proteins to be significantly enriched (MKRN1 RINGmut over MKRN1 wt with FDR
    Alexa Fluor 488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 71255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti alpha tubulin antibody
    Interactome of <t>GFP-MKRN1</t> RINGmut reveals putative ubiquitylation substrates. Experiments were performed using SILAC-based MS. Asymmetrical z-scores of combined SILAC ratios (n = 3 replicates) are shown. Proteins are detected in at least two out of three replicates. ( A ) Protein interactome of GFP-MKRN1 RINGmut in HEK293T cells analysed by quantitative mass spectrometry. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,097 protein groups were quantified in at least two out of three replicates ( Supplemental Table S1 ). MKRN1 and interesting candidate ubiquitylation targets are highlighted. ( B ) Quantitative comparison of the interactome of GFP-MKRN1 wt and GFP-MKRN1 RINGmut shows that potential ubiquitylation candidates identified in ( A ) are enriched in GFP-MKRN1 RINGmut over GFP-MKRN1 wt . Comparison reveals 137 proteins to be significantly enriched (MKRN1 RINGmut over MKRN1 wt with FDR
    Monoclonal Anti Alpha Tubulin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 19851 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Localization of Aub and Ago3 in the egg-chamber and embryos depends on Vasa A) Box plot showing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Aub positive pole plasm, as determined by immunohistochemical detection of Aub. Experiments were performed in 3 independent replicates. B) Box plot representing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Ago3 positive pole plasm, as determined by immunohistochemical detection of Ago3 protein. Experiments were performed in 3 independent replicates. C) Mass spectormetry analysis of GFP-Vas WT (left panel) and GFP-Vas DQAD (right panel) co-IPs. Comparison of the fold-change in abundance of proteins based on spectral count ratio (enrichment > 2 fold), and statistical significance (p

    Journal: bioRxiv

    Article Title: Induction of Chk2 signaling in the germarium is sufficient to cause oogenesis arrest in Drosophila

    doi: 10.1101/611798

    Figure Lengend Snippet: Localization of Aub and Ago3 in the egg-chamber and embryos depends on Vasa A) Box plot showing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Aub positive pole plasm, as determined by immunohistochemical detection of Aub. Experiments were performed in 3 independent replicates. B) Box plot representing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Ago3 positive pole plasm, as determined by immunohistochemical detection of Ago3 protein. Experiments were performed in 3 independent replicates. C) Mass spectormetry analysis of GFP-Vas WT (left panel) and GFP-Vas DQAD (right panel) co-IPs. Comparison of the fold-change in abundance of proteins based on spectral count ratio (enrichment > 2 fold), and statistical significance (p

    Article Snippet: Western blotting was performed using antibodies against Vasa (rat; 1:3000; ( )), Aub (rabbit; 1:1000; , Ago3 (mouse; 1:500; gift of Mikiko Siomi), GFP (rabbit; 1:5000; Chemokine TP401), and Tub (mouse; 1:10000; Sigma T5168).

    Techniques: Immunohistochemistry

    Proximity ligation assay shows distinct proximity between histones (old versus new) and lagging strand-enriched DNA replication machinery components in GSCs. ( a ) A representative GSC showing PLA signals between lagging-strand-specific ligase-HA and new H3-mKO (top), and a representative GSC showing PLA signals between ligase-HA and old H3-GFP (bottom). ( b ) Quantification of the number of PLA puncta per nucleus between ligase and histones (old versus new) in GSCs and SGs. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. In GSCs, PLA puncta between ligase and new H3-mKO: 26.5; ( n =35); between ligase and old H3-GFP: 18.5; ( n =53). In SGs, PLA puncta between ligase and new H3-mKO: 16.7; ( n =24); between ligase and old H3-GFP: 21.9; ( n =21) **: P

    Journal: Nature structural & molecular biology

    Article Title: Asymmetric histone inheritance via strand-specific incorporation and biased replication fork movement

    doi: 10.1038/s41594-019-0269-z

    Figure Lengend Snippet: Proximity ligation assay shows distinct proximity between histones (old versus new) and lagging strand-enriched DNA replication machinery components in GSCs. ( a ) A representative GSC showing PLA signals between lagging-strand-specific ligase-HA and new H3-mKO (top), and a representative GSC showing PLA signals between ligase-HA and old H3-GFP (bottom). ( b ) Quantification of the number of PLA puncta per nucleus between ligase and histones (old versus new) in GSCs and SGs. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. In GSCs, PLA puncta between ligase and new H3-mKO: 26.5; ( n =35); between ligase and old H3-GFP: 18.5; ( n =53). In SGs, PLA puncta between ligase and new H3-mKO: 16.7; ( n =24); between ligase and old H3-GFP: 21.9; ( n =21) **: P

    Article Snippet: Primary antibodies used were mouse anti-Fas III (1:200, DSHB, 7G10), anti-HA (1:200; Sigma-Aldrich H3663), anti-PCNA (1:200; Santa Cruz sc-56), anti-GFP (1:1,000; Abcam ab 13970), anti-mKO (1:200; MBL PM051M), anti-mCherry (1:1000; Invitrogen ), anti-H3K27me3 (1:200; Millipore 07–449), anti-H4K20me2/3 (1:400; Abcam ab7817), anti-ssDNA (1:100, DSHB) and anti-BrdU (1:200; Abcam ab6326).

    Techniques: Proximity Ligation Assay

    Asymmetric H3 and symmetric H2A distribution on replicating sister chromatids. ( a,b ) Airyscan images of chromatin fibers labeled with EdU showing distribution of old H2A-eGFP and new H2A-mCherry (a) or old H3-eGFP and new H3-mCherry (b) on nonreplicated regions lacking EdU label (dashed line boxes) and replicating regions labeled with EdU (solid line boxes). Line-plots show old and new histone distribution across unreplicated (top) or replicated (bottom) regions. ( c ) Two-color STED image of chromatin fiber showing old H3-GFP and new H3-mKO distribution across unreplicated (dashed line box) and replicating (solid line box) chromatin regions. The transition from single to double-fiber occurs at the point where new histone incorporation begins (white arrow). Line-plots show old H3-GFP and new H3-mKO distribution across unreplicated region without new H3 (top) or on replicated region with new H3 (bottom). ( d,e ) Quantification of distribution of old H2A and H3 (d) or new H2A and H3 (e) between sister chromatids at replication regions on chromatin fibers. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. *** P

    Journal: Nature structural & molecular biology

    Article Title: Asymmetric histone inheritance via strand-specific incorporation and biased replication fork movement

    doi: 10.1038/s41594-019-0269-z

    Figure Lengend Snippet: Asymmetric H3 and symmetric H2A distribution on replicating sister chromatids. ( a,b ) Airyscan images of chromatin fibers labeled with EdU showing distribution of old H2A-eGFP and new H2A-mCherry (a) or old H3-eGFP and new H3-mCherry (b) on nonreplicated regions lacking EdU label (dashed line boxes) and replicating regions labeled with EdU (solid line boxes). Line-plots show old and new histone distribution across unreplicated (top) or replicated (bottom) regions. ( c ) Two-color STED image of chromatin fiber showing old H3-GFP and new H3-mKO distribution across unreplicated (dashed line box) and replicating (solid line box) chromatin regions. The transition from single to double-fiber occurs at the point where new histone incorporation begins (white arrow). Line-plots show old H3-GFP and new H3-mKO distribution across unreplicated region without new H3 (top) or on replicated region with new H3 (bottom). ( d,e ) Quantification of distribution of old H2A and H3 (d) or new H2A and H3 (e) between sister chromatids at replication regions on chromatin fibers. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. *** P

    Article Snippet: Primary antibodies used were mouse anti-Fas III (1:200, DSHB, 7G10), anti-HA (1:200; Sigma-Aldrich H3663), anti-PCNA (1:200; Santa Cruz sc-56), anti-GFP (1:1,000; Abcam ab 13970), anti-mKO (1:200; MBL PM051M), anti-mCherry (1:1000; Invitrogen ), anti-H3K27me3 (1:200; Millipore 07–449), anti-H4K20me2/3 (1:400; Abcam ab7817), anti-ssDNA (1:100, DSHB) and anti-BrdU (1:200; Abcam ab6326).

    Techniques: Labeling

    Histone H4 shows asymmetric inheritance pattern during Drosophila GSC asymmetric divisions. ( a ) A cartoon depicting the experimental design. ( b ) H4 distribution in a post-mitotic GSC-GB pair labeled with EdU, showing H4-GFP is distributed asymmetrically towards the GSC, whereas H4-mKO is distributed more evenly between the GSC and the GB. ( c ) H4 distribution patterns in a post-mitotic SG pair, showing both H4-GFP and H4-mKO are symmetrically distributed between the two SG nuclei. ( d ) Quantification of H4- GFP and H4-mKO distributions in GSC-GB pairs ( n =44) and SG1-SG2 pairs ( n for additional statistical information. ( e ) An anaphase and telophase GSC showing asymmetric segregation of H4-GFP towards the GSC and H4-mKO towards the GB. Scale bars for panels b, c and e, 5μm; asterisk: hub.

    Journal: Nature structural & molecular biology

    Article Title: Asymmetric histone inheritance via strand-specific incorporation and biased replication fork movement

    doi: 10.1038/s41594-019-0269-z

    Figure Lengend Snippet: Histone H4 shows asymmetric inheritance pattern during Drosophila GSC asymmetric divisions. ( a ) A cartoon depicting the experimental design. ( b ) H4 distribution in a post-mitotic GSC-GB pair labeled with EdU, showing H4-GFP is distributed asymmetrically towards the GSC, whereas H4-mKO is distributed more evenly between the GSC and the GB. ( c ) H4 distribution patterns in a post-mitotic SG pair, showing both H4-GFP and H4-mKO are symmetrically distributed between the two SG nuclei. ( d ) Quantification of H4- GFP and H4-mKO distributions in GSC-GB pairs ( n =44) and SG1-SG2 pairs ( n for additional statistical information. ( e ) An anaphase and telophase GSC showing asymmetric segregation of H4-GFP towards the GSC and H4-mKO towards the GB. Scale bars for panels b, c and e, 5μm; asterisk: hub.

    Article Snippet: Primary antibodies used were mouse anti-Fas III (1:200, DSHB, 7G10), anti-HA (1:200; Sigma-Aldrich H3663), anti-PCNA (1:200; Santa Cruz sc-56), anti-GFP (1:1,000; Abcam ab 13970), anti-mKO (1:200; MBL PM051M), anti-mCherry (1:1000; Invitrogen ), anti-H3K27me3 (1:200; Millipore 07–449), anti-H4K20me2/3 (1:400; Abcam ab7817), anti-ssDNA (1:100, DSHB) and anti-BrdU (1:200; Abcam ab6326).

    Techniques: Labeling

    Histones H2A and H2B show symmetric distribution during Drosophila GSC asymmetric division. ( a ) Symmetric H2A inheritance pattern in a post-mitotic GSC-GB (top), mitotic GSC (middle) and post-mitotic spermatogonial (bottom) pairs. ( b ) Quantification of H2A-GFP and H2A-mKO distribution in GSC-GB pairs ( n =20) and SG1-SG2 pairs ( n =20). Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. * P

    Journal: Nature structural & molecular biology

    Article Title: Asymmetric histone inheritance via strand-specific incorporation and biased replication fork movement

    doi: 10.1038/s41594-019-0269-z

    Figure Lengend Snippet: Histones H2A and H2B show symmetric distribution during Drosophila GSC asymmetric division. ( a ) Symmetric H2A inheritance pattern in a post-mitotic GSC-GB (top), mitotic GSC (middle) and post-mitotic spermatogonial (bottom) pairs. ( b ) Quantification of H2A-GFP and H2A-mKO distribution in GSC-GB pairs ( n =20) and SG1-SG2 pairs ( n =20). Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. * P

    Article Snippet: Primary antibodies used were mouse anti-Fas III (1:200, DSHB, 7G10), anti-HA (1:200; Sigma-Aldrich H3663), anti-PCNA (1:200; Santa Cruz sc-56), anti-GFP (1:1,000; Abcam ab 13970), anti-mKO (1:200; MBL PM051M), anti-mCherry (1:1000; Invitrogen ), anti-H3K27me3 (1:200; Millipore 07–449), anti-H4K20me2/3 (1:400; Abcam ab7817), anti-ssDNA (1:100, DSHB) and anti-BrdU (1:200; Abcam ab6326).

    Techniques:

    Stem cell potential and molecular characteristics of PDX1+ A undiff . a Isolation of PDX1+ and PDX1− A undiff from Plzf-mC/CreER; Pdx1 GFP/+ adults by flow cytometry. A undiff are mCherry+ CD9+ c-KIT−. Gates for GFP+ and GFP− cells were set according to Plzf-mC/CreER control (left profile). Percentage of cells in GFP+ gate from representative sample is shown ( n = 7). b Pdx1-GFP+ and GFP− adult A undiff fractions were transplanted into recipient testis and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative donor colonies. Panels on right show higher magnification details of indicated areas and grayscale panels show individual immunostaining. Scale bar, 100 μm. c Colony-forming efficiency of Pdx1-GFP+ and GFP− A undiff fractions in transplantation assays from b . Data is presented as mean number of colonies per 10 5 donor cells ± s.e.m. ( n = 15 recipient testes for Pdx1-GFP− cells and n = 13 for Pdx1-GFP+ cells). Donor cells were pooled from a total of 7 Plzf-mC/CreER; Pdx1 GFP/+ adults. d Mean length ± s.e.m. of donor colonies was measured from tiled microscope images from experiment of c . e Mean number of GFP+ cells/donor colony ± s.e.m. were calculated for a set of whole-mount samples from transplant assay of c ( n = 58 colonies from Pdx1-GFP− cells and n = 63 from Pdx1-GFP+). f Heatmap illustrates expression of indicated genes from RNA-Seq analysis of Pdx1-GFP+ and GFP− A undiff fractions isolated as in a ( n = 4 mice). Differentially expressed genes (DEG) are in bold. Cut-off for DEG is false discovery rate (FDR)

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: Stem cell potential and molecular characteristics of PDX1+ A undiff . a Isolation of PDX1+ and PDX1− A undiff from Plzf-mC/CreER; Pdx1 GFP/+ adults by flow cytometry. A undiff are mCherry+ CD9+ c-KIT−. Gates for GFP+ and GFP− cells were set according to Plzf-mC/CreER control (left profile). Percentage of cells in GFP+ gate from representative sample is shown ( n = 7). b Pdx1-GFP+ and GFP− adult A undiff fractions were transplanted into recipient testis and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative donor colonies. Panels on right show higher magnification details of indicated areas and grayscale panels show individual immunostaining. Scale bar, 100 μm. c Colony-forming efficiency of Pdx1-GFP+ and GFP− A undiff fractions in transplantation assays from b . Data is presented as mean number of colonies per 10 5 donor cells ± s.e.m. ( n = 15 recipient testes for Pdx1-GFP− cells and n = 13 for Pdx1-GFP+ cells). Donor cells were pooled from a total of 7 Plzf-mC/CreER; Pdx1 GFP/+ adults. d Mean length ± s.e.m. of donor colonies was measured from tiled microscope images from experiment of c . e Mean number of GFP+ cells/donor colony ± s.e.m. were calculated for a set of whole-mount samples from transplant assay of c ( n = 58 colonies from Pdx1-GFP− cells and n = 63 from Pdx1-GFP+). f Heatmap illustrates expression of indicated genes from RNA-Seq analysis of Pdx1-GFP+ and GFP− A undiff fractions isolated as in a ( n = 4 mice). Differentially expressed genes (DEG) are in bold. Cut-off for DEG is false discovery rate (FDR)

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Isolation, Flow Cytometry, Cytometry, Expressing, Immunostaining, Transplantation Assay, Microscopy, RNA Sequencing Assay, Mouse Assay

    A undiff heterogeneity during culture. a Oct4-GFP− and GFP+ A undiff from Plzf-mC/CreER; Oct4-GFP adults placed in culture and analysed 2–3 weeks later. Right: IF of colonies ( n = 2 per fraction). Scale bar, 50 μm. b Mean colony-for ming efficiency of Oct4-GFP− and GFP+ A undiff ± s.e.m. from a ( n = 6 mice). c Cultures from Oct4-GFP− and GFP+ A undiff plated at 25 × 10 3 per well and counted at indicated timepoints. Mean recovery ± s.e.m. shown. d Cultures from Oct4-GFP− and GFP+ A undiff transplanted and analysed 8 weeks later by IF. Representative colonies shown (2 sets of lines) ( n = 11 testes Oct4-GFP− and n = 9 testes Oct4-GFP+). Scale bar, 100 μm. e Cultures from Oct4-GFP− and GFP+ A undiff sorted by GFP and plated at 25 × 10 3 per well. GFP+ cells determined by flow cytometry. Mean ± s.e.m. shown ( n = 6 cultures). f qRT-PCR of GFP+ and GFP− cells from Oct4-GFP− and GFP+ A undiff cultures. Expression corrected to β-actin and normalized so mean of GFP− or GFP+ fractions equals 1. Mean ± s.e.m. shown ( n = 4 cultures). g Representative IF of primary colonies (P0) and passage 5 (P5) cultures from Oct4-GFP− and GFP+ A undiff ( n = 4 lines). Scale bar, 50 μm. h Cultures from Oct4-GFP− and GFP+ A undiff plated at increasing densities (10 × 10 3 , 100 × 10 3 and 200 × 10 3 cells/well) and analysed 7–10 days later. Representative IF shown ( n = 4 lines). Scale bar, 50 μm. i Cultures from Oct4-GFP− and GFP+ A undiff plated at low and high densities (20 × 10 3 , 200 × 10 3 cells/well) and cultured for 2 weeks. Conditioned media was collected at indicated times (hours) after media replenishment for ELISA. Mean GDNF levels are shown as percentage of starting levels ± s.e.m. ( n = 4 cultures). j Cultures from Oct4-GFP− A undiff (20 × 10 3 cells/well) switched to media containing reduced GDNF and bFGF (1 ng/ml, left) or maintained with regular media (right) for 4 days. Representative flow cytometry shown. k Cultures from Oct4-GFP− and GFP+ A undiff sorted according to GFP and plated (10 × 10 3 cells/well) in media with GDNF but no bFGF (GDNF) or bFGF without GDNF (bFGF). Cells analysed at indicated timepoints by flow cytometry. Mean percentage of cells GFP+ ± standard deviation (s.d.) ( n = 3 replicates) from representative experiment shown. Grey plots: mean values in regular media (GDNF+ bFGF). l Cultures from Oct4-GFP− and GFP+ A undiff (20 × 10 3 cells/well) grown 3 days in regular media switched to media without GDNF or bFGF (−), bFGF without GDNF (bFGF), GDNF without bFGF (GDNF) or regular medium (GDNF+ bFGF) then analysed by IF after 2 weeks. Representative images shown ( n = 2 lines). Scale bar, 50 μm. m Plzf-mC/CreER cultures incubated in media containing indicated inhibitors (Inh) for 4 days prior to IF. Representative images shown ( n = 2 lines). Scale bar, 50 μm. Two-tailed Student’s t -test used (* P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: A undiff heterogeneity during culture. a Oct4-GFP− and GFP+ A undiff from Plzf-mC/CreER; Oct4-GFP adults placed in culture and analysed 2–3 weeks later. Right: IF of colonies ( n = 2 per fraction). Scale bar, 50 μm. b Mean colony-for ming efficiency of Oct4-GFP− and GFP+ A undiff ± s.e.m. from a ( n = 6 mice). c Cultures from Oct4-GFP− and GFP+ A undiff plated at 25 × 10 3 per well and counted at indicated timepoints. Mean recovery ± s.e.m. shown. d Cultures from Oct4-GFP− and GFP+ A undiff transplanted and analysed 8 weeks later by IF. Representative colonies shown (2 sets of lines) ( n = 11 testes Oct4-GFP− and n = 9 testes Oct4-GFP+). Scale bar, 100 μm. e Cultures from Oct4-GFP− and GFP+ A undiff sorted by GFP and plated at 25 × 10 3 per well. GFP+ cells determined by flow cytometry. Mean ± s.e.m. shown ( n = 6 cultures). f qRT-PCR of GFP+ and GFP− cells from Oct4-GFP− and GFP+ A undiff cultures. Expression corrected to β-actin and normalized so mean of GFP− or GFP+ fractions equals 1. Mean ± s.e.m. shown ( n = 4 cultures). g Representative IF of primary colonies (P0) and passage 5 (P5) cultures from Oct4-GFP− and GFP+ A undiff ( n = 4 lines). Scale bar, 50 μm. h Cultures from Oct4-GFP− and GFP+ A undiff plated at increasing densities (10 × 10 3 , 100 × 10 3 and 200 × 10 3 cells/well) and analysed 7–10 days later. Representative IF shown ( n = 4 lines). Scale bar, 50 μm. i Cultures from Oct4-GFP− and GFP+ A undiff plated at low and high densities (20 × 10 3 , 200 × 10 3 cells/well) and cultured for 2 weeks. Conditioned media was collected at indicated times (hours) after media replenishment for ELISA. Mean GDNF levels are shown as percentage of starting levels ± s.e.m. ( n = 4 cultures). j Cultures from Oct4-GFP− A undiff (20 × 10 3 cells/well) switched to media containing reduced GDNF and bFGF (1 ng/ml, left) or maintained with regular media (right) for 4 days. Representative flow cytometry shown. k Cultures from Oct4-GFP− and GFP+ A undiff sorted according to GFP and plated (10 × 10 3 cells/well) in media with GDNF but no bFGF (GDNF) or bFGF without GDNF (bFGF). Cells analysed at indicated timepoints by flow cytometry. Mean percentage of cells GFP+ ± standard deviation (s.d.) ( n = 3 replicates) from representative experiment shown. Grey plots: mean values in regular media (GDNF+ bFGF). l Cultures from Oct4-GFP− and GFP+ A undiff (20 × 10 3 cells/well) grown 3 days in regular media switched to media without GDNF or bFGF (−), bFGF without GDNF (bFGF), GDNF without bFGF (GDNF) or regular medium (GDNF+ bFGF) then analysed by IF after 2 weeks. Representative images shown ( n = 2 lines). Scale bar, 50 μm. m Plzf-mC/CreER cultures incubated in media containing indicated inhibitors (Inh) for 4 days prior to IF. Representative images shown ( n = 2 lines). Scale bar, 50 μm. Two-tailed Student’s t -test used (* P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Quantitative RT-PCR, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation, Incubation, Two Tailed Test

    PDX1+ and EOMES+ spermatogonia during development and regeneration. a Representative whole-mount IF of tubules from WT mice of indicated ages (PND; postnatal day) ( n = 2 mice per age). Scale bars, 50 μm. b Representative IF of testis sections from WT mice of indicated ages ( n = 3 mice per timepoint). Insets show details of indicated areas. Arrowheads: EOMES+ PDX1+ cells. Asterisks: EOMES+ PDX1− cells. Scale bars, 50 μm. c , d Flow cytometry of fixed and permeabilized testis cells from WT mice of indicated ages. Mean percentages of PLZF+ c-KIT− A undiff expressing PDX1 and EOMES are shown ± s.e.m. ( n = 3–5 mice/time point). e WT adults were treated with busulfan (10 mg/kg) and tubules analysed by whole-mount IF 14 days later ( n = 2 mice). Top panels: regenerative areas with GFRα1+ A al . Bottom: non-regenerative areas lacking GFRα1+ A al . Insets show details of indicated regions. Scale bar, 50 μm. f Regeneration assay of e 4 weeks post busulfan (2 areas shown). Arrowheads: PDX1+ A undiff . Scale bar, 50 μm. g Representative flow cytometry of fixed and permeabilized testis cells from WT adults untreated or busulfan treated as in e ( n = 4 busulfan-treated mice and n = 5 untreated). PLZF+ c-KIT− A undiff are shown. Percentage of A undiff KI67+ and EOMES+ are indicated. h Mean percentages of PLZF+ c-KIT− A undiff expressing PDX1, EOMES and KI67 from g are shown ± s.e.m. i Representative flow cytometry of PDX1 in indicated populations from g ( n = 4 mice per condition). Percentages of cells PDX1+ are indicated. j PDX1 levels (median fluorescent intensity) in EOMES+ PLZF+ cells from i . Mean values ± s.e.m. shown. k Quantitative RT-PCR of A undiff from Plzf-mC/CreER mice of indicated ages or 10 days post busulfan as in e . Expression levels are corrected to β-actin and normalized to an adult sample. Mean values ± s.e.m. are indicated ( n = 4 PND10 and busulfan-treated, n = 5 PND20, n = 6 adults). Selected significance values are shown. l Model of A undiff functional states. Self-renewing (curved arrows) GFRα1+ A undiff adopt different states identified by variable expression of Pdx1 , Eomes and Lhx1 . A fraction of GFRα1+ A undiff lacks PDX1, EOMES and LHX1 (grey in panel) and may represent a transitional state destined to become differentiation-primed. Niche signals differentially support self-renewing states. Given dynamic niche properties, different states predominate in development and homeostatic plus regenerative testis. The state marked by PDX1, EOMES and LHX1 is specific to homeostatic testis and potentially optimized for life-long germline maintenance. Pdx1 is downregulated and Eomes plus Lhx1 upregulated in states suited for short-term expansion during development and regeneration. Oct4-GFP+ differentiation-primed A undiff convert back to a self-renewing state under optimised culture conditions or by transplantation into recipient testis with vacant niches. Significance was calculated by two-tailed Student’s t -test (* P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: PDX1+ and EOMES+ spermatogonia during development and regeneration. a Representative whole-mount IF of tubules from WT mice of indicated ages (PND; postnatal day) ( n = 2 mice per age). Scale bars, 50 μm. b Representative IF of testis sections from WT mice of indicated ages ( n = 3 mice per timepoint). Insets show details of indicated areas. Arrowheads: EOMES+ PDX1+ cells. Asterisks: EOMES+ PDX1− cells. Scale bars, 50 μm. c , d Flow cytometry of fixed and permeabilized testis cells from WT mice of indicated ages. Mean percentages of PLZF+ c-KIT− A undiff expressing PDX1 and EOMES are shown ± s.e.m. ( n = 3–5 mice/time point). e WT adults were treated with busulfan (10 mg/kg) and tubules analysed by whole-mount IF 14 days later ( n = 2 mice). Top panels: regenerative areas with GFRα1+ A al . Bottom: non-regenerative areas lacking GFRα1+ A al . Insets show details of indicated regions. Scale bar, 50 μm. f Regeneration assay of e 4 weeks post busulfan (2 areas shown). Arrowheads: PDX1+ A undiff . Scale bar, 50 μm. g Representative flow cytometry of fixed and permeabilized testis cells from WT adults untreated or busulfan treated as in e ( n = 4 busulfan-treated mice and n = 5 untreated). PLZF+ c-KIT− A undiff are shown. Percentage of A undiff KI67+ and EOMES+ are indicated. h Mean percentages of PLZF+ c-KIT− A undiff expressing PDX1, EOMES and KI67 from g are shown ± s.e.m. i Representative flow cytometry of PDX1 in indicated populations from g ( n = 4 mice per condition). Percentages of cells PDX1+ are indicated. j PDX1 levels (median fluorescent intensity) in EOMES+ PLZF+ cells from i . Mean values ± s.e.m. shown. k Quantitative RT-PCR of A undiff from Plzf-mC/CreER mice of indicated ages or 10 days post busulfan as in e . Expression levels are corrected to β-actin and normalized to an adult sample. Mean values ± s.e.m. are indicated ( n = 4 PND10 and busulfan-treated, n = 5 PND20, n = 6 adults). Selected significance values are shown. l Model of A undiff functional states. Self-renewing (curved arrows) GFRα1+ A undiff adopt different states identified by variable expression of Pdx1 , Eomes and Lhx1 . A fraction of GFRα1+ A undiff lacks PDX1, EOMES and LHX1 (grey in panel) and may represent a transitional state destined to become differentiation-primed. Niche signals differentially support self-renewing states. Given dynamic niche properties, different states predominate in development and homeostatic plus regenerative testis. The state marked by PDX1, EOMES and LHX1 is specific to homeostatic testis and potentially optimized for life-long germline maintenance. Pdx1 is downregulated and Eomes plus Lhx1 upregulated in states suited for short-term expansion during development and regeneration. Oct4-GFP+ differentiation-primed A undiff convert back to a self-renewing state under optimised culture conditions or by transplantation into recipient testis with vacant niches. Significance was calculated by two-tailed Student’s t -test (* P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR, Functional Assay, Transplantation Assay, Two Tailed Test

    Characterization of Plzf-mC/CreER transgenic mice. a , b Representative IF of adult Plzf-mC/CreER testis sections ( n = 3 mice). Tubule stages and populations are indicated. Scale bar, 50 μm. c Representative flow cytometry of fixed and permeabilized testis from Plzf-mC/CreER and wildtype (WT) adult testis ( n = 3 mice per genotype). PLZF+ cells are shown. d Plzf-mC/CreER; Z/EG mice injected daily with TAM for 5 days were harvested at indicated days after treatment. e Representative IF of testis sections from d ( n = 3 testes per time point). Insets show details of indicated areas. Scale bar, 50 μm. f Representative whole-mount IF from d . Inset shows detail of indicated area. Arrowheads: unlabelled GFRα1+ cells. Scale bar, 50 μm. g Flow cytometry of fixed and permeabilized testis cells from d . Graph indicates mean fraction of A undiff (PLZF+ c-KIT−) and PLZF+ c-KIT+ early differentiating cells expressing GFP ± standard error of mean (s.e.m.) ( n = 4 testes D3, D10 and D90, n = 6 testes D30). h Representative flow cytometry of live Plzf-mC/CreER testis cells. SSC is side scatter. mCherry+ gate was set according to WT. i Quantitative RT-PCR for spermatogonial markers from Plzf-mC/CreER cell fractions sorted as in h . mC− indicates mCherry−. Expression levels are corrected to β-actin and normalized so mean value of fraction #2 equals 1. Mean values ± s.e.m. are indicated ( n = 3 sorts, 2 mice pooled per sort). Significance vs. mCherry− cells is shown. j Violin plots of gene expression in 150 single cells of fraction #1 cells from h . Cells were gated according to Plzf and Vasa expression. k Left: mean in vitro colony-forming activity of Plzf-mC/CreER fractions ± s.e.m. isolated as in h ( n = 3 mice). mC− indicates mCherry−. Significance vs. mCherry− fraction is indicated. Right: representative IF of passaged cells from fraction #1 treated with vehicle or retinoic acid for 48 h ( n = 3). Scale bar, 50 μm. l Left: transplantation of cultured cells established from Plzf-mC/CreER fraction #1. Right: representative whole-mount IF of tubules 8 weeks post transplant demonstrating formation of mCherry+ colonies ( n = 5 recipients). Comparable spermatogenic capacity was observed upon transplantation of an independent line (3.90 colonies/10 5 cells; n = 4 recipient testes). Scale bar, 100 μm. Significance was calculated by two-tailed Student’s t -test (** P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: Characterization of Plzf-mC/CreER transgenic mice. a , b Representative IF of adult Plzf-mC/CreER testis sections ( n = 3 mice). Tubule stages and populations are indicated. Scale bar, 50 μm. c Representative flow cytometry of fixed and permeabilized testis from Plzf-mC/CreER and wildtype (WT) adult testis ( n = 3 mice per genotype). PLZF+ cells are shown. d Plzf-mC/CreER; Z/EG mice injected daily with TAM for 5 days were harvested at indicated days after treatment. e Representative IF of testis sections from d ( n = 3 testes per time point). Insets show details of indicated areas. Scale bar, 50 μm. f Representative whole-mount IF from d . Inset shows detail of indicated area. Arrowheads: unlabelled GFRα1+ cells. Scale bar, 50 μm. g Flow cytometry of fixed and permeabilized testis cells from d . Graph indicates mean fraction of A undiff (PLZF+ c-KIT−) and PLZF+ c-KIT+ early differentiating cells expressing GFP ± standard error of mean (s.e.m.) ( n = 4 testes D3, D10 and D90, n = 6 testes D30). h Representative flow cytometry of live Plzf-mC/CreER testis cells. SSC is side scatter. mCherry+ gate was set according to WT. i Quantitative RT-PCR for spermatogonial markers from Plzf-mC/CreER cell fractions sorted as in h . mC− indicates mCherry−. Expression levels are corrected to β-actin and normalized so mean value of fraction #2 equals 1. Mean values ± s.e.m. are indicated ( n = 3 sorts, 2 mice pooled per sort). Significance vs. mCherry− cells is shown. j Violin plots of gene expression in 150 single cells of fraction #1 cells from h . Cells were gated according to Plzf and Vasa expression. k Left: mean in vitro colony-forming activity of Plzf-mC/CreER fractions ± s.e.m. isolated as in h ( n = 3 mice). mC− indicates mCherry−. Significance vs. mCherry− fraction is indicated. Right: representative IF of passaged cells from fraction #1 treated with vehicle or retinoic acid for 48 h ( n = 3). Scale bar, 50 μm. l Left: transplantation of cultured cells established from Plzf-mC/CreER fraction #1. Right: representative whole-mount IF of tubules 8 weeks post transplant demonstrating formation of mCherry+ colonies ( n = 5 recipients). Comparable spermatogenic capacity was observed upon transplantation of an independent line (3.90 colonies/10 5 cells; n = 4 recipient testes). Scale bar, 100 μm. Significance was calculated by two-tailed Student’s t -test (** P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Transgenic Assay, Mouse Assay, Flow Cytometry, Cytometry, Injection, Expressing, Quantitative RT-PCR, In Vitro, Activity Assay, Isolation, Transplantation Assay, Cell Culture, Two Tailed Test

    Comparative analysis of reporter gene expression in spermatogonia. a , b Representative whole-mount IF of adult (8–10 weeks post natal) Plzf-mC/CreER; Sox2 GFP ( a ) and Plzf-mC/CreER; Oct4-GFP ( b ) seminiferous tubules. Inset panels show individual immunostaining within indicated area at higher magnification. Tubule staging and select A s and A pr are indicated. Scale bars, 50 μm. c Representative flow cytometry analysis of fixed and permeabilized testis cells from 1 of 3 Oct4-GFP and wild-type (WT) control adults. PLZF+ cell population is shown. Percentages of cells contained within gates are indicated. d Quantification of flow cytometry results from c . Graph indicates percentage of A undiff (PLZF+ c-KIT−) and cells initiating differentiation (PLZF+ c-KIT+) expressing GFP in Oct4-GFP adults. Horizontal bars indicate mean values ( n = 3 mice). e Graph shows percentage of GFRα1+ and SOX3+ spermatogonia positive for GFP in whole-mount seminiferous tubules of Oct4-GFP adults. Spermatogonial identity was confirmed by SALL4 counterstain. Horizontal bars indicate mean values ( n = 3 mice, > 200 cells scored per data point). f Representative whole-mount IF of adult Oct4-GFP seminiferous tubules for indicated markers ( n = 3 mice). Select A undiff cells are indicated. Scale bar, 50 μm. g Scheme summarizing expression patterns of indicated genes and transgenic reporters plus changes in cell morphology during spermatogonial differentiation. Markers used to isolate different spermatogonial populations are indicated. h Isolation of Oct4-GFP− and Oct4-GFP+ A undiff from Plzf-mC/CreER; Oct4-GFP adults by flow cytometry. Percentage of cells in each gate from a representative sample is indicated ( n = 6 mice). i Oct4-GFP− and GFP+ adult A undiff fractions were transplanted into recipients and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative colonies. PLZF counterstain confirms A undiff and spermatogonial identity. Panels show higher magnification details of indicated areas. Scale bar, 100 μm. Graph shows colony-forming efficiency of Oct4-GFP+ and GFP− A undiff fractions. Data is presented as mean number of colonies per 10 5 donor cells ± s.e.m. ( n = 7 recipient testes for Oct4-GFP− cells and n = 6 for Oct4-GFP+ cells). Donor cells were pooled from 2 Plzf-mC/CreER; Oct4-GFP adults. Significance was calculated by two-tailed Student’s t -test (* P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: Comparative analysis of reporter gene expression in spermatogonia. a , b Representative whole-mount IF of adult (8–10 weeks post natal) Plzf-mC/CreER; Sox2 GFP ( a ) and Plzf-mC/CreER; Oct4-GFP ( b ) seminiferous tubules. Inset panels show individual immunostaining within indicated area at higher magnification. Tubule staging and select A s and A pr are indicated. Scale bars, 50 μm. c Representative flow cytometry analysis of fixed and permeabilized testis cells from 1 of 3 Oct4-GFP and wild-type (WT) control adults. PLZF+ cell population is shown. Percentages of cells contained within gates are indicated. d Quantification of flow cytometry results from c . Graph indicates percentage of A undiff (PLZF+ c-KIT−) and cells initiating differentiation (PLZF+ c-KIT+) expressing GFP in Oct4-GFP adults. Horizontal bars indicate mean values ( n = 3 mice). e Graph shows percentage of GFRα1+ and SOX3+ spermatogonia positive for GFP in whole-mount seminiferous tubules of Oct4-GFP adults. Spermatogonial identity was confirmed by SALL4 counterstain. Horizontal bars indicate mean values ( n = 3 mice, > 200 cells scored per data point). f Representative whole-mount IF of adult Oct4-GFP seminiferous tubules for indicated markers ( n = 3 mice). Select A undiff cells are indicated. Scale bar, 50 μm. g Scheme summarizing expression patterns of indicated genes and transgenic reporters plus changes in cell morphology during spermatogonial differentiation. Markers used to isolate different spermatogonial populations are indicated. h Isolation of Oct4-GFP− and Oct4-GFP+ A undiff from Plzf-mC/CreER; Oct4-GFP adults by flow cytometry. Percentage of cells in each gate from a representative sample is indicated ( n = 6 mice). i Oct4-GFP− and GFP+ adult A undiff fractions were transplanted into recipients and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative colonies. PLZF counterstain confirms A undiff and spermatogonial identity. Panels show higher magnification details of indicated areas. Scale bar, 100 μm. Graph shows colony-forming efficiency of Oct4-GFP+ and GFP− A undiff fractions. Data is presented as mean number of colonies per 10 5 donor cells ± s.e.m. ( n = 7 recipient testes for Oct4-GFP− cells and n = 6 for Oct4-GFP+ cells). Donor cells were pooled from 2 Plzf-mC/CreER; Oct4-GFP adults. Significance was calculated by two-tailed Student’s t -test (* P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Expressing, Immunostaining, Flow Cytometry, Cytometry, Mouse Assay, Transgenic Assay, Isolation, Two Tailed Test

    Characterization of PDX1+ spermatogonia. a Representative whole-mount IF of WT (top 3 rows, n = 3 mice), Oct4-GFP and Pdx1 GFP adult tubules ( n = 2 mice per genotype). Antibodies to PDX1 and GFRα1 are goat polyclonals and distinguished by nuclear vs . cell surface staining respectively. Scale bars, 50 μm. b Mean percentage of GFRα1+ cells/chains PDX1+ ± s.e.m. from WT analysis in a ( n = 4 mice, > 250 cells/chains per sample). c Representative whole-mount IF of adult Plzf-mC/CreER; Z/EG tubules D3 post-TAM ( n = 2 mice). Arrowheads: PDX1+ A s . Inset shows detail of indicated area. Scale bar, 50 μm. d Representative IF of adult WT testis sections ( n = 4 mice). Insets: details of selected areas. EOMES+ GFRα1+ (arrowheads) and EOMES− GFRα1+ cells (bracket) are shown. Tubule stage is indicated. Scale bar, 50 μm. e Representative IF of adult Oct4-GFP testis section ( n = 2 mice). Insets: immunostaining within indicated area. EOMES+ GFP− (arrowheads) and EOMES− GFP+ (bracket) spermatogonia are indicated. Asterisks: autofluorescent interstitium. Scale bar, 50 μm. f Representative flow cytometry of fixed and permeabilized testis cells from Oct4-GFP adults ( n = 2). PLZF+ cells shown. g Representative IF of adult WT testis sections ( n = 4 mice). Insets: immunostaining within indicated area. PDX1+ EOMES+ spermatogonia are indicated (arrowheads). Tubule stage is shown. Asterisk: autofluorescent interstitium. Scale bar, 50 μm. h Representative flow cytometry of fixed and permeabilized WT adult testis cells ( n = 4). Mean numbers of EOMES+ and PDX1+ cells in PDX1+ and EOMES+ gates respectively are shown ± s.e.m. i Representative flow cytometry for KI67 in indicated PLZF+ populations of fixed and permeabilized adult WT testis cells ( n = 4 mice). j Quantification of flow cytometry from i . Percentage of indicated PLZF+ fractions KI67+ . Horizontal bars: mean values ( n = 4 mice). k Representative IF of adult WT testis sections demonstrating PDX1+ A undiff localisation (arrowheads) within tubules ( n = 4 mice). Asterisks: autofluorescent interstitium. Tubule stages are shown. Scale bar, 50 μm. l Quantification of spermatogonial localisation from k . Mean values ± s.e.m. are shown ( n = 4 mice, 56–95 tubule sections per mouse). Significance vs. tubule-tubule localisation is indicated. m Representative IF of Id4 IRES-GFP adult testis sections ( n = 4 mice). Insets show immunostaining within indicated area. Arrowheads: PDX1+ EOMES+ GFP+ spermatogonia. Asterisk: PDX1 low EOMES+ GFP+ cell. Tubule stage is shown. Scale bar, 50 μm. n Representative flow cytometry of indicated PLZF+ populations of fixed and permeabilized adult Id4 IRES-GFP testis cells ( n = 4 mice). Mean numbers of PDX1+ and EOMES+ A undiff expressing GFP ± s.e.m. o Flow cytometry of Id4 IRES-GFP testis cells as in n . PLZF+ fractions are shown. Mean numbers of PLZF+ GFP+ cells positive for EOMES and PDX1 are indicated ± s.e.m. ( n = 4 mice). Significance was calculated by two-tailed Student’s t -test (*** P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: Characterization of PDX1+ spermatogonia. a Representative whole-mount IF of WT (top 3 rows, n = 3 mice), Oct4-GFP and Pdx1 GFP adult tubules ( n = 2 mice per genotype). Antibodies to PDX1 and GFRα1 are goat polyclonals and distinguished by nuclear vs . cell surface staining respectively. Scale bars, 50 μm. b Mean percentage of GFRα1+ cells/chains PDX1+ ± s.e.m. from WT analysis in a ( n = 4 mice, > 250 cells/chains per sample). c Representative whole-mount IF of adult Plzf-mC/CreER; Z/EG tubules D3 post-TAM ( n = 2 mice). Arrowheads: PDX1+ A s . Inset shows detail of indicated area. Scale bar, 50 μm. d Representative IF of adult WT testis sections ( n = 4 mice). Insets: details of selected areas. EOMES+ GFRα1+ (arrowheads) and EOMES− GFRα1+ cells (bracket) are shown. Tubule stage is indicated. Scale bar, 50 μm. e Representative IF of adult Oct4-GFP testis section ( n = 2 mice). Insets: immunostaining within indicated area. EOMES+ GFP− (arrowheads) and EOMES− GFP+ (bracket) spermatogonia are indicated. Asterisks: autofluorescent interstitium. Scale bar, 50 μm. f Representative flow cytometry of fixed and permeabilized testis cells from Oct4-GFP adults ( n = 2). PLZF+ cells shown. g Representative IF of adult WT testis sections ( n = 4 mice). Insets: immunostaining within indicated area. PDX1+ EOMES+ spermatogonia are indicated (arrowheads). Tubule stage is shown. Asterisk: autofluorescent interstitium. Scale bar, 50 μm. h Representative flow cytometry of fixed and permeabilized WT adult testis cells ( n = 4). Mean numbers of EOMES+ and PDX1+ cells in PDX1+ and EOMES+ gates respectively are shown ± s.e.m. i Representative flow cytometry for KI67 in indicated PLZF+ populations of fixed and permeabilized adult WT testis cells ( n = 4 mice). j Quantification of flow cytometry from i . Percentage of indicated PLZF+ fractions KI67+ . Horizontal bars: mean values ( n = 4 mice). k Representative IF of adult WT testis sections demonstrating PDX1+ A undiff localisation (arrowheads) within tubules ( n = 4 mice). Asterisks: autofluorescent interstitium. Tubule stages are shown. Scale bar, 50 μm. l Quantification of spermatogonial localisation from k . Mean values ± s.e.m. are shown ( n = 4 mice, 56–95 tubule sections per mouse). Significance vs. tubule-tubule localisation is indicated. m Representative IF of Id4 IRES-GFP adult testis sections ( n = 4 mice). Insets show immunostaining within indicated area. Arrowheads: PDX1+ EOMES+ GFP+ spermatogonia. Asterisk: PDX1 low EOMES+ GFP+ cell. Tubule stage is shown. Scale bar, 50 μm. n Representative flow cytometry of indicated PLZF+ populations of fixed and permeabilized adult Id4 IRES-GFP testis cells ( n = 4 mice). Mean numbers of PDX1+ and EOMES+ A undiff expressing GFP ± s.e.m. o Flow cytometry of Id4 IRES-GFP testis cells as in n . PLZF+ fractions are shown. Mean numbers of PLZF+ GFP+ cells positive for EOMES and PDX1 are indicated ± s.e.m. ( n = 4 mice). Significance was calculated by two-tailed Student’s t -test (*** P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Mouse Assay, Staining, Immunostaining, Flow Cytometry, Cytometry, Expressing, Two Tailed Test

    Identification and characterization of distinct A undiff populations. a Oct4-GFP− and Oct4-GFP+ A undiff fractions were isolated from Plzf-mC/CreER; Oct4-GFP adults for gene expression profiling by microarray. A undiff fraction is mCherry+ CD9+ c-KIT−. b Confirmation of gene expression signatures of Oct4-GFP− and Oct4-GFP+ A undiff by quantitative RT-PCR. Candidate genes were selected from microarray analysis of a . Expression levels are corrected to those of β-actin and normalized so mean value of GFP− or GFP+ fractions equals 1. Mean values from 3-5 mice ± s.e.m. are indicated. Genes enriched in Oct4-GFP− and Oct4-GFP+ populations are shown in separate groups. Control genes Plzf and Vasa are shown. Significance was calculated by two-tailed Student’s t -test (* P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: Identification and characterization of distinct A undiff populations. a Oct4-GFP− and Oct4-GFP+ A undiff fractions were isolated from Plzf-mC/CreER; Oct4-GFP adults for gene expression profiling by microarray. A undiff fraction is mCherry+ CD9+ c-KIT−. b Confirmation of gene expression signatures of Oct4-GFP− and Oct4-GFP+ A undiff by quantitative RT-PCR. Candidate genes were selected from microarray analysis of a . Expression levels are corrected to those of β-actin and normalized so mean value of GFP− or GFP+ fractions equals 1. Mean values from 3-5 mice ± s.e.m. are indicated. Genes enriched in Oct4-GFP− and Oct4-GFP+ populations are shown in separate groups. Control genes Plzf and Vasa are shown. Significance was calculated by two-tailed Student’s t -test (* P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Isolation, Expressing, Microarray, Quantitative RT-PCR, Mouse Assay, Two Tailed Test

    Interactome of GFP-MKRN1 RINGmut reveals putative ubiquitylation substrates. Experiments were performed using SILAC-based MS. Asymmetrical z-scores of combined SILAC ratios (n = 3 replicates) are shown. Proteins are detected in at least two out of three replicates. ( A ) Protein interactome of GFP-MKRN1 RINGmut in HEK293T cells analysed by quantitative mass spectrometry. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,097 protein groups were quantified in at least two out of three replicates ( Supplemental Table S1 ). MKRN1 and interesting candidate ubiquitylation targets are highlighted. ( B ) Quantitative comparison of the interactome of GFP-MKRN1 wt and GFP-MKRN1 RINGmut shows that potential ubiquitylation candidates identified in ( A ) are enriched in GFP-MKRN1 RINGmut over GFP-MKRN1 wt . Comparison reveals 137 proteins to be significantly enriched (MKRN1 RINGmut over MKRN1 wt with FDR

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: Interactome of GFP-MKRN1 RINGmut reveals putative ubiquitylation substrates. Experiments were performed using SILAC-based MS. Asymmetrical z-scores of combined SILAC ratios (n = 3 replicates) are shown. Proteins are detected in at least two out of three replicates. ( A ) Protein interactome of GFP-MKRN1 RINGmut in HEK293T cells analysed by quantitative mass spectrometry. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,097 protein groups were quantified in at least two out of three replicates ( Supplemental Table S1 ). MKRN1 and interesting candidate ubiquitylation targets are highlighted. ( B ) Quantitative comparison of the interactome of GFP-MKRN1 wt and GFP-MKRN1 RINGmut shows that potential ubiquitylation candidates identified in ( A ) are enriched in GFP-MKRN1 RINGmut over GFP-MKRN1 wt . Comparison reveals 137 proteins to be significantly enriched (MKRN1 RINGmut over MKRN1 wt with FDR

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: Mass Spectrometry, Transformation Assay

    MKRN1 binds upstream of A-stretches in 3’ UTRs. ( A ) MKRN1 predominantly binds in the 3’ UTR of protein-coding genes. Piecharts summarising the distribution of MKRN1 binding sites to different RNA biotypes (7,331 binding sites, left) and different regions within protein-coding transcripts (6,913 binding sites, right). ( B ) MKRN1 binding sites display a downstream enrichment of AAAA homopolymers. Frequency per nucleotide (nt) for four homopolymeric 4-mers in a 101-nt window around the midpoints of the top 20% MKRN1 binding sites (according to signal-over-background; see Material and methods). ( C ) MKRN1 crosslink events accumulate upstream of A-stretches. Metaprofile (top) shows the mean crosslink events per nt in a 201-nt window around the start position of 1,412 MKRN1-associated A-stretches in 3’ UTRs. Heatmap visualisation (bottom) displays crosslink events per nt (see colour scale) in a 101-nt window around the MKRN1-associated A-stretches. ( D ) MKRN1 binding site strength (signal-over-background, SOB) increases with length of longest continuous run of A’s (LCA) within the A-stretch. Mean and standard deviation of MKRN1 binding sites associated with A-stretches harbouring LCAs of increasing length (x-axis). MKRN1 binding sites without associated A-stretches are shown for comparison on the left. Number of binding sites in each category indicated as barchart above. ( E ) MKRN1 binds upstream of A-stretches in the 3’ UTR of the LARP1 gene. Genome browser view of GFP-MKRN1 iCLIP data showing crosslink events per nt (merged replicates, turquois) together with binding sites (lilac) and associated A-stretches (dark green).

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: MKRN1 binds upstream of A-stretches in 3’ UTRs. ( A ) MKRN1 predominantly binds in the 3’ UTR of protein-coding genes. Piecharts summarising the distribution of MKRN1 binding sites to different RNA biotypes (7,331 binding sites, left) and different regions within protein-coding transcripts (6,913 binding sites, right). ( B ) MKRN1 binding sites display a downstream enrichment of AAAA homopolymers. Frequency per nucleotide (nt) for four homopolymeric 4-mers in a 101-nt window around the midpoints of the top 20% MKRN1 binding sites (according to signal-over-background; see Material and methods). ( C ) MKRN1 crosslink events accumulate upstream of A-stretches. Metaprofile (top) shows the mean crosslink events per nt in a 201-nt window around the start position of 1,412 MKRN1-associated A-stretches in 3’ UTRs. Heatmap visualisation (bottom) displays crosslink events per nt (see colour scale) in a 101-nt window around the MKRN1-associated A-stretches. ( D ) MKRN1 binding site strength (signal-over-background, SOB) increases with length of longest continuous run of A’s (LCA) within the A-stretch. Mean and standard deviation of MKRN1 binding sites associated with A-stretches harbouring LCAs of increasing length (x-axis). MKRN1 binding sites without associated A-stretches are shown for comparison on the left. Number of binding sites in each category indicated as barchart above. ( E ) MKRN1 binds upstream of A-stretches in the 3’ UTR of the LARP1 gene. Genome browser view of GFP-MKRN1 iCLIP data showing crosslink events per nt (merged replicates, turquois) together with binding sites (lilac) and associated A-stretches (dark green).

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: Binding Assay, Standard Deviation

    MKRN1 interacts with translational regulators and other RBPs. ( A ) Overlap of the 53 significant interaction partners of GFP-MKRN1 wt in human HEK293T cells with previously published interactors of MKRN1 in mouse embryonic stem cells (mESC) ( Cassar et al. 2015 ). ( B ) GO terms enriched for the 53 MKRN1 interactors. P values (modified Fisher exact test, Benjamini-Hochberg correction) are depicted for all significant GO terms (corrected P value

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: MKRN1 interacts with translational regulators and other RBPs. ( A ) Overlap of the 53 significant interaction partners of GFP-MKRN1 wt in human HEK293T cells with previously published interactors of MKRN1 in mouse embryonic stem cells (mESC) ( Cassar et al. 2015 ). ( B ) GO terms enriched for the 53 MKRN1 interactors. P values (modified Fisher exact test, Benjamini-Hochberg correction) are depicted for all significant GO terms (corrected P value

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: Modification

    MKRN1 stalls ribosomes at poly(A) sequences. ( A ) The dual fluorescence reporter harbours an N-terminal GFP, followed by a FLAG-SR-X linker and a C-terminal RFP, which are separated by P2A sites to ensure translation into three separate proteins ( Juszkiewicz and Hegde 2017 ). The resulting GFP:RFP ratio was determined using flow cytometry. The inserted fragment K(AAA) 20 encodes 20 lysines by repeating the codon AAA. The starting vector without insert (K 0 ) served as control. Schematic ribosomes illustrate translation of the respective reporter segments. ( B ) Ribosomes are efficiently stalled at K(AAA) 20 in HEK293T cells. Median RFP:GFP ratios, normalised to K 0 , are shown. Error bars represent standard deviation of the mean (s.d.m., n = 6 replicates). P value indicated above (paired two-tailed t-test). ( C ) Ribosomes fail to stall in the absence of MKRN1. HEK293T cells were transfected with control siRNA or siRNAs targeting MKRN1 (KD1 and KD2) or ZNF598 for 24 h, followed by transfection of the reporter plasmids for 48 h. Western blots for KDs are shown in Supplemental Fig. S6B . RFP and GFP signals were analysed by flow cytometry. Median RFP:GFP ratios, normalised to K 0 in control, are shown. Error bars represent s.d.m.; P values indicated above (paired two-tailed t-test, Benjamini-Hochberg correction, n ≥ 6 replicates; ns, not significant).

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: MKRN1 stalls ribosomes at poly(A) sequences. ( A ) The dual fluorescence reporter harbours an N-terminal GFP, followed by a FLAG-SR-X linker and a C-terminal RFP, which are separated by P2A sites to ensure translation into three separate proteins ( Juszkiewicz and Hegde 2017 ). The resulting GFP:RFP ratio was determined using flow cytometry. The inserted fragment K(AAA) 20 encodes 20 lysines by repeating the codon AAA. The starting vector without insert (K 0 ) served as control. Schematic ribosomes illustrate translation of the respective reporter segments. ( B ) Ribosomes are efficiently stalled at K(AAA) 20 in HEK293T cells. Median RFP:GFP ratios, normalised to K 0 , are shown. Error bars represent standard deviation of the mean (s.d.m., n = 6 replicates). P value indicated above (paired two-tailed t-test). ( C ) Ribosomes fail to stall in the absence of MKRN1. HEK293T cells were transfected with control siRNA or siRNAs targeting MKRN1 (KD1 and KD2) or ZNF598 for 24 h, followed by transfection of the reporter plasmids for 48 h. Western blots for KDs are shown in Supplemental Fig. S6B . RFP and GFP signals were analysed by flow cytometry. Median RFP:GFP ratios, normalised to K 0 in control, are shown. Error bars represent s.d.m.; P values indicated above (paired two-tailed t-test, Benjamini-Hochberg correction, n ≥ 6 replicates; ns, not significant).

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: Fluorescence, Flow Cytometry, Plasmid Preparation, Standard Deviation, Two Tailed Test, Transfection, Western Blot

    Interaction with PABP is required for MKRN1 RNA binding. ( A,B ) UV crosslinking experiments to measure the RNA binding capacity of GFP-MKRN1 wt and GFP-MKRN1 PAM2mut . Autoradiographs (top) and Western blots (bottom) show GFP-MKRN1/RNA complexes and GFP-MKRN1 protein, respectively, in the eluates from replicates 2 (with 4SU and UV crosslinking at 365 nm) ( A ) and 3 (with conventional UV crosslinking at 254 nm) ( B ). For calibration, input samples for GFP-MKRN1 wt were diluted to 75%, 50% and 25% prior to GFP AP. Note that samples were loaded in different order in ( B ). Quantifications are given below. Uncropped gel images are shown in Supplemental Fig. S10 .

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: Interaction with PABP is required for MKRN1 RNA binding. ( A,B ) UV crosslinking experiments to measure the RNA binding capacity of GFP-MKRN1 wt and GFP-MKRN1 PAM2mut . Autoradiographs (top) and Western blots (bottom) show GFP-MKRN1/RNA complexes and GFP-MKRN1 protein, respectively, in the eluates from replicates 2 (with 4SU and UV crosslinking at 365 nm) ( A ) and 3 (with conventional UV crosslinking at 254 nm) ( B ). For calibration, input samples for GFP-MKRN1 wt were diluted to 75%, 50% and 25% prior to GFP AP. Note that samples were loaded in different order in ( B ). Quantifications are given below. Uncropped gel images are shown in Supplemental Fig. S10 .

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: RNA Binding Assay, Western Blot

    MKRN1 is required to stall ribosomes at K(AAA) 20 in reporter assays. ( A ) Translation of dual fluorescence reporter plasmids was assessed by flow cytometry upon MKRN1 and/or ZNF598 KD. Median RFP:GFP ratios (normalised to K 0 in control KD) are shown for the reporter plasmids K 0 , K(AAA) 12 , K(AAA) 20 , and R(CGA) 10 . Error bars represent standard deviation of the mean (s.d.m., n ≥ 6 replicates; paired two-tailed t-test, Benjamini-Hochberg correction). Density plot of median RFP:GFP ratios of one replicate experiment with K(AAA) 20 with control or MKRN1 KD (two independent siRNAs, KD1 and KD2) or ZNF598 is shown on the right. KDs of MKRN1 and ZNF598 were assessed by Western blot (n = 3 replicates). Black arrowhead indicates ZNF598. Replicates 2 and 3, and uncropped gel images are shown in Supplemental Fig. S11A,B . ( C ) MKRN1 KD2 also reduces MKRN2 levels. MKRN1 KD1 and KD2 were performed for 72 h. Expression levels of MKRN1 and MKRN2 were assessed in relation to ß-actin levels by qPCR in MKRN1 KD (siRNA 1 and 2) and control KD. Error bars indicate s.d.m. (n = 2 replicates). ( D,E ) Cross-regulation of MKRN1 and ZNF598. ( D ) MKRN1 KD1 reduces endogenous ZNF598 protein levels. Effect of MKRN1 KD (KD1, siRNA 1 and KD2, siRNA 2) and ZNF598 KD for 72 h was assessed by Western blot for endogenous MKRN1 and ZNF598. Quantifications depict MKRN1 or ZNF598 expression levels in MKRN1 or ZNF598 KD over control KD condition, normalised to tubulin levels (n = 3 replicates). Replicates 2 and 3, and uncropped gel images are shown in Supplemental Fig. S11C,D . ( E ) ZNF598 overexpression reduces MKRN1 protein levels. Effect of ZNF598 and MKRN1 (wt and mutants) overexpression was tested after 48 h. Quantification as in ( D ). Uncropped gel images for all replicates are shown in Supplemental Fig. S11E,F .

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: MKRN1 is required to stall ribosomes at K(AAA) 20 in reporter assays. ( A ) Translation of dual fluorescence reporter plasmids was assessed by flow cytometry upon MKRN1 and/or ZNF598 KD. Median RFP:GFP ratios (normalised to K 0 in control KD) are shown for the reporter plasmids K 0 , K(AAA) 12 , K(AAA) 20 , and R(CGA) 10 . Error bars represent standard deviation of the mean (s.d.m., n ≥ 6 replicates; paired two-tailed t-test, Benjamini-Hochberg correction). Density plot of median RFP:GFP ratios of one replicate experiment with K(AAA) 20 with control or MKRN1 KD (two independent siRNAs, KD1 and KD2) or ZNF598 is shown on the right. KDs of MKRN1 and ZNF598 were assessed by Western blot (n = 3 replicates). Black arrowhead indicates ZNF598. Replicates 2 and 3, and uncropped gel images are shown in Supplemental Fig. S11A,B . ( C ) MKRN1 KD2 also reduces MKRN2 levels. MKRN1 KD1 and KD2 were performed for 72 h. Expression levels of MKRN1 and MKRN2 were assessed in relation to ß-actin levels by qPCR in MKRN1 KD (siRNA 1 and 2) and control KD. Error bars indicate s.d.m. (n = 2 replicates). ( D,E ) Cross-regulation of MKRN1 and ZNF598. ( D ) MKRN1 KD1 reduces endogenous ZNF598 protein levels. Effect of MKRN1 KD (KD1, siRNA 1 and KD2, siRNA 2) and ZNF598 KD for 72 h was assessed by Western blot for endogenous MKRN1 and ZNF598. Quantifications depict MKRN1 or ZNF598 expression levels in MKRN1 or ZNF598 KD over control KD condition, normalised to tubulin levels (n = 3 replicates). Replicates 2 and 3, and uncropped gel images are shown in Supplemental Fig. S11C,D . ( E ) ZNF598 overexpression reduces MKRN1 protein levels. Effect of ZNF598 and MKRN1 (wt and mutants) overexpression was tested after 48 h. Quantification as in ( D ). Uncropped gel images for all replicates are shown in Supplemental Fig. S11E,F .

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: Fluorescence, Flow Cytometry, Standard Deviation, Two Tailed Test, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Over Expression

    MKRN1 binds at poly(A) tails. ( A ) MKRN1 binds near the polyadenylation site of the SRSF4 gene. Genome browser view as in Fig. 2E . ( B ) Unmapped MKRN1 iCLIP reads display increased A-content (more than half of all nucleotides in the read), evidencing poly(A) tail binding. Cumulative fraction of iCLIP reads (y-axis, merged replicates) that could not be mapped to the human genome (see Materials and methods) and show at least a given A-content (x-axis). iCLIP data for the unrelated RBP HNRNPH ( Braun et al. 2018 ) are shown for comparison. ( C ) MKRN1 crosslink events increase towards 3’UTR ends. Metaprofile shows the sum of crosslink events per nt in a 2001-nt window around annotated polyadenylation sites of transcripts with > 1 kb 3’ UTRs (n = 11,257). ( D ) Overall RNA binding of MKRN1 is strongly reduced when abrogating PABP interaction. Audioradiograph (left) of UV crosslinking experiments (replicate 1, with 4SU and UV crosslinking at 365 nm; replicates 2 and 3 in Supplemental Fig. S5 ) comparing GFP-MKRN1 PAM2mut with GFP-MKRN1 wt at different dilution steps for calibration. Quantification of radioactive signal of protein-RNA complexes and corresponding Western blots shown on the right. Uncropped gel images are shown in Supplemental Fig. S10 .

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: MKRN1 binds at poly(A) tails. ( A ) MKRN1 binds near the polyadenylation site of the SRSF4 gene. Genome browser view as in Fig. 2E . ( B ) Unmapped MKRN1 iCLIP reads display increased A-content (more than half of all nucleotides in the read), evidencing poly(A) tail binding. Cumulative fraction of iCLIP reads (y-axis, merged replicates) that could not be mapped to the human genome (see Materials and methods) and show at least a given A-content (x-axis). iCLIP data for the unrelated RBP HNRNPH ( Braun et al. 2018 ) are shown for comparison. ( C ) MKRN1 crosslink events increase towards 3’UTR ends. Metaprofile shows the sum of crosslink events per nt in a 2001-nt window around annotated polyadenylation sites of transcripts with > 1 kb 3’ UTRs (n = 11,257). ( D ) Overall RNA binding of MKRN1 is strongly reduced when abrogating PABP interaction. Audioradiograph (left) of UV crosslinking experiments (replicate 1, with 4SU and UV crosslinking at 365 nm; replicates 2 and 3 in Supplemental Fig. S5 ) comparing GFP-MKRN1 PAM2mut with GFP-MKRN1 wt at different dilution steps for calibration. Quantification of radioactive signal of protein-RNA complexes and corresponding Western blots shown on the right. Uncropped gel images are shown in Supplemental Fig. S10 .

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: Binding Assay, RNA Binding Assay, Western Blot

    MKRN1 interacts with PABP and other regulators of translation and RNA stability. ( A ) Protein interactome of GFP-MKRN1 wt in HEK293T cells analysed by quantitative MS-based proteomics. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,100 protein groups were quantified in at least two out of three replicate experiments. MKRN1 and significant interactors are highlighted (FDR

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: MKRN1 interacts with PABP and other regulators of translation and RNA stability. ( A ) Protein interactome of GFP-MKRN1 wt in HEK293T cells analysed by quantitative MS-based proteomics. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,100 protein groups were quantified in at least two out of three replicate experiments. MKRN1 and significant interactors are highlighted (FDR

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: qMS Based, Transformation Assay