Figure 8B , C . 293T cells were transfected with the indicated DNAs and immunoprecipitated with an anti - Flag antibody or pulled down with streptavidin - binding beads . D , E . 293T cells were transfected as indicated and immunoprecipitated with an anti - HA antibody . LATS2 immune complexes were subjected to a kinase assay with His - YAP and cold ATP . F . Lats1 - / - ; Lats2 fl / fl ; SV40 mouse embryonic fibroblasts complemented with vector , LATS2 WT or ΔUBA mutant were infected with empty ( control ) or Zeocin - Cre retroviruses and selected with 400 μg / ml of Zeocin for 4 days . Harvested cells were analyzed by Western blotting for the indicated proteins .

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Figure 8B, C. 293T cells were transfected with the indicated DNAs and immunoprecipitated with an anti-Flag antibody or pulled down with streptavidin-binding beads.D, E. 293T cells were transfected as indicated and immunoprecipitated with an anti-HA antibody. LATS2 immune complexes were subjected to a kinase assay with His-YAP and cold ATP.F. Lats1 -/-; Lats2 fl/fl; SV40 mouse embryonic fibroblasts complemented with vector, LATS2 WT or ΔUBA mutant were infected with empty (control) or Zeocin-Cre retroviruses and selected with 400 μg/ml of Zeocin for 4 days. Harvested cells were analyzed by Western blotting for the indicated proteins.


Figure 1Paneth cell quantification on Lgr5 - DTReGFP duodenum ( Duo ) and Ileum ( Ile ) . Left panel : Representative images of Lendrum ' s staining that evidence Paneth cell granules . Arrows show differentiated Paneth cells . Right panel : quantification of the Paneth cells per 10 intervilli regions . Each symbol indicates the value for a given embryo . Expression analysis by qRT - PCR of the indicated Paneth cell markers in Lgr5 - DTReGFP ileums ( n = 8 WT , 12 HE , 10 KO ) . X - gal staining in Axin2Lac / + - Lgr5 - GFP - CreERT2 WT or KO ileums . Gene expression analysis by qRT - PCR of stem cell markers in Lgr5 - DTReGFP ileums ( n = 3 WT , 6 HE , 6 KO ) . FACS quantification of the number of eGFP + ve ( ISC ) cells per small intestine at the indicated developmental stages in Lgr5 HEs and Lgr5 KOs . Each symbol indicates the value for a given embryo . Mean value for each group is depicted on the graph . Plating efficiency of ex vivo cultured E18 . 5 small intestines quantified 6 days after initial seeding . Each symbol indicates the value for a given embryo . Influence of mouse Rspondin 1 concentration on growth of replated Lgr5 - DTReGFP WT and Lgr5 KO organoids after 5 days of culture .

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Figure 1Paneth cell quantification on Lgr5-DTReGFP duodenum (Duo) and Ileum (Ile). Left panel: Representative images of Lendrum's staining that evidence Paneth cell granules. Arrows show differentiated Paneth cells. Right panel: quantification of the Paneth cells per 10 intervilli regions. Each symbol indicates the value for a given embryo.Expression analysis by qRT-PCR of the indicated Paneth cell markers in Lgr5-DTReGFP ileums (n= 8 WT, 12 HE, 10 KO).X-gal staining in Axin2Lac/+-Lgr5-GFP-CreERT2 WT or KO ileums.Gene expression analysis by qRT-PCR of stem cell markers in Lgr5-DTReGFP ileums (n= 3 WT, 6 HE, 6 KO).FACS quantification of the number of eGFP+ve (ISC) cells per small intestine at the indicated developmental stages in Lgr5 HEs and Lgr5 KOs. Each symbol indicates the value for a given embryo. Mean value for each group is depicted on the graph.Plating efficiency of ex vivo cultured E18.5 small intestines quantified 6 days after initial seeding. Each symbol indicates the value for a given embryo.Influence of mouse Rspondin 1 concentration on growth of replated Lgr5-DTReGFP WT and Lgr5 KO organoids after 5 days of culture.


Figure 2Paneth cell quantification on Lgr5 - DTReGFP ileums . Each symbol indicates the value for a given embryo . Immunofluorescence showing Olfm4 + ve cells in Lgr5 - DTReGFP duodenums . Cell membranes are shown with b - catenin and nuclei were counterstained with DAPI . Quantification of Olfm4 + ve cells per intervilli regions in duodenum and ileum . Each symbol indicates the value for a given embryo . Axin2 expression levels detected by RNAscope on Lgr5 - DTReGFP proximal intestine . Gene expression analysis by qRT - PCR of stem cell and differentiation markers in Lgr5 - DTReGFP ileums ( vehicle - treated : 7 WT and 7 KO ; LGK974 - treated : 3 WT and 7 KO ) .

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Figure 2Paneth cell quantification on Lgr5-DTReGFP ileums. Each symbol indicates the value for a given embryo.Immunofluorescence showing Olfm4+ve cells in Lgr5-DTReGFP duodenums. Cell membranes are shown with b-catenin and nuclei were counterstained with DAPI. Quantification of Olfm4+ve cells per intervilli regions in duodenum and ileum. Each symbol indicates the value for a given embryo.Axin2 expression levels detected by RNAscope on Lgr5-DTReGFP proximal intestine.Gene expression analysis by qRT-PCR of stem cell and differentiation markers in Lgr5-DTReGFP ileums (vehicle-treated: 7 WT and 7 KO; LGK974-treated: 3 WT and 7 KO).


Figure 3Representative pictures of lineage tracing in Lgr5 - expressing or Lgr5 cKO intestine after 10 days of chase and quantification of the number of traced clones per recombined surface at PN18 ( ileum ) . Each symbol indicates the value for a given mouse . Representative immunofluorescence pictures showing Paneth cells ( Plyz + ve ) in traced clones ( RFP + ve ) from control ( Lgr5Cre / + ) and cKO ( Lgr5Cre / flox ) PN18 and adult ileums . Arrow heads point to Plyz + ve cells in traced clones . Quantification of the number of Paneth cell number per recombined RFP + ve crypt on control and cKO ileums in PN18 . Each symbol indicates the value for a given mouse .

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Figure 3Representative pictures of lineage tracing in Lgr5-expressing or Lgr5 cKO intestine after 10 days of chase and quantification of the number of traced clones per recombined surface at PN18 (ileum). Each symbol indicates the value for a given mouse.Representative immunofluorescence pictures showing Paneth cells (Plyz+ve) in traced clones (RFP+ve) from control (Lgr5Cre/+) and cKO (Lgr5Cre/flox) PN18 and adult ileums. Arrow heads point to Plyz+ve cells in traced clones. Quantification of the number of Paneth cell number per recombined RFP+ve crypt on control and cKO ileums in PN18. Each symbol indicates the value for a given mouse.


Figure 4ISC ( eGFP + ve ) cells were FACS - sorted from Lgr5 - DTReGFP embryos at E16 . 5 and subjected to RNAseq analysis . Heatmap of differentially regulated genes in HE and KO ISCs at E16 . 5 . Biological replicates for RNAseq experiments on ISC Lgr5 - DTReGFP E16 . 5 independent embryonic pools , each obtained from independent litters ( n = 2 HE , 2 KO ) Compared Hallmarks for upregulated and downregulated genes in the transcriptome of E16 . 5 Lgr5 KOs vs E16 . 5 HEs , adult HEs vs E18 . 5 HE embryos and adult Lgr5 - Cre cKO vs HEs . * Asterisks refer to given pathways for which differentially modulated genes were found upregulated and downregulated . Right panels : GSEA showing enrichment of the epithelial to mesenchymal transition ( EMT ) data set in E16 . 5 Lgr5 KOs vs E16 . 5 HEs , adult HEs vs E18 . 5 HE embryos , and adult Lgr5 cKO vs adult HE modulated genes . NES : Normalized Enrichment Score . Biological replicates for RNAseq experiments on ISC Lgr5 - DTReGFP E16 . 5 independent embryonic pools , each obtained from independent litters ( n = 2 HE , 2 KO ) ; Lgr5 - DTReGFP E18 . 5 individual embryos ( n = 2 HE ) and Lgr5 - DTReGFP adult individual animals ( n = 2 HE ) ; Lgr5 - Cre / flox adult individual animals ( n = 2 HE , 3 cKO ) . Expression levels of genes commonly downregulated in ISCs of Lgr5 KOs vs Lgr5 HEs at E16 . 5 and Lgr5 adult vs E18 . 5 HEs . RPKM : reads per kilobase of transcript per million mapped reads . Below : GeneOntology analysis of downregulated genes identified in the Epithelial Mesenchymal Transition Gene Set ( Hallmark MSigDB collection ) . Biological replicates for RNAseq experiments on ISC Lgr5 - DTReGFP E16 . 5 independent embryonic pools , each obtained from independent litters ( n = 2 HE , 2 KO ) ; Lgr5 - DTReGFP E18 . 5 individual embryos ( n = 2 HE ) and Lgr5 - DTReGFP adult individual animals ( n = 2 HE ) ; Lgr5 - Cre / flox adult individual animals ( n = 2 HE , 3 cKO ) . Data are represented as means ± sem ( C ) . Expression profile of Col1a1 , Zeb2 and Cxcl10 genes detected by RNAscope in embryonic and adult intestines . Insets with higher magnification are depicted on the right of each corresponding picture , boundaries between epithelium and mesenchyme are shown with dotted lines and arrows point to epithelial expression .

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Figure 4ISC (eGFP+ve) cells were FACS-sorted from Lgr5-DTReGFP embryos at E16.5 and subjected to RNAseq analysis. Heatmap of differentially regulated genes in HE and KO ISCs at E16.5. Biological replicates for RNAseq experiments on ISC Lgr5-DTReGFP E16.5 independent embryonic pools, each obtained from independent litters (n= 2 HE, 2 KO)Compared Hallmarks for upregulated and downregulated genes in the transcriptome of E16.5 Lgr5 KOs vs E16.5 HEs, adult HEs vs E18.5 HE embryos and adult Lgr5-Cre cKO vs HEs. * Asterisks refer to given pathways for which differentially modulated genes were found upregulated and downregulated. Right panels: GSEA showing enrichment of the epithelial to mesenchymal transition (EMT) data set in E16.5 Lgr5 KOs vs E16.5 HEs, adult HEs vs E18.5 HE embryos, and adult Lgr5 cKO vs adult HE modulated genes. NES: Normalized Enrichment Score. Biological replicates for RNAseq experiments on ISC Lgr5-DTReGFP E16.5 independent embryonic pools, each obtained from independent litters (n= 2 HE, 2 KO); Lgr5-DTReGFP E18.5 individual embryos (n=2 HE) and Lgr5-DTReGFP adult individual animals (n=2 HE); Lgr5-Cre/flox adult individual animals (n= 2 HE, 3 cKO).Expression levels of genes commonly downregulated in ISCs of Lgr5 KOs vs Lgr5 HEs at E16.5 and Lgr5 adult vs E18.5 HEs. RPKM: reads per kilobase of transcript per million mapped reads. Below: GeneOntology analysis of downregulated genes identified in the Epithelial Mesenchymal Transition Gene Set (Hallmark MSigDB collection). Biological replicates for RNAseq experiments on ISC Lgr5-DTReGFP E16.5 independent embryonic pools, each obtained from independent litters (n= 2 HE, 2 KO); Lgr5-DTReGFP E18.5 individual embryos (n=2 HE) and Lgr5-DTReGFP adult individual animals (n=2 HE); Lgr5-Cre/flox adult individual animals (n= 2 HE, 3 cKO). Data are represented as means ± sem (C).Expression profile of Col1a1, Zeb2 and Cxcl10 genes detected by RNAscope in embryonic and adult intestines. Insets with higher magnification are depicted on the right of each corresponding picture, boundaries between epithelium and mesenchyme are shown with dotted lines and arrows point to epithelial expression.


Figure 5Expression of Axin2 , Lgr5 , Lgr4 , Rspo1 , Rspo2 , Rspo3 and Rspo4 genes was analyzed on E15 , E16 . 5 and adult stages by RNAscope . Arrows evidence expressing cells .

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Figure 5Expression of Axin2, Lgr5, Lgr4, Rspo1, Rspo2, Rspo3 and Rspo4 genes was analyzed on E15, E16.5 and adult stages by RNAscope. Arrows evidence expressing cells.


Figure 6Representative pictures showing the influence of mouse Rspondin type and concentration on growth of replated Lgr5 - DTReGFP WT and Lgr5 KO organoids after 4 days of culture . Arrows show alive elements and arrow heads dead elements . Organoid morphology depends on Rspondin ligand type and concentration . Left panel : growth quantified as the percentage of elements present at day1 surviving at day4 . A mean of 100 elements were analyzed per sample and per condition ( n : 4 WT , 2 KO organoid culture samples , each originating from a given embryo ) . Right panel : organoid complexity measured by branching coefficient . Each symbol indicates the value of a given organoid ( analysis was done on 4 WT and 2 KO organoid culture samples , each originating from a given embryo ) . Heatmap showing the impact of Rspondin type and concentration ( ng / ml ) on relative Wnt target gene expression ( list from the Sansom APC data set ) in Lgr5 - DTReGFP WT and KO organoids analyzed by RNAseq ( n = 3 WT and 2 organoid culture samples , each originating from a given embryo ) . Gene expression analysis by qRT - PCR of stem cell markers in Lgr5 - DTREeGFP WT and KO organoids after 5 days of culture ( expression relative to reference genes ) . Each symbol indicates the value for an organoid culture originated from a given embryo . Immunofluorescence showing Olfm4 + ve cells in Lgr5 - DTReGFP WT and KO organoids cultured in Rspo2 conditions . Epithelium is delineated with b - catenin and nuclei counterstained with DAPI ( Merge ) . Insets : white arrowheads evidence Olfm4 + ve cells concentrated in organoid protrusions . Quantification of Olfm4 + ve area with respect to the total epithelial area . Each symbol indicates the value for an organoid culture originating from a given embryo .

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Figure 6Representative pictures showing the influence of mouse Rspondin type and concentration on growth of replated Lgr5-DTReGFP WT and Lgr5 KO organoids after 4 days of culture. Arrows show alive elements and arrow heads dead elements.Organoid morphology depends on Rspondin ligand type and concentration. Left panel: growth quantified as the percentage of elements present at day1 surviving at day4. A mean of 100 elements were analyzed per sample and per condition (n: 4 WT, 2 KO organoid culture samples, each originating from a given embryo). Right panel: organoid complexity measured by branching coefficient. Each symbol indicates the value of a given organoid (analysis was done on 4 WT and 2 KO organoid culture samples, each originating from a given embryo).Heatmap showing the impact of Rspondin type and concentration (ng/ml) on relative Wnt target gene expression (list from the Sansom APC data set) in Lgr5-DTReGFP WT and KO organoids analyzed by RNAseq (n= 3 WT and 2 organoid culture samples, each originating from a given embryo).Gene expression analysis by qRT-PCR of stem cell markers in Lgr5-DTREeGFP WT and KO organoids after 5 days of culture (expression relative to reference genes). Each symbol indicates the value for an organoid culture originated from a given embryo.Immunofluorescence showing Olfm4+ve cells in Lgr5-DTReGFP WT and KO organoids cultured in Rspo2 conditions. Epithelium is delineated with b-catenin and nuclei counterstained with DAPI (Merge). Insets: white arrowheads evidence Olfm4+ve cells concentrated in organoid protrusions. Quantification of Olfm4+ve area with respect to the total epithelial area. Each symbol indicates the value for an organoid culture originating from a given embryo.


Figure 1A Representative images of alkaline phosphatase ( ALP ) , Alizarin Red S ( ARS ) and Van Gieson ' s staining of human MSCs B , C Quantitative analyses of the ALP activity and calcium mineralization . Results are shown as mean ± SEM ; n = 6 ; * * : p < 0 . 01 and * * * : p < 0 . 001 by t test D , E Quantitative RT - PCR and western blot analyses of the expression of osteogenic markers . Cells were cultured in osteogenic medium for 5 days . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by t test F H & E staining of the MSCs - mediated ectopic bone formation . Scale bar , 50 μm G Quantitative analysis of bone volume versus total tissue volume ( BV / TV ) . Results are shown as mean ± SEM ; n = 6 . * : p < 0 . 05 by t

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Figure 1A Representative images of alkaline phosphatase (ALP), Alizarin Red S (ARS) and Van Gieson's staining of human MSCs B, C Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=6; **: p<0.01 and ***: p<0.001 by t test D, E Quantitative RT-PCR and western blot analyses of the expression of osteogenic markers. Cells were cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by t test F H&E staining of the MSCs-mediated ectopic bone formation. Scale bar, 50 μm G Quantitative analysis of bone volume versus total tissue volume (BV/TV). Results are shown as mean ± SEM; n=6. *: p<0.05 by t test


Figure 4 A Scatter plot of RNA - seq expression analysis . 508 genes were upregulated and 391 genes were downregulated . Human MSCs were transfected with USP34 or control siRNAs and cultured in osteogenic medium for 2 days . Two biological replicates per group C Heatmap of representative genes associated with BMP2 signaling pathway D Relative activity of BRE luciferase assay . 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours . Results are shown as mean ± SEM ; n = 3 . * * * : p < 0 . 001 by t testE Immunoblot analysis of BMP2 - induce Sp7 expression . Prx1 - Cre ; Usp34f / f and Usp34f / f control MSCs were starved overnight and then stimulated with 100 ng / ml BMP2 for 24 hoursF Quantitative RT - PCR analysis of BMP2 - induce Sp7 transcription . Cells were depleted from serum overnight before BMP2 stimulation . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 and * * : p < 0 . 01 by t testG Representative images of ALP and ARS staining . MSCs were cultured with normal medium supplemented with 100 ng / ml BMP2 for 10 and 21 days , respectivelyH , I Quantitative analyses of the ALP activity ( n = 4 ) and calcium mineralization ( n = 5 ) . Results are shown as mean ± SEM ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by t test J , K Representative microCT images and quantification of newly formed bone in response to BMP2 . Results are shown as mean ± SEM ; n = 8 . * : p < 0 . 05 by t test . Scale bar , 2500 μm L H & E staining of BMP2 - induced newly formed bone . Scale bar , 50 μmM Quantitative analysis of bone volume versus total tissue volume ( BV / TV ) . Results are shown as mean ± SEM ; n = 8 ; * * : p < 0 . 01 by t test N H & E staining of calvarial organ cultures stimulated with or without 100 ng / ml BMP2 . Red dotted lines indicate the interface between original and newly formed bone . Scale bar , 50 μmO Quantitative analysis of newly formed bone volume versus original bone volume ( % ) . Results are shown as mean ± SEM ; n = 8 ; * * : p < 0 . 01 by t

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 A Scatter plot of RNA-seq expression analysis. 508 genes were upregulated and 391 genes were downregulated. Human MSCs were transfected with USP34 or control siRNAs and cultured in osteogenic medium for 2 days. Two biological replicates per group  C Heatmap of representative genes associated with BMP2 signaling pathway D Relative activity of BRE luciferase assay. 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours. Results are shown as mean ± SEM; n=3. ***: p<0.001 by t testE Immunoblot analysis of BMP2-induce Sp7 expression. Prx1-Cre;Usp34f/f and Usp34f/f control MSCs were starved overnight and then stimulated with 100 ng/ml BMP2 for 24 hoursF Quantitative RT-PCR analysis of BMP2-induce Sp7 transcription. Cells were depleted from serum overnight before BMP2 stimulation. Results are shown as mean ± SEM; n=3; *: p<0.05 and **: p<0.01 by t testG Representative images of ALP and ARS staining. MSCs were cultured with normal medium supplemented with 100 ng/ml BMP2 for 10 and 21 days, respectivelyH, I Quantitative analyses of the ALP activity (n=4) and calcium mineralization (n=5). Results are shown as mean ± SEM; *: p<0.05, **: p<0.01 and ***: p<0.001 by t test J, K Representative microCT images and quantification of newly formed bone in response to BMP2. Results are shown as mean ± SEM; n=8. *: p<0.05 by t test. Scale bar, 2500 μm L H&E staining of BMP2-induced newly formed bone. Scale bar, 50 μmM Quantitative analysis of bone volume versus total tissue volume (BV/TV). Results are shown as mean ± SEM; n=8; **: p<0.01 by t test N H&E staining of calvarial organ cultures stimulated with or without 100 ng/ml BMP2. Red dotted lines indicate the interface between original and newly formed bone. Scale bar, 50 μmO Quantitative analysis of newly formed bone volume versus original bone volume (%). Results are shown as mean ± SEM; n=8; **: p<0.01 by t test


Figure 6A Co - immunoprecipitation of USP34 with ectopically expressed FLAG - tagged Smad1 in 293T cells B Immunoblot analysis showing decreased FLAG - Smad1 after USP34 knockdown C Immunoblot of Smad1 - linked polyUb . 293T cells were co - transfected with FLAG - Smad1 overexpression plasmid and USP34 siRNA , and treated with 10 µM MG132 for 4 hours before collection D Immunoblot analysis of the degradation of FLAG - Smad1 protein . 293T cells were co - transfected with FLAG - Smad1 overexpression plasmid and USP34 siRNA , and treated with 50 µg / ml cycloheximide ( CHX ) before collection at designated time points . Right : relative quantification of FLAG - Smad1 protein levels at different time pointsE Co - immunoprecipitation of USP34 with ectopically expressed FLAG - tagged RUNX2 in 293T cellsF Co - immunoprecipitation of USP34 with endogenous RUNX2 in mouse MSCsG Immunoblot of RUNX2 - linked polyUb . Differentiated mouse MSCs were treated with 10 µM MG132 for 4 hours before collection . Prx1 - Cre ( + ) indicates MSCs isolated from Prx1 - Cre ; Usp34f / f mice , and Prx1 - Cre ( - ) indicates those from Usp34f / f control miceH Immunoblot analysis of the degradation of endogenous RUNX2 protein in MSCs . Prx1 - Cre ; Usp34f / f and Usp34f / f control MSCs were stimulated with BMP2 , treated with 50 µg / ml CHX , and collected at designated time points . Right : relative quantification of RUNX2 protein levels at different time pointsI Representative images of ALP and ARS staining . Usp34f / f and Prx1 - Cre ; Usp34f / f MSCs were transduced with GFP , Smad1 , Runx2 or Smad1 + Runx2 adenoviruses , and then cultured in osteogenic medium for 5 and 10 days , respectivelyJ , K Quantitative analyses of the ALP activity and calcium mineralization . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc testL , M H & E staining and quantitative analysis of MSCs - mediated ectopic bone formation . Scale bar , 50 μm . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 and * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Co-immunoprecipitation of USP34 with ectopically expressed FLAG-tagged Smad1 in 293T cells B Immunoblot analysis showing decreased FLAG-Smad1 after USP34 knockdown  C Immunoblot of Smad1-linked polyUb. 293T cells were co-transfected with FLAG-Smad1 overexpression plasmid and USP34 siRNA, and treated with 10 µM MG132 for 4 hours before collection  D Immunoblot analysis of the degradation of FLAG-Smad1 protein. 293T cells were co-transfected with FLAG-Smad1 overexpression plasmid and USP34 siRNA, and treated with 50 µg/ml cycloheximide (CHX) before collection at designated time points. Right: relative quantification of FLAG-Smad1 protein levels at different time pointsE Co-immunoprecipitation of USP34 with ectopically expressed FLAG-tagged RUNX2 in 293T cellsF Co-immunoprecipitation of USP34 with endogenous RUNX2 in mouse MSCsG Immunoblot of RUNX2-linked polyUb. Differentiated mouse MSCs were treated with 10 µM MG132 for 4 hours before collection. Prx1-Cre(+) indicates MSCs isolated from Prx1-Cre;Usp34f/f mice, and Prx1-Cre(-) indicates those from Usp34f/f control miceH Immunoblot analysis of the degradation of endogenous RUNX2 protein in MSCs. Prx1-Cre;Usp34f/f and Usp34f/f control MSCs were stimulated with BMP2, treated with 50 µg/ml CHX, and collected at designated time points. Right: relative quantification of RUNX2 protein levels at different time pointsI Representative images of ALP and ARS staining. Usp34f/f and Prx1-Cre;Usp34f/f MSCs were transduced with GFP, Smad1, Runx2 or Smad1+Runx2 adenoviruses, and then cultured in osteogenic medium for 5 and 10 days, respectivelyJ, K Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=8; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc testL, M H&E staining and quantitative analysis of MSCs-mediated ectopic bone formation. Scale bar, 50 μm. Results are shown as mean ± SEM; n=8; *: p<0.05 and **: p<0.01 by ANOVA with Tukey's post hoc test


Figure 7A Relative activity of BRE luciferase assay . 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours . Results are shown as mean ± SEM ; n = 3 . * : p < 0 . 05 by ANOVA with Tukey ' s post hoc testB Immunoblot analysis of BMP2 - induce Sp7 expression . Prx1 - Cre ; Usp34f / f MSCs were treated with Smurf1 or control siRNAs , starved overnight and then stimulated with 100 ng / ml BMP2 for 24 hoursC Quantitative RT - PCR analysis of BMP2 - induce Sp7 transcription . Prx1 - Cre ; Usp34f / f MSCs were treated with Smurf1 or control siRNAs , and depleted from serum overnight before BMP2 stimulation . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 and * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc testD ALP staining and quantitative measurement of ALP activity . MSCs were cultured with normal medium supplemented with 100 ng / ml BMP2 for 5 days . Results are shown as mean ± SEM ; n = 8 ; * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc test E Representative images of ARS staining and quantification . Cells were cultured with normal medium supplemented with 100 ng / ml BMP2 for 14 days . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 , and * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc test F Representative images of ALP and ARS staining . Mouse MSCs were treated with Smurf1 or control siRNAs , and cultured in osteogenic medium for 5 and 10 days , respectivelyG , H Quantitative analyses of the ALP activity and calcium mineralization . Results are shown as mean ± SEM ; n = 8 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc test I , J Quantitative RT - PC analyses of the expression of osteogenic markers . Cells were treated with Smurf1 or control siRNAs , and cultured in osteogenic medium for 5 days . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc testwestern blot analyses of the expression of osteogenic markers . Cells were treated with Smurf1 or control siRNAs , and cultured in osteogenic medium for 5 days . Results are shown as mean ± SEM ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 and * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A Relative activity of BRE luciferase assay. 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours. Results are shown as mean ± SEM; n=3. *: p<0.05 by ANOVA with Tukey's post hoc testB Immunoblot analysis of BMP2-induce Sp7 expression. Prx1-Cre;Usp34f/f MSCs were treated with Smurf1 or control siRNAs, starved overnight and then stimulated with 100 ng/ml BMP2 for 24 hoursC Quantitative RT-PCR analysis of BMP2-induce Sp7 transcription. Prx1-Cre;Usp34f/f MSCs were treated with Smurf1 or control siRNAs, and depleted from serum overnight before BMP2 stimulation. Results are shown as mean ± SEM; n=3; *: p<0.05 and **: p<0.01 by ANOVA with Tukey's post hoc testD ALP staining and quantitative measurement of ALP activity. MSCs were cultured with normal medium supplemented with 100 ng/ml BMP2 for 5 days. Results are shown as mean ± SEM; n=8; **: p<0.01 by ANOVA with Tukey's post hoc test E Representative images of ARS staining and quantification. Cells were cultured with normal medium supplemented with 100 ng/ml BMP2 for 14 days. Results are shown as mean ± SEM; n=8; *: p<0.05, and **: p<0.01 by ANOVA with Tukey's post hoc test  F Representative images of ALP and ARS staining. Mouse MSCs were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 and 10 days, respectivelyG, H Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=8; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test I, J Quantitative RT-PC analyses of the expression of osteogenic markers. Cells were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc testwestern blot analyses of the expression of osteogenic markers. Cells were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test


Figure 1A , B , Western blotting analysis of proteins co - precipitated with anti - FLAG antibody from testis extracts of E15 . 5 wild - type embryos and transgenic embryos expressing FLAG - tagged NANOS2 with or without RNase ( A ) or from extracts of HeLa cells transfected with FLAG - tagged DND1 and with or without HA - tagged NANOS2 ( B ) . Precipitates were analyzed with the indicated antibodies . C , GST pull - down assay using E . coli extracts expressing GST , GST - fused NANOS2 , or NANOS3 mixed with MBP - tagged LacZ or DND1 . Precipitates were analyzed by Coomassie Brilliant Blue ( CBB ) staining or western blotting with an anti - MBP antibody . An arrowhead indicates MBP - DND1 co - precipitated with GST - tagged NANOS2 or NANOS3 . D , E , Western blotting analyses of proteins co - precipitated with anti - FLAG antibody from extracts of HeLa cells transfected with FLAG - tagged NANOS2 , NANOS2 ( ΔN10 ) , NANOS2 ( ΔC10 ) , NANOS2 ( C61A , C96A ) or NANOS3 , and HA - tagged DND1 ( D ) D , E , Western blotting analyses of proteins co - precipitated with anti - FLAG antibody from extracts of HeLa cells transfected with FLAG - tagged NANOS2 , NANOS2 ( ΔN10 ) , NANOS2 ( ΔC10 ) , NANOS2 ( C61A , C96A ) or NANOS3 , and HA - tagged DND1 ( D ) or with FLAG - tagged NANOS2 , NANOS2 ( C61A ) , NANOS2 ( C96A ) or NANOS2 ( C61A , C96A ) , and HA - tagged DND1 ( E ) . Precipitates were analyzed with the indicated antibodies . F , GST pull - down assay using E . coli extracts expressing GST - fused DND1 mixed with MBP - tagged LacZ , NANOS2 , NANOS2 ( C61A , C96A ) or NANOS2 ( 60 - 114 ) . Arrowheads indicate MBP - tagged NANOS2 ( lane 2 ) or NANOS2 ( 60 - 114 ) ( lane 4 ) .

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Figure 1A, B, Western blotting analysis of proteins co-precipitated with anti-FLAG antibody from testis extracts of E15.5 wild-type embryos and transgenic embryos expressing FLAG-tagged NANOS2 with or without RNase (A) or from extracts of HeLa cells transfected with FLAG-tagged DND1 and with or without HA-tagged NANOS2 (B).Precipitates were analyzed with the indicated antibodies. C, GST pull-down assay using E. coli extracts expressing GST, GST-fused NANOS2, or NANOS3 mixed with MBP-tagged LacZ or DND1. Precipitates were analyzed by Coomassie Brilliant Blue (CBB) staining or western blotting with an anti-MBP antibody. An arrowhead indicates MBP-DND1 co-precipitated with GST-tagged NANOS2 or NANOS3.D, E, Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with FLAG-tagged NANOS2, NANOS2 (ΔN10), NANOS2 (ΔC10), NANOS2 (C61A, C96A) or NANOS3, and HA-tagged DND1 (D)D, E, Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with FLAG-tagged NANOS2, NANOS2 (ΔN10), NANOS2 (ΔC10), NANOS2 (C61A, C96A) or NANOS3, and HA-tagged DND1 (D) or with FLAG-tagged NANOS2, NANOS2 (C61A), NANOS2 (C96A) or NANOS2 (C61A, C96A), and HA-tagged DND1 (E).Precipitates were analyzed with the indicated antibodies. F, GST pull-down assay using E. coli extracts expressing GST-fused DND1 mixed with MBP-tagged LacZ, NANOS2, NANOS2 (C61A, C96A) or NANOS2 (60-114). Arrowheads indicate MBP-tagged NANOS2 (lane 2) or NANOS2 (60-114) (lane 4).


Figure 2Western blotting analyses of proteins co - precipitated with anti - FLAG antibody from extracts of HeLa cells transfected with HA - tagged NANOS1 , NANOS2 or NANOS3 , and FLAG - tagged DND1 ( A ) Western blotting analyses of proteins co - precipitated with anti - FLAG antibody from extracts of HeLa cells transfected with HA - tagged NANOS1 , NANOS2 or NANOS3 , and FLAG - tagged DND1 ( A ) or with FLAG - tagged PUMILIO1 or PUMILIO2 , and HA - tagged NANOS1 , NANOS2 or NANOS3 with or without MYC - tagged DND1 ( B ) . C , Characterization of antibodies against PUMILIO1 and PUMILIO2 . Western blotting analyses of Flag - tagged PUMILIO1 and PUMILIO2 in HeLa cells transfected with FLAG - tagged PUMILIO1 or PUMILIO2 using antibodies against FLAG , PUMILIO1 or PUMILIO2 . Note that the antibodies against PUMILIO1 specifically recognized FLAG - tagged PUMILIO1 while the anti - PUMILIO2 antibody could detect both Flag - tagged PUMILIO1 and PUMILIO2 but less actively . D , FLAG - tagged NANOS2 was immunoprecipitated with an anti - FLAG antibody from E15 . 5 testicular extracts from transgenic mice expressing FLAG - tagged NANOS2 . Precipitates were analyzed by western blotting with the indicated antibodies . Note that co - precipitation of both PUMILIO1 and PUMILIO2 were not detected even though both CNOT1 and DND1 were clearly co - precipitated .

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Figure 2Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with HA-tagged NANOS1, NANOS2 or NANOS3, and FLAG-tagged DND1 (A)Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with HA-tagged NANOS1, NANOS2 or NANOS3, and FLAG-tagged DND1 (A) or with FLAG-tagged PUMILIO1 or PUMILIO2, and HA-tagged NANOS1, NANOS2 or NANOS3 with or without MYC-tagged DND1 (B).C, Characterization of antibodies against PUMILIO1 and PUMILIO2. Western blotting analyses of Flag-tagged PUMILIO1 and PUMILIO2 in HeLa cells transfected with FLAG-tagged PUMILIO1 or PUMILIO2 using antibodies against FLAG, PUMILIO1 or PUMILIO2. Note that the antibodies against PUMILIO1 specifically recognized FLAG-tagged PUMILIO1 while the anti-PUMILIO2 antibody could detect both Flag-tagged PUMILIO1 and PUMILIO2 but less actively.D, FLAG-tagged NANOS2 was immunoprecipitated with an anti-FLAG antibody from E15.5 testicular extracts from transgenic mice expressing FLAG-tagged NANOS2. Precipitates were analyzed by western blotting with the indicated antibodies. Note that co-precipitation of both PUMILIO1 and PUMILIO2 were not detected even though both CNOT1 and DND1 were clearly co-precipitated.


Figure 3Figure 3 | DND1 co - localizes with NANOS2 in P - bodies . A − C , Sections of male gonads from E15 . 5embryos were immunostained with antibodies against DND1 ( green ) and DCP1a ( red ) . D − F , Squash preparation of a male gonocyte from E16 . 5embryoimmunostained with antibodies against DND1 ( red ) and DCP1a ( green ) . Arrowheads indicate co - localization of DND1 and DCP1a . G − I , Sections of male gonads from E15 . 5embryos were immunostained with antibodies against DND1 ( red ) and NANOS2 ( green ) . Arrowheads indicate co - localization of DND1 and NANOS2 . J − M , NIH3T3 cells transfected with HA - tagged Dnd1 and FLAG - tagged Nanos2 were then immunostained with antibodies against DND1 ( J ) ( magenta ) , NANOS2 ( K ) ( red ) , and DCP1a ( L ) ( green ) . N − Y , Biomolecular fluorescence complementation assay . NIH3T3 cells transfected with VENUS - C - fused Dnd1 and VENUS - N - fused Nanos2 ( N − U ) or Nanos2 ( C61A , C96A ) ( V - γ ) were immunostained with antibodies against DND1 ( N , S , V ) , NANOS2 ( O , W , α ) or DDX6 ( R , Z ) . Then , the signals of VENUS fusion protein were visualized ( P , T , X , β ) . Arrowheads in Z indicate P - bodies . DNA was labeled with DAPI ( blue ) . Scale bars : 50 m in A for A − C ; 10 m in D for D - F , G for G − I , J for J − M , N for N − γ . Insets in A - C and J - γ show an enlarged vision of each picture to better depict localization of each protein . See also Fig . EV2T - Y .

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Figure 3Figure 3 | DND1 co-localizes with NANOS2 in P-bodies. A−C, Sections of male gonads from E15.5embryos were immunostained with antibodies against DND1 (green) and DCP1a (red). D−F, Squash preparation of a male gonocyte from E16.5embryoimmunostained with antibodies against DND1 (red) and DCP1a (green). Arrowheads indicate co-localization of DND1 and DCP1a. G−I, Sections of male gonads from E15.5embryos were immunostained with antibodies against DND1 (red) and NANOS2 (green). Arrowheads indicate co-localization of DND1 and NANOS2. J−M, NIH3T3 cells transfected with HA-tagged Dnd1 and FLAG-tagged Nanos2 were then immunostained with antibodies against DND1 (J) (magenta), NANOS2 (K) (red), and DCP1a (L) (green). N−Y, Biomolecular fluorescence complementation assay. NIH3T3 cells transfected with VENUS-C-fused Dnd1 and VENUS-N-fused Nanos2 (N−U) or Nanos2 (C61A, C96A) (V-γ) were immunostained with antibodies against DND1 (N, S, V), NANOS2 (O, W, α) or DDX6 (R, Z). Then, the signals of VENUS fusion protein were visualized (P, T, X, β). Arrowheads in Z indicate P-bodies. DNA was labeled with DAPI (blue). Scale bars: 50 m in A for A−C; 10 m in D for D-F, G for G−I, J for J−M, N for N−γ. Insets in A-C and J-γ show an enlarged vision of each picture to better depict localization of each protein. See also Fig. EV2T-Y.


Figure 4A , Western blotting analyses of proteins in testes from E13 . 5 to E16 . 5 embryos of Dnd1flox / flox or Dnd1flox / flox _ Tg ( Oct4PE - CreERT2 ) each administered with tamoxifen at E13 . 5 and E15 . 5 embryos of Nanos2 + / - or Nanos2 - / - with the indicated antibodies . B - P , Sections of testes from Dnd1flox / flox ( B , E , H , K , N ) , Dnd1flox / flox _ Tg ( Oct4PE - CreERT2 ) ( C , F , I , L , O ) or Nanos2 - / - ( D , G , J , M , P ) embryos were prepared at E16 . 5 and then immunostained with antibodies against pH3 ( B - D ) , STRA8 ( E - G ) , SYCP3 ( H - J ) , activated - Caspase3 ( K - M ) or LAMININ ( N - P ) ( green ) . Germ cells were immunostained with TRA98 ( B - D ) or DAZL ( N - P ) ( red ) , and DNA was labeled with DAPI ( blue ) . Tamoxifen was administered at E13 . 5 . Scale bars : 50 m in B for B − D and K - P , 50 m in E for E − J . See also Fig . EV3 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A, Western blotting analyses of proteins in testes from E13.5 to E16.5 embryos of Dnd1flox/flox or Dnd1flox/flox_Tg(Oct4PE-CreERT2) each administered with tamoxifen at E13.5 and E15.5 embryos of Nanos2+/- or Nanos2-/- with the indicated antibodies.B-P, Sections of testes from Dnd1flox/flox (B, E, H, K, N), Dnd1flox/flox_Tg(Oct4PE-CreERT2) (C, F, I, L, O) or Nanos2-/- (D, G, J, M, P) embryos were prepared at E16.5 and then immunostained with antibodies against pH3 (B-D), STRA8 (E-G), SYCP3 (H-J), activated-Caspase3 (K-M) or LAMININ (N-P) (green). Germ cells were immunostained with TRA98 (B-D) or DAZL (N-P) (red), and DNA was labeled with DAPI (blue). Tamoxifen was administered at E13.5. Scale bars: 50 m in B for B−D and K-P, 50 m in E for E−J. See also Fig. EV3.


Figure 5A - L , Sections of testes from Dnd1flox / flox ( A , B , C , G , H , I , M , N , O ) and Dnd1flox / flox _ Tg ( Oct4PE - CreERT2 ) ( D , E , F , J , K , L , P , Q , R ) embryos were prepared at E16 . 5 and then immunostained with antibodies against NANOS2 ( A , D ) ( green ) , DCP1a ( G , J ) ( green ) , DDX6 ( M , P ) ( green ) and TRA98 ( B , E , H , K , N , Q ) . DNA was labeled with DAPI ( blue ) . Tamoxifen was administered at E13 . 5 . Scale bars : 50 m in A for A − F ; 50 m in G for G - L ; 50 m in M for M - R . Insets show an enlarged vision of each picture to better depict localization of NANOS2 , DCP1a and DDX6 . S - V , Immunoprecipitation with an anti - FLAG antibody from E15 . 5 male gonadal extracts of wild - type and the transgenic mouse line expressing FLAG - tagged DND1 ( S , T ) , or with an anti - NANOS2 antibody from E15 . 5 male gonadal extracts of Dnd1flox / flox mice with or without Oct4PE - CreERT2 ( U , V ) . Precipitates were analyzed by western blotting ( S , U ) or by RT - qPCR ( T , V ) . The RT - qPCR data are shown as average relative mRNA levels ± SEs ( n = 3 ) . W , X , Western blotting analyses of proteins in NANOS2 - depleted ( W ) or DND1 - depleted ( X ) testis extracts from E15 . 5 embryos . Proteins were analyzed with the indicated antibodies .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-L, Sections of testes from Dnd1flox/flox (A, B, C, G, H, I, M, N, O) and Dnd1flox/flox_Tg(Oct4PE-CreERT2) (D, E, F, J, K, L, P, Q, R) embryos were prepared at E16.5 and then immunostained with antibodies against NANOS2 (A, D) (green), DCP1a (G, J) (green), DDX6 (M, P) (green) and TRA98 (B, E, H, K, N, Q). DNA was labeled with DAPI (blue). Tamoxifen was administered at E13.5. Scale bars: 50 m in A for A−F; 50 m in G for G-L; 50 m in M for M-R. Insets show an enlarged vision of each picture to better depict localization of NANOS2, DCP1a and DDX6.S-V, Immunoprecipitation with an anti-FLAG antibody from E15.5 male gonadal extracts of wild-type and the transgenic mouse line expressing FLAG-tagged DND1 (S, T), or with an anti-NANOS2 antibody from E15.5 male gonadal extracts of Dnd1flox/flox mice with or without Oct4PE-CreERT2 (U, V). Precipitates were analyzed by western blotting (S, U) or by RT-qPCR (T, V). The RT-qPCR data are shown as average relative mRNA levels ± SEs (n=3).W, X, Western blotting analyses of proteins in NANOS2-depleted (W) or DND1-depleted (X) testis extracts from E15.5 embryos. Proteins were analyzed with the indicated antibodies.


Figure 1A . Whole chromosomes imaged by conventional TEM . Data information : Scale bars : 1µmB . Slice from a large tomographic volume ; part of the carbon film surrounding a hole with vitrified ice containing the plates is indicated with an asterisk ; the inset illustrates that the slice of a plate corresponds to a line in the x - y plane ( perpendicular to the direction of the electron beam ) . Data information : Scale bars : 200 nmC - G Slices from different tomograms and the corresponding 3D segmentations showing plates with different sizes and shapes . In addition to a typical slice through the x - y plane ( yellow ) , two slices through the x - z ( red ) and y - z ( green ) planes ( orthogonal to the x - y plane ) are shown in ( C ) . Data information : Scale bars : 200 nm D ) , 100 nm ( E - G ) , 50 nm ( C ) .

O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Whole chromosomes imaged by conventional TEM. Data information: Scale bars: 1µmB. Slice from a large tomographic volume; part of the carbon film surrounding a hole with vitrified ice containing the plates is indicated with an asterisk; the inset illustrates that the slice of a plate corresponds to a line in the x-y plane (perpendicular to the direction of the electron beam). Data information: Scale bars: 200 nmC-G Slices from different tomograms and the corresponding 3D segmentations showing plates with different sizes and shapes. In addition to a typical slice through the x-y plane (yellow), two slices through the x-z (red) and y-z (green) planes (orthogonal to the x-y plane) are shown in (C). Data information: Scale bars: 200 nm D), 100 nm (E-G), 50 nm (C).


Figure 2A . Nucleosomes ( N ) decorating a relaxed plate ( P1 ) ; insets in the upper right show additional examples . Nucleosomes are not visible as individual units in typical compact plates ( P2 ) . Short compact interdigitated solenoids are shown in the main image ( S ) and in the bottom - left insets . Data information : Scale bars : 50 nmB - E Slice ( B ) and segmentations in three different orientations ( C ) of a large relaxed plate decorated with many nucleosomes on its right side . Slice ( D ) and segmentation ( E ) of a relaxed plate forming a tube decorated with nucleosomes . Data information : Scale bars : 50 nm insets , B - E )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Nucleosomes (N) decorating a relaxed plate (P1); insets in the upper right show additional examples. Nucleosomes are not visible as individual units in typical compact plates (P2). Short compact interdigitated solenoids are shown in the main image (S) and in the bottom-left insets. Data information: Scale bars: 50 nmB-E Slice (B) and segmentations in three different orientations (C) of a large relaxed plate decorated with many nucleosomes on its right side. Slice (D) and segmentation (E) of a relaxed plate forming a tube decorated with nucleosomes. Data information: Scale bars: 50 nm insets, B-E)


Figure 3A , B Plates with several layers that are not closely appressed . C - E Large multilayer plates having the size of human metaphase chromatids [ ~ 0 . 6 µm diameter ] ( C ) ; the inset schematically shows the perpendicular orientation of chromatin layers with respect to the chromatid axes proposed in the thin - plate model In other slices ( D , E ) , the multilayer structures shown in ( C ) are more compact and the individual layers are not visible as separate elements . Data information : Scale bars : 50 nm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A,B Plates with several layers that are not closely appressed. C-E Large multilayer plates having the size of human metaphase chromatids [~0.6 µm diameter ] (C); the inset schematically shows the perpendicular orientation of chromatin layers with respect to the chromatid axes proposed in the thin-plate model In other slices (D,E), the multilayer structures shown in (C) are more compact and the individual layers are not visible as separate elements. Data information: Scale bars: 50 nm.


Figure 4A - C Slices showing lateral association between two plates and the corresponding 3D segmentations . D . Edge - to - edge interactions form cylindrical structures . Data information : Scale bars : 50 nm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-C Slices showing lateral association between two plates and the corresponding 3D segmentations. D. Edge-to-edge interactions form cylindrical structures. Data information: Scale bars: 50 nm.


Figure 5A - C The cation concentrations used are indicated for each experiment ; AU , arbitrary units .

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Figure 5A-C The cation concentrations used are indicated for each experiment; AU, arbitrary units.


Figure 1A . Spike - binding antibodies in serum . Binding by antibodies of classes M , G , and A is detected with secondary antibodies conjugated to different fluorochromes , and the results are shown on the left , middle , and right plots , respectively . Binding ( geometric mean fluorescence intensity - GMFI - of corresponding secondary antibody ) to TE cells expressing SARS - CoV - 2 spike protein is plotted on the vertical axis , and binding to untransfected cells on the horizontal axis . Each point represents a value from one donor . Samples from donors with no known exposure to SARS - CoV - 2 antigens ( n = 86 ) are plotted with blue circles , recently infected donors ( n = 34 ) with red circles , and vaccinated donors ( n = 14 ) with green diamonds . Red circles with black triangles correspond to donors from whose B cells monoclonal antibodies were isolated ( n = 5 , P values are derived from a two - way analysis of variance followed by Tukey ' s test to compare the specific binding , i . e . , ( binding to spike - expressing cells ) / ( binding to untransfected cells ) between conditions ( convalescent or vaccinated against unexposed ) within each antibody class . The experiment was independently repeated three times , and results shown come from the third replicate . B . Change in antibody binding following vaccination against SARS - CoV - 2 . Binding of antibodies from sera assessed in the experiment shown in ( A ) , is shown for two samples of serum from each of 7 donors . Data are plotted as in ( A ) with binding to spike - expressing cells on the vertical axis , and to untransfected cells on the horizontal , using blue symbols for samples from before vaccination , and red symbols for samples from 15 - 29 days after the first vaccination ( but before any second vaccination ) . Black lines connect points corresponding to pre / post pairs of samples from each donor . Left plot shows results for IgM and right plot for IgG . C . Comparison of post - vaccination increase in binding to untransfected cells between IgG and IgM . The increase in binding to untransfected TE 0 cells between pre - and post - vaccination samples is shown on the left with blue symbols for IgG and on the right with black symbols for IgM . The horizontal line at y = 0 is the expected result when no increased binding is observed . P value is derived from a two - tailed , Wilcoxon matched pairs signed rank test . D . Comparison of spike - specific IgM , as measured by flow cytometry or ELISA . The left two " FC " are the results of flow cytometry , and the left verical axis shows the ratio of serum IgM binding to spike - expressing cells , vs binding to non - expressing control cells , as plottted in A . The right two columns show spike - RBD - specific IgM as measured by an IgM - capture ELISA , and the right vertical axis shows the optical densities ( OD ) . P values are calculated by two - way analysis of variance , followed by Sidak ' s multiple comparisons test . The entire experiment was repeated three times , and the data shown are derived from the third replicate .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Spike-binding antibodies in serum. Binding by antibodies of classes M, G, and A is detected with secondary antibodies conjugated to different fluorochromes, and the results are shown on the left, middle, and right plots, respectively. Binding (geometric mean fluorescence intensity - GMFI- of corresponding secondary antibody) to TE cells expressing SARS-CoV-2 spike protein is plotted on the vertical axis, and binding to untransfected cells on the horizontal axis. Each point represents a value from one donor. Samples from donors with no known exposure to SARS-CoV-2 antigens (n = 86) are plotted with blue circles, recently infected donors (n = 34) with red circles, and vaccinated donors (n = 14) with green diamonds. Red circles with black triangles correspond to donors from whose B cells monoclonal antibodies were isolated (n = 5, P values are derived from a two-way analysis of variance followed by Tukey's test to compare the specific binding, i.e., (binding to spike-expressing cells)/(binding to untransfected cells) between conditions (convalescent or vaccinated against unexposed) within each antibody class. The experiment was independently repeated three times, and results shown come from the third replicate.B. Change in antibody binding following vaccination against SARS-CoV-2. Binding of antibodies from sera assessed in the experiment shown in (A), is shown for two samples of serum from each of 7 donors. Data are plotted as in (A) with binding to spike-expressing cells on the vertical axis, and to untransfected cells on the horizontal, using blue symbols for samples from before vaccination, and red symbols for samples from 15-29 days after the first vaccination (but before any second vaccination). Black lines connect points corresponding to pre/post pairs of samples from each donor. Left plot shows results for IgM and right plot for IgG. C. Comparison of post-vaccination increase in binding to untransfected cells between IgG and IgM. The increase in binding to untransfected TE 0 cells between pre- and post-vaccination samples is shown on the left with blue symbols for IgG and on the right with black symbols for IgM. The horizontal line at y=0 is the expected result when no increased binding is observed. P value is derived from a two-tailed, Wilcoxon matched pairs signed rank test.D. Comparison of spike-specific IgM, as measured by flow cytometry or ELISA. The left two "FC" are the results of flow cytometry, and the left verical axis shows the ratio of serum IgM binding to spike-expressing cells, vs binding to non-expressing control cells, as plottted in A. The right two columns show spike-RBD-specific IgM as measured by an IgM-capture ELISA, and the right vertical axis shows the optical densities (OD). P values are calculated by two-way analysis of variance, followed by Sidak's multiple comparisons test. The entire experiment was repeated three times, and the data shown are derived from the third replicate.


Figure 2Phenotypes of blood B cells . B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads , exposed to adherent cells expressing spike - mCherry for 3 hours , then retrieved and labeled with fluorescent antibodies and measured by flow cytometry . ( A ) UMAP algorithm ( 5000 randomly selected cells / sample ) was used to depict the major B cell subsets clustered according to marker expression . Phenotypes of blood B cells . B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads , exposed to adherent cells expressing spike - mCherry for 3 hours , then retrieved and labeled with fluorescent antibodies and measured by flow cytometry . FlowSOM - based B cell subpopulations are overlaid as a color dimension , and the colors of the clusters are shown on the left of heatmap ( B ) . B . Heatmap showing mean population expression levels of markers used for UMAP visualization and FlowSOM - clustering . Colors shown in the legend on the left are also used in the UMAP representation in ( A ) . Phenotypes of blood B cells . B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads , exposed to adherent cells expressing spike - mCherry for 3 hours , then retrieved and labeled with fluorescent antibodies and measured by flow cytometry . C . Gating strategy to define CD69 - high , mCherry - high , spike - capturing B cells ( i . e . , membrane antigen capture activated cells , MACAC , red box ) . Dot plot shows CD69 and mCherry fluorescence for B cells from one of three donors gated by forward and side scatter , and negative for the dye used to mark the antigen donor cells . Phenotypes of blood B cells . B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads , exposed to adherent cells expressing spike - mCherry for 3 hours , then retrieved and labeled with fluorescent antibodies and measured by flow cytometry . D . Relative fractions of different B cell subpopulations , from three donors , as shown in A - B , in all B cells compared to within MACAC B cells . P values are based on two - tailed t - tests between the groups . Correlation coefficients ( r ) were calculated from the z - statistic of the Wilcoxon - Mann - Whitney test . A black horizontal line represents the median . Boxplots represent the interquartile range ( IQR ) . Whiskers extend to the farthest data point within a maximum of 1 . 5 × IQR . Every point represents one donor . E . Gating strategy in MACAC sorting . Single cells are selected based on scatter , antigen - expressing TE spike - mCherry cells excluded on Cell Trace Violet label , and the spike - capturing ( mCherry - high ) , activated ( CD69 - high ) B cells ( population labeled " MACAC " in red on the right - most plot ) are sorted . The middle plot shows B cells that did not adhere to the TE - spike - cherry antigen - expressing cells ( putatively antigen - irrelevant ) , and the right plot those that did ( putatively antigen - recognizing ) . Plots show data from one of five convalescent donors Cells in the MACAC gate were singly distributed into wells of 384 - well plates and cultured for 9 days with IL - 21 and CD40L , and then the single well culture supernatants were screened for anti - spike antibody binding and virus neutralization as described below . F . Results of single well supernatant screening for antibody binding to SARS - CoV - 2 spike protein . Results from 3266 wells from 5 donors are shown for IgM ( left ) , IgG ( middle ) , and IgA ( right ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. (A) UMAP algorithm (5000 randomly selected cells/sample) was used to depict the major B cell subsets clustered according to marker expression.Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. FlowSOM-based B cell subpopulations are overlaid as a color dimension, and the colors of the clusters are shown on the left of heatmap (B). B. Heatmap showing mean population expression levels of markers used for UMAP visualization and FlowSOM-clustering. Colors shown in the legend on the left are also used in the UMAP representation in (A).Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. C. Gating strategy to define CD69-high, mCherry-high, spike-capturing B cells (i.e., membrane antigen capture activated cells, MACAC, red box). Dot plot shows CD69 and mCherry fluorescence for B cells from one of three donors gated by forward and side scatter, and negative for the dye used to mark the antigen donor cells.Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. D. Relative fractions of different B cell subpopulations, from three donors, as shown in A-B, in all B cells compared to within MACAC B cells. P values are based on two-tailed t-tests between the groups. Correlation coefficients (r) were calculated from the z-statistic of the Wilcoxon-Mann-Whitney test. A black horizontal line represents the median. Boxplots represent the interquartile range (IQR). Whiskers extend to the farthest data point within a maximum of 1.5× IQR. Every point represents one donor.E. Gating strategy in MACAC sorting. Single cells are selected based on scatter, antigen-expressing TE spike-mCherry cells excluded on Cell Trace Violet label, and the spike-capturing (mCherry-high), activated (CD69-high) B cells (population labeled "MACAC" in red on the right-most plot) are sorted. The middle plot shows B cells that did not adhere to the TE-spike-cherry antigen-expressing cells (putatively antigen-irrelevant), and the right plot those that did (putatively antigen-recognizing). Plots show data from one of five convalescent donors Cells in the MACAC gate were singly distributed into wells of 384-well plates and cultured for 9 days with IL-21 and CD40L, and then the single well culture supernatants were screened for anti-spike antibody binding and virus neutralization as described below. F. Results of single well supernatant screening for antibody binding to SARS-CoV-2 spike protein. Results from 3266 wells from 5 donors are shown for IgM (left), IgG (middle), and IgA (right).


Figure 3A . Example of neutralization of chimeric VSV * ∆ G - S ∆ 21 by donor - derived IgG , IgM and IgA antibodies . 100 pfu VSV * ∆ G - S ∆ 21 was mixed with antibody at specified concentrations for 1 hour , then added to Vero cells . After 24 hours , the cells were imaged , and infected wells identified by GFP expression , here pseudocolored with GFP - bright shown in black against a white background . Virus neutralization is manifested as reduction in GFP . Images shown here come from wells infected with virus alone ( left column ) , virus pre - incubated with the moderately neutralizing IgG 1I12 ( second and third columns ) , the potent neutralizing IgM 2J17 ( fourth and fitfh columns ) , or with the moderately potent IgA 1B17 ( sixth and seventh columns ) . Each antibody was tested in the range of concentrations shown to the left of the images , from 10000 down to 0 . 1 ng / ml . B . Neutralization curves from each of the cloned antibodies using plaque reduction assay of VSV * ∆ G - S ∆ 21 . The horizontal axis shows the serial dilution of the antibodies . The vertical axis shows , for each well exposed to antibody , the ratio of infected cell plaque count to the corresponding value from control wells without antibody . Each point is the mean of 9 values pooled from three independent experiments each with triplicate wells . Error bars indicate standard error . Antibodies tested are listed below the figure and include 3 IgM ( black lines and symbols ) , 11 IgG ( blue lines and symbols ) and 6 IgA ( red lines and symbols ) . C . Quantification of results from ( B ) . Area under the curve is plotted for every antibody and compared between different antibody classes by one - way ANOVA followed by Tukey ' s test ( mean from three biological replicates and SEM ) . Area under the curve was chosen as a measure of neutralization capacity , rather than ND50 ( as used in subsequent figures ) , because some antibodies included in this figure exhibited no neutralization at the concentrations tested , precluding the calculation of an ND50 . E . Flow cytometric determination of antibody - dependent complement deposition on cells . Cells expressing SARS - CoV - 2 spike protein and untransfected control cells were incubated with various concentrations of antibody in the presence of fresh human serum ( from a SARS - CoV - 2 unexposed donor ) . Activation of the complement cascade was measured by flow cytometric assessment of complement component C3b deposition on the surface of the cells . Results for the 3 IgM , ( black ) , 11 IgG , ( blue ) , and 6 IgA antibodies ( red ) are shown . P values were calculated by two - way analysis of variance , followed by Tukey ' s test . F . Influence of complement on virus neutralization . A plaque reduction neutralization assay like that shown in Fig 3A - C was conducted with IgM 2J17 in the presence of either fresh , complement - sufficient human serum , or heat inactivated serum . The concentration of 2J17 is shown on the horizontal axis . The vertical axis shows , for each well exposed to antibody , the ratio of infected cell plaque count to the corresponding value from control wells with serum ( either fresh or inactivated ) but without antibody . The points plotted with filled red symbols correspond to the condition with heat inactivated serum , and the open blue symbols results with fresh serum . Each symbol corresponds to the mean of nine wells pooled from three independent experiments , each with triplicate wells . Error bars correspond to standard error . P value was calculated using a paired , two - tailed t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Example of neutralization of chimeric VSV*∆G-S∆21 by donor-derived IgG, IgM and IgA antibodies. 100 pfu VSV*∆G-S∆21 was mixed with antibody at specified concentrations for 1 hour, then added to Vero cells. After 24 hours, the cells were imaged, and infected wells identified by GFP expression, here pseudocolored with GFP-bright shown in black against a white background. Virus neutralization is manifested as reduction in GFP. Images shown here come from wells infected with virus alone (left column), virus pre-incubated with the moderately neutralizing IgG 1I12 (second and third columns), the potent neutralizing IgM 2J17 (fourth and fitfh columns), or with the moderately potent IgA 1B17 (sixth and seventh columns). Each antibody was tested in the range of concentrations shown to the left of the images, from 10000 down to 0.1 ng/ml.B. Neutralization curves from each of the cloned antibodies using plaque reduction assay of VSV*∆G-S∆21. The horizontal axis shows the serial dilution of the antibodies. The vertical axis shows, for each well exposed to antibody, the ratio of infected cell plaque count to the corresponding value from control wells without antibody. Each point is the mean of 9 values pooled from three independent experiments each with triplicate wells. Error bars indicate standard error. Antibodies tested are listed below the figure and include 3 IgM (black lines and symbols), 11 IgG (blue lines and symbols) and 6 IgA (red lines and symbols).C. Quantification of results from (B). Area under the curve is plotted for every antibody and compared between different antibody classes by one-way ANOVA followed by Tukey's test (mean from three biological replicates and SEM). Area under the curve was chosen as a measure of neutralization capacity, rather than ND50 (as used in subsequent figures), because some antibodies included in this figure exhibited no neutralization at the concentrations tested, precluding the calculation of an ND50.E. Flow cytometric determination of antibody-dependent complement deposition on cells. Cells expressing SARS-CoV-2 spike protein and untransfected control cells were incubated with various concentrations of antibody in the presence of fresh human serum (from a SARS-CoV-2 unexposed donor). Activation of the complement cascade was measured by flow cytometric assessment of complement component C3b deposition on the surface of the cells. Results for the 3 IgM, (black), 11 IgG, (blue), and 6 IgA antibodies (red) are shown. P values were calculated by two-way analysis of variance, followed by Tukey's test.F. Influence of complement on virus neutralization. A plaque reduction neutralization assay like that shown in Fig 3A-C was conducted with IgM 2J17 in the presence of either fresh, complement-sufficient human serum, or heat inactivated serum. The concentration of 2J17 is shown on the horizontal axis. The vertical axis shows, for each well exposed to antibody, the ratio of infected cell plaque count to the corresponding value from control wells with serum (either fresh or inactivated) but without antibody. The points plotted with filled red symbols correspond to the condition with heat inactivated serum, and the open blue symbols results with fresh serum. Each symbol corresponds to the mean of nine wells pooled from three independent experiments, each with triplicate wells. Error bars correspond to standard error. P value was calculated using a paired, two-tailed t-test.


Figure 4A . The effect of competition on concentration versus binding curves of three spike - specific , class - switched antibodies . Antibodies were expressed either as IgM , as originally isolated ( black symbols and lines ) , or artificially switched to IgG1 ( blue lines and symbols ) . For each antibody , identified by name at the top of each plot , the binding of the antibody alone ( " alone " , open symbols ) , or the competitive binding of the antibody in a mixture of IgM and IgG1 ( " compet . " , filled symbols ) is shown . In the competition scenario , each of the two classes of the antibody were added together at the concentration shown on the horizontal axis . The vertical axis shows the ratio of geometric mean fluorescence of human IgM , or IgG on spike - expressing cells divided by the corresponding signal on spike - non - expressing control cells . Each point shows the mean of 3 values from three independent experiments . Error bars show standard error . P value was calculated by comparing the area under the curve of each antibody class in the " alone " condition with the binding of the " competition " condition by paired , two - tailed t - test . B . Virus neutralization by antibodies expressed as IgM or IgG1 . A plaque reduction neutralization assay as described in Fig 3A - B was used to measure neutralization by the three , donor - derived IgM ( black lines and open symbols ) and their artificially class - switched IgG1 equivalents ( blue lines and filled symbols ) . Horizontal axis shows the antibody concentration and the vertical axis shows the level of infection expressed as percentage GFP expression compared to control wells with no antibody added . Each point shows the mean of nine replicate values pooled from three independent experiments , each with triplicate wells . Error bars show standard error . C . Comparison of virus neutralization by antibodies expressed as IgM or IgG1 shown in B . Area under the curve was calculated from each of the three independently performed experiments . P was calculated by paired , two - tailed t - test . Virus neutralization by antibodies artificially class switched from IgA or IgG1 to IgM . A plaque reduction neutralization assay as described in Fig 3A - B was used to measure neutralization by the two donor - derived IgA ( pink and light blue lines ) and one donor - derived IgG ( bordeaux lines ) . The antibodies expressed in their original classes ( IgA or IgG ) are plotted with filled symbols of the same colors as the lines , and their artificially class - switched IgM equivalents are plotted with open symbols . Horizontal axis shows the antibody concentration and the vertical axis shows the level of infection expressed as percentage GFP expression compared to control wells with no antibody added . Each point shows the mean of nine replicate values pooled from three independent experiments , each with triplicate wells . ND50 was calculated from each of the three independently performed experiments . P was calculated by paired , two - tailed t - test for each antibody pair . Affinities of donor - derived spike - binding IgG1 ( n = 8 ) and donor - derived IgM ( n = 3 ) expressed as IgG1 , measured by surface plasmon resonance . P value was calculated by unpaired two - tailed t - test .

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Figure 4A. The effect of competition on concentration versus binding curves of three spike-specific, class-switched antibodies. Antibodies were expressed either as IgM, as originally isolated (black symbols and lines), or artificially switched to IgG1 (blue lines and symbols). For each antibody, identified by name at the top of each plot, the binding of the antibody alone ("alone", open symbols), or the competitive binding of the antibody in a mixture of IgM and IgG1 ("compet.", filled symbols) is shown. In the competition scenario, each of the two classes of the antibody were added together at the concentration shown on the horizontal axis. The vertical axis shows the ratio of geometric mean fluorescence of human IgM, or IgG on spike-expressing cells divided by the corresponding signal on spike-non-expressing control cells. Each point shows the mean of 3 values from three independent experiments. Error bars show standard error. P value was calculated by comparing the area under the curve of each antibody class in the "alone" condition with the binding of the "competition" condition by paired, two-tailed t-test.B. Virus neutralization by antibodies expressed as IgM or IgG1. A plaque reduction neutralization assay as described in Fig 3A-B was used to measure neutralization by the three, donor-derived IgM (black lines and open symbols) and their artificially class-switched IgG1 equivalents (blue lines and filled symbols). Horizontal axis shows the antibody concentration and the vertical axis shows the level of infection expressed as percentage GFP expression compared to control wells with no antibody added. Each point shows the mean of nine replicate values pooled from three independent experiments, each with triplicate wells. Error bars show standard error. C. Comparison of virus neutralization by antibodies expressed as IgM or IgG1 shown in B. Area under the curve was calculated from each of the three independently performed experiments. P was calculated by paired, two-tailed t-test.Virus neutralization by antibodies artificially class switched from IgA or IgG1 to IgM. A plaque reduction neutralization assay as described in Fig 3A-B was used to measure neutralization by the two donor-derived IgA (pink and light blue lines) and one donor-derived IgG (bordeaux lines). The antibodies expressed in their original classes (IgA or IgG) are plotted with filled symbols of the same colors as the lines, and their artificially class-switched IgM equivalents are plotted with open symbols. Horizontal axis shows the antibody concentration and the vertical axis shows the level of infection expressed as percentage GFP expression compared to control wells with no antibody added. Each point shows the mean of nine replicate values pooled from three independent experiments, each with triplicate wells. ND50 was calculated from each of the three independently performed experiments. P was calculated by paired, two-tailed t-test for each antibody pair.Affinities of donor-derived spike-binding IgG1 (n = 8) and donor-derived IgM (n = 3) expressed as IgG1, measured by surface plasmon resonance. P value was calculated by unpaired two-tailed t-test.


Figure 5A . Heatmap of antibody binding to spike protein subdomains ( complete spike extracellular domain " spike " , S1 , S2 , RBD and NTD in ELISA . Color gradient shown on right represents optical density at 450 nm ( OD ) . Values are mean OD from two independent experiments . Analogous ELISA results for 3 IgM and their IgG1 - class - switched derivativesC . Flow - cytometric epitope binning of monoclonal IgM , utilizing competitive displacement of IgG by IgM with identical variable region Cells expressing SARS - CoV - 2 spike protein and untransfected control cells were incubated with each of 3 neutralizing IgM antibodies artificially switched to IgG1 , either alone , or mixed with one of the 3 antibodies expressed as IgM . The binding of each IgG1 alone is shown in the last column of the heatmap , and in competition with each other IgM in columns 1 - 3 . Color shade represents the specific IgG binding ( ratio of GMFI on TE spike - mCherry cells to TE 0 cells ) . Values are mean GMFI ratios from three independent experiments . When IgM binds the same epitope as the IgG ( automatically the case when the source antibody is the same ) , the IgM will displace the IgG , resulting in a reduction in the IgG signal . Asterisks mark those combinations of antibodies with a statistically significant decrease in binding of the IgG1 in the presence of IgM , compared to the IgG alone across 3 independent experiments , ( * p < 0 . 05 , two - way analysis of variance , followed by Tukey ' s test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Heatmap of antibody binding to spike protein subdomains (complete spike extracellular domain "spike", S1, S2, RBD and NTD in ELISA. Color gradient shown on right represents optical density at 450 nm (OD). Values are mean OD from two independent experiments. Analogous ELISA results for 3 IgM and their IgG1-class-switched derivativesC. Flow-cytometric epitope binning of monoclonal IgM, utilizing competitive displacement of IgG by IgM with identical variable region Cells expressing SARS-CoV-2 spike protein and untransfected control cells were incubated with each of 3 neutralizing IgM antibodies artificially switched to IgG1, either alone, or mixed with one of the 3 antibodies expressed as IgM. The binding of each IgG1 alone is shown in the last column of the heatmap, and in competition with each other IgM in columns 1-3. Color shade represents the specific IgG binding (ratio of GMFI on TE spike-mCherry cells to TE 0 cells). Values are mean GMFI ratios from three independent experiments. When IgM binds the same epitope as the IgG (automatically the case when the source antibody is the same), the IgM will displace the IgG, resulting in a reduction in the IgG signal. Asterisks mark those combinations of antibodies with a statistically significant decrease in binding of the IgG1 in the presence of IgM, compared to the IgG alone across 3 independent experiments, (* p<0.05, two-way analysis of variance, followed by Tukey's test).


Figure 1 ( D ) Measurement of intracellular ROS levels by fluorescent probe DCFH - DA , and the fluorescence intensity of ROS was calculated ; All scale bars are 20 μm . n = 3 biological replicates . ( G ) Liver tissues were obtained from the indicated mice and then examined using transmission electron microscopy ( Arrowheads indicate mitochondria ) ; Scale bars are 1 μm and 5 μm . n = 3 biological replicates .

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Figure 1(D) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated; All scale bars are 20 μm. n=3 biological replicates.(G) Liver tissues were obtained from the indicated mice and then examined using transmission electron microscopy (Arrowheads indicate mitochondria); Scale bars are 1 μm and 5 μm. n = 3 biological replicates.


Figure 2 ( B ) Gpx4 protein was measured in liver of WT and PPARα - / - mice fed blank solvent or GW7647 ; n = 4 biological replicates . ( E ) Chromatin immunoprecipitation assays were performed on soluble formaldehyde - crosslinked chromatin isolated from untreated and GW7647 - treated WT or PPARα - / - livers with polyclonal anti - PPARα antibodies ( anti - PPARα ) or control IgG . The final DNA extraction was polymerase chain reaction - amplified with a primer pair that covered the sequence in intron 3 of Gpx4 .

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Figure 2(B) Gpx4 protein was measured in liver of WT and PPARα-/- mice fed blank solvent or GW7647; n = 4 biological replicates.(E) Chromatin immunoprecipitation assays were performed on soluble formaldehyde-crosslinked chromatin isolated from untreated and GW7647-treated WT or PPARα-/- livers with polyclonal anti-PPARα antibodies (anti-PPARα) or control IgG. The final DNA extraction was polymerase chain reaction-amplified with a primer pair that covered the sequence in intron 3 of Gpx4.


Figure 3 ( B ) Liver sections were obtained from the indicated mice and stained with probe1 . All scale bars are 20 μm . n = 3 biological replicates . ( D ) TRF protein was measured in liver of WT and PPARα - / - mice fed blank solvent or GW7647 . n = 4 biological replicates .

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Figure 3(B) Liver sections were obtained from the indicated mice and stained with probe1. All scale bars are 20 μm. n = 3 biological replicates.(D) TRF protein was measured in liver of WT and PPARα-/- mice fed blank solvent or GW7647. n = 4 biological replicates.


Figure 4 ( E ) Liver tissue was obtained from the indicated mice and then examined using transmission electron microscopy ( Arrowheads indicate mitochondria ) . Scale bars are 1 μm and 5 μm . n = 4 biological replicates . ( J ) Measurement of intracellular ROS levels by fluorescent probe DCFH - DA , and the fluorescence intensity of ROS was calculated . All scale bars are 20 μm . n = 3 biological replicates .

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Figure 4(E) Liver tissue was obtained from the indicated mice and then examined using transmission electron microscopy (Arrowheads indicate mitochondria). Scale bars are 1 μm and 5 μm. n = 4 biological replicates.(J) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated. All scale bars are 20 μm. n = 3 biological replicates.


Figure 5 ( E ) Measurement of intracellular ROS levels by fluorescent probe DCFH - DA , and the fluorescence intensity of ROS was calculated . n = 3 biological replicates . All scale bars are 20 μm . ( J ) Measurement of intracellular ROS levels by fluorescent probe DCFH - DA , and the fluorescence intensity of ROS was calculated . n = 3 biological replicates . All scale bars are 20 μm .

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Figure 5(E) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated. n = 3 biological replicates. All scale bars are 20 μm.(J) Measurement of intracellular ROS levels by fluorescent probe DCFH-DA, and the fluorescence intensity of ROS was calculated. n = 3 biological replicates. All scale bars are 20 μm.


Figure 1 ( D ) HaloTag pulse - chase analysis in WT MEF monitoring fluorescent Halo - AATNNN during up to 6 h of chase in the presence of BafA1 . The fluorescent JF646 - Halo ligand and the polymer - specific 2C1 antibody stains are shown . ( E ) Same as ( D ) for fluorescent Halo - ATZNNN . ( F ) Quantification of accumulation of fluorescent Halo - AATNNN and Halo - ATZNNN within LAMP1 - positive endolysosomes . Mean ± SEM , n = 20 , 11 , 10 , 13 for Halo - AATNNN , n = 18 , 12 , 12 , 13 for Halo - ATZNNN . Two - way ANOVA and Sidak ' s multiple comparison test , ns P > 0 . 05 , * * * P < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(D) HaloTag pulse-chase analysis in WT MEF monitoring fluorescent Halo-AATNNN during up to 6 h of chase in the presence of BafA1. The fluorescent JF646-Halo ligand and the polymer-specific 2C1 antibody stains are shown. (E) Same as (D) for fluorescent Halo-ATZNNN. (F) Quantification of accumulation of fluorescent Halo-AATNNN and Halo-ATZNNN within LAMP1-positive endolysosomes. Mean ± SEM, n=20, 11, 10, 13 for Halo-AATNNN, n= 18, 12, 12, 13 for Halo-ATZNNN. Two-way ANOVA and Sidak's multiple comparison test, ns P>0.05, *** P<0.001.