( E ) ChIP - seq of HEK2935xUAS expressing full GAL4DBDCBX2 . 1 DaxxMycHis showing CBX2 . 1 dependent recruitment of DAXX to promoters of CCND2 RUNX1 genes .

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(E) ChIP-seq analysis of HEK2935xUAS cells expressing full length GAL4DBDCBX2.1 and DaxxMycHis showing CBX2.1 dependent recruitment of DAXX to promoters of CCND2 and RUNX1 genes.


( F ) ChIP - seq of GAL4DBDCBX2 . 1 occupancy in HEK2935xUAS expressing DAXXMycHis ( X - axis ) or GAL4DBDCBX2 . 1 DAXXMycHis ( Y - axis ) , showing ( log2 ) enrichment for GAL4DBDCBX2 . 1 at many TSSs ( ± 1 kb ) in expressing CBX2 . 1 ( Y - axis ) .

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(F) ChIP-seq analysis of GAL4DBDCBX2.1 occupancy in HEK2935xUAS cells expressing DAXXMycHis (X-axis) or GAL4DBDCBX2.1 and DAXXMycHis (Y-axis), showing (log2) enrichment for GAL4DBDCBX2.1 at many TSSs (± 1 kb) in cells expressing the CBX2.1 protein (Y-axis).


( G ) ChIP - seq of GAL4DBDCBX2 . 1 versus DAXXMycHis occupancy in HEK2935xUAS expressing GAL4DBDCBX2 . 1 DAXXMycHis , revealing very comparable enrichment ( log2 ) of both at TSSs . TSSs highlighted in red show enrichment with anti - Myc antibody independently of GAL4DBDCBX2 . 1 occupancy .

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(G) ChIP-seq analysis of GAL4DBDCBX2.1 versus DAXXMycHis occupancy in HEK2935xUAS cells expressing GAL4DBDCBX2.1 and DAXXMycHis, revealing very comparable enrichment levels (log2) of both proteins at TSSs. TSSs highlighted in red show enrichment with anti-Myc antibody independently of GAL4DBDCBX2.1 occupancy.


( H ) ChIP - seq of DAXXMycHis occupancy in HEK2935xUAS expressing DAXXMycHis ( X - axis ) versus GAL4DBDCBX2 . 1 DAXXMycHis ( Y - axis ) showing strong enrichment ( log2 ) for DAXXMycHis at many TSSs of genes , but only when both are expressed ( Y - axis ) , indicating that CBX2 . 1 targets DAXX to . TSSs highlighted in red show enrichment with anti - Myc antibody independently of GAL4DBDCBX2 . 1 occupancy .

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(H) ChIP-seq analysis of DAXXMycHis occupancy in HEK2935xUAS cells expressing DAXXMycHis (X-axis) versus GAL4DBDCBX2.1 and DAXXMycHis (Y-axis) showing strong enrichment (log2) for DAXXMycHis at many TSSs of genes, but only when both proteins are expressed (Y-axis), indicating that CBX2.1 targets DAXX to chromatin. TSSs highlighted in red show enrichment with anti-Myc antibody independently of GAL4DBDCBX2.1 occupancy.


( A ) IF of CBX2 SUMO2 / 3 in PN0 ( n = 10 ) PN5 ( n = 12 ) zygotes . Data information : Scale bars , 10µm .

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(A) IF of CBX2 and SUMO2/3 in PN0 (n=10) and PN5 (n=12) zygotes. Data information: Scale bars, 10µm.


( B ) ChIP - qPCR analyses of HEK2935xUAS co - expressing GAL4DBDCBX2 . 1 either with DAXXMycHis or DAXXΔSIM1 / 2MycHis . Data are means ± ( n = 3 ) .

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(B) ChIP-qPCR analyses of HEK2935xUAS cells co-expressing GAL4DBDCBX2.1 either with DAXXMycHis or DAXXΔSIM1/2MycHis. Data are means ± SEM (n = 3).


( C ) IF of EGFP DAXX in Daxxm - z + PN5 zygotes micro - injected with Daxx - EGFP or DaxxΔSIM1 / 2 - EGFP mRNA ( 100ng / µl ) . Data information : Scale bars , 10µm .

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(C) IF of EGFP and DAXX in Daxxm-z+ PN5 zygotes micro-injected with Daxx-EGFP or DaxxΔSIM1/2-EGFP mRNA (100ng/µl). Data information: Scale bars, 10µm.


( D ) Left : Boxplot displaying EGFP signal intensities at PCH relative to euchromatin in Daxxm - z + zygotes micro - injected with Daxx - EGFP or DaxxΔSIM1 / 2 - EGFP mRNA . Right : Boxplot displaying signal of EGFP at euchromatin in same samples . In boxplots , enter lines show medians ; box limits indicate 25th 75th percentiles as determined by R software ; whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Datapoints are indicated by circles . Experiments were replicated 3 times . * * * P < 0 . 001 , N . S . : no statistical difference , P > 0 . 05 , t - .

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(D) Left: Boxplot displaying EGFP signal intensities at PCH relative to euchromatin in Daxxm-z+ zygotes micro-injected with Daxx-EGFP or DaxxΔSIM1/2-EGFP mRNA. Right: Boxplot displaying signal intensity of EGFP at euchromatin in same samples. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 3 times. ***P<0.001, N.S.: no statistical difference, P>0.05, t-test.


( F ) Ni - pulldown of 6xHisSUMO1 / 2 / 3 - conjugated from HEK293 extracts expressing 6xHis - tagged SUMO1 , SUMO2 or SUMO3 in combination with CBX2 - EGFP . CBX2 SUMO - conjugated CBX2 were detected with an anti - GFP antibody .

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(F) Ni-pulldown of 6xHisSUMO1/2/3-conjugated proteins from HEK293 extracts expressing 6xHis-tagged SUMO1, SUMO2 or SUMO3 in combination with CBX2-EGFP. CBX2 and SUMO-conjugated CBX2 were detected with an anti-GFP antibody.


( G ) Ni - pulldown of 6xHis - SUMO2 conjugated from HEK293 extracts transiently co - expressing 6xHis - SUMO2 with CBX2 - EGFP or CBX24KR - EGFP . Detection of

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(G) Ni-pulldown of 6xHis-SUMO2 conjugated proteins from HEK293 extracts transiently co-expressing 6xHis-SUMO2 with CBX2-EGFP or CBX24KR-EGFP. Detection of CBX2


( H ) IF of DAXX EGFP in wild - type PN5 zygotes , micro - injected with Cbx2 or Cbx24KR - EGFP mRNA ( 100ng / µl ) . Data information : Scale bars , 10µm .

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(H) IF of DAXX and EGFP in wild-type PN5 zygotes, micro-injected with Cbx2 or Cbx24KR-EGFP mRNA (100ng/µl). Data information: Scale bars, 10µm.


( I ) Left : Boxplot displaying signal of DAXX at PCH relative to mean at pat - PCH in wild - type zygotes micro - injected with Cbx2 or Cbx24KR - EGFP mRNA . Right : Absolute intensities of EGFP in same samples . In boxplots , enter lines show medians ; box limits indicate 25th 75th percentiles as determined by R software ; whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Datapoints are indicated by circles . Experiments were replicated 2 times . * * * P < 0 . 001 , t - .

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(I) Left: Boxplot displaying signal intensity of DAXX at PCH relative to mean intensity at pat-PCH in wild-type zygotes micro-injected with Cbx2 or Cbx24KR-EGFP mRNA. Right: Absolute intensities of EGFP in same samples. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2 times. ***P<0.001, t-test.


( A ) IF of DAXX ATRX in PN5 stage pronuclei of wild - type ( n = 19 ) , Hp1βm - z + ( n = 10 ) Suv39h2m - z + ( n = 9 ) zygotes . Data information : Panels Paternal maternal pronuclei of a given condition originate from same zygotes . scale bars 10µm .

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(A) IF of DAXX and ATRX in PN5 stage pronuclei of wild-type (n=19), Hp1βm-z+ (n=10) and Suv39h2m-z+ (n=9) zygotes. Data information: Panels Paternal and maternal pronuclei of a given condition originate from same zygotes. All scale bars 10µm.


( B ) Boxplots displaying signal intensities of DAXX or ATRX at pat - mat - PCH in wild - type ( n = 5 ) Hp1βm - z + ( n = 10 ) mouse zygotes at PN5 . In boxplots , enter lines show medians ; box limits indicate 25th 75th percentiles as determined by R software ; whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Datapoints are indicated by circles . Experiments were replicated 2 times . * P < 0 . 05 , * * P < 0 . 01 , t - .

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(B) Boxplots displaying signal intensities of DAXX or ATRX at pat- and mat-PCH in wild-type (n=5) and Hp1βm-z+ (n=10) mouse zygotes at PN5. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2 times. *P<0.05, **P<0.01, t-test.


( C ) IF of DAXX in PN5 stage pronuclei of zygotes expressing CBX2 - EGFP ( n = 9 ) or CBX2F12A - EGFP ( n = 17 ) . mRNAs ( 100ng / µl ) were injected into mouse PN2 stage zygotes . Yellow dashed circles represent contours of pronuclei . . Data information : Panels C : Paternal maternal pronuclei of a given condition originate from same zygotes . scale bars 10µm .

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(C) IF of DAXX in PN5 stage pronuclei of zygotes expressing CBX2-EGFP (n=9) or CBX2F12A-EGFP (n=17). mRNAs (100ng/µl) were injected into mouse PN2 stage zygotes. Yellow dashed circles represent the contours of the pronuclei.. Data information: Panels C: Paternal and maternal pronuclei of a given condition originate from same zygotes. All scale bars 10µm.


( A ) Developmental progression of Daxxm + z + , Daxxm - z + Daxxm - z - early .

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(A) Developmental progression of Daxxm+z+, Daxxm-z+ and Daxxm-z- early embryos.


( B ) Examples of morula blastocyst with normal large polyploid nuclei . Data information : scale bars 10µm .

(B) Examples of morula and blastocyst embryos with normal and large polyploid nuclei. Data information: All scale bars 10µm.


( C ) Percentages of with normal or polyploid nuclei .

(C) Percentages of embryos with normal or polyploid nuclei.


( D ) Still of time - lapse imaging of first cleavage division in wild - type maternally deficient zygotes expressing H2B - mCherry . Time points represent minutes after prometaphase . Lagging chromosomes micronuclei are indicated by yellow arrowheads . Data information : scale bars 10µm .

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(D) Still images of time-lapse imaging of the first cleavage division in wild-type and maternally deficient zygotes expressing H2B-mCherry. Time points represent minutes after prometaphase. Lagging chromosomes and micronuclei are indicated by yellow arrowheads. Data information: All scale bars 10µm.


( E ) of zygotes with lagging chromosomes during 1st cleavage division . Data represent mean ± s . e . m . in n ≥ 3 replicate experiments .

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(E) Percentage of zygotes with lagging chromosomes during 1st cleavage division. Data represent mean ± s.e.m. in n≥3 replicate experiments.


( F ) IF of 5 - methyl 5 - hydroxy - methyl cytosine of wild - type ( n = 42 ) , Daxxm - z + ( n = 14 ) Ring1m - z + ; Rnf2m - z + ( n = 6 ) zygotes with lagging chromosomes . Yellow arrowheads indicate lagging chromosomes . Data information : scale bars 10µm .

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(F) IF of 5-methyl and 5-hydroxy-methyl cytosine staining of wild-type (n=42), Daxxm-z+ (n=14) and Ring1m-z+; Rnf2m-z+ (n=6) zygotes with lagging chromosomes. Yellow arrowheads indicate lagging chromosomes. Data information: All scale bars 10µm.


( G ) - FISH for major ( green ) minor ( red ) satellites on cleavage chromosomes of a Daxxm - z + zygote with a chromosome broken within major satellite region . Data information : scale bars 10µm .

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(G) DNA-FISH for major (green) and minor (red) satellites on cleavage chromosomes of a Daxxm-z+ zygote with a chromosome broken within the major satellite region. Data information: All scale bars 10µm.


( H ) H3 . 3 - EGFP live - imaging signal in pat - mat - PCH of control Daxxm + z + ( n = 18 ) , Daxxm - z + ( n = 8 ) Ring1m - z + ; Rnf2m - z + ( n = 12 ) zygotes . Scale bar 10µm .

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(H) H3.3-EGFP live-imaging signal in pat- and mat-PCH of control Daxxm+z+ (n=18), Daxxm-z+ (n=8) and Ring1m-z+; Rnf2m-z+ (n=12) zygotes. Scale bar 10µm.


( I ) Left : IF of endogenous H3 ATRX at pat - PCH in control Daxxm - z + PN5 zygotes . Right : profiles of H3 ATRX along in left panel at pat - PCH of zygotes . Scale bars 5 µm . Data information : scale bars 10µm .

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(I) Left: IF of endogenous H3 and ATRX at pat-PCH in control and Daxxm-z+ PN5 zygotes. Right: Fluorescence intensity profiles of H3 and ATRX along white line in left panel at pat-PCH of zygotes. Scale bars 5 µm. Data information: All scale bars 10µm.


( J ) Boxplot displaying H3 signal at pat - PCH of Daxxm + z + Daxxm - z + PN5 zygotes . In boxplots , enter lines show medians ; box limits indicate 25th 75th percentiles as determined by R software ; whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Datapoints are indicated by circles . Experiments were replicated 2 times . * * * P < 0 . 001 , t - , n = 12 for both groups .

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(J) Boxplot displaying H3 signal intensity at pat-PCH of Daxxm+z+ and Daxxm-z+ PN5 zygotes. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2 times. ***P<0.001, t-test, n=12 for both groups.


( K ) IP of DAXX - EGFP DAXXR244A - EGFP with an anti - GFP antibody from extract of HEK293 . Immuno - blot detection was performed with antibodies recognizing GFP , histone variant H3 . 3 CBX2 .

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(K) IP of DAXX-EGFP and DAXXR244A-EGFP proteins with an anti-GFP antibody from nuclear extract of HEK293 cells. Immuno-blot detection was performed with antibodies recognizing GFP, histone variant H3.3 and CBX2.


( L ) of Daxxm - z + zygotes with lagging chromosomes following injection of Tubulin - EGFP , Daxx - EGFP or DaxxR244A - EGFP mRNAs ( 20 or 100 ng / µl ) at PN2 / 3 stage . Data represent mean ± s . e . m . ( n ≥ 2 injection experiments ) . * * * P < 0 . 001 High Daxx - EGFP versus other 3 groups ; Fisher ' s exact ( on pooled ) .

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(L) Percentage of Daxxm-z+ zygotes with lagging chromosomes following injection of Tubulin-EGFP, Daxx-EGFP or DaxxR244A-EGFP mRNAs (20 or 100 ng/µl) at PN2/3 stage. Data represent mean ± s.e.m. (n ≥2 injection experiments). *** P<0.001 High Daxx-EGFP versus other 3 groups; Fisher's exact test (on pooled embryos).


( M ) Left : IF of EGFP endogenous H3 at pat - PCH in Daxxm - z + PN5 zygotes expressing DAXX - EGFP or DAXXR244A - EGFP or nothing exogenously . Right : profiles of H3 EGFP along lines in left panels at pat - PCH of zygotes . Scale bars 5 µm . Data information : scale bars 10µm .

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(M) Left: IF of EGFP and endogenous H3 at pat-PCH in Daxxm-z+ PN5 zygotes expressing DAXX-EGFP or DAXXR244A-EGFP or nothing exogenously. Right: Fluorescence intensity profiles of H3 and EGFP along white lines in left panels at pat-PCH of zygotes. Scale bars 5 µm. Data information: All scale bars 10µm.


( N ) Boxplot displaying 3D of H3 signal at pat - PCH in Daxxm - z + zygotes expressing exogenous DAXX - EGFP ( n = 14 ) or DAXXR244A - EGFP ( n = 13 ) . In boxplots , enter lines show medians ; box limits indicate 25th 75th percentiles as determined by R software ; whiskers extend 1 . 5 times interquartile range from 25th 75th percentiles . Datapoints are indicated by circles . Experiments were replicated 2 - 3 times . * * * P < 0 . 001 Daxx - EGFP versus other 2 groups ; t - .

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(N) Boxplot displaying 3D quantification of H3 signal intensity at pat-PCH in Daxxm-z+ zygotes expressing exogenous DAXX-EGFP (n=14) or DAXXR244A-EGFP (n=13). In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2-3 times. ***P<0.001 Daxx-EGFP versus other 2 groups; t-test.


( A ) FISH of forward reverse strand major satellite sequences in Daxxm + z + Daxxm - z + PN5 zygotes . Scale bars , 10µm .

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(A) RNA FISH of forward and reverse strand major satellite sequences in Daxxm+z+ and Daxxm-z+ PN5 zygotes. Scale bars, 10µm.


( B ) Box plots indicating foci number ( left ) total ( right ) of forward ( top ) reverse ( bottom ) transcription as measured by FISH on 3D paternal pronuclei in Daxxm + z + Daxxm - z + PN5 zygotes . Lower hinge , central upper hinge in boxplots represent 25th , 50th ( median ) 75th percentiles respectively . Upper / lower whiskers extend to largest / smallest values no further than 1 . 5 * IQR from upper / lower hinge , where IQR is inter - quartile range or between 25th 75th percentiles . Datapoints are indicated by circles . Experiments were replicated 3 times . * * * P < 0 . 001 ; t - .

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(B) Box plots indicating the foci number (left) and total intensity (right) of forward (top) and reverse (bottom) transcription as measured by RNA FISH on 3D paternal pronuclei in Daxxm+z+ and Daxxm-z+ PN5 zygotes. Lower hinge, central line and upper hinge in boxplots represent 25th, 50th (median) and 75th percentiles respectively. Upper/lower whiskers extend to the largest/smallest values no further than 1.5 * IQR from the upper/lower hinge, where IQR is inter-quartile range or distance between 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 3 times. ***P<0.001; t-test.


( C , D ) MA - plots showing Fold Change ( FC ) ( in log2 ) in gene expression between indicated samples as a function of averaged expression across samples . Dotted lines indicated log2 ( FC ) of 1 .

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(C, D) MA-plots showing Fold Change (FC) (in log2) in gene expression between indicated samples as a function of averaged expression across all samples. Dotted lines indicated log2(FC) of 1.


( E ) Histogram showing classification of genes into 3 groups according to distribution of ChIP enrichment ( log2 ) of H3K27me3 around TSS in MII

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(E) Histogram showing classification of genes into 3 groups according to distribution of ChIP enrichment (log2) of H3K27me3 around TSS in MII oocytes


( F ) MA - plot showing FC ( in log2 ) in gene expression between indicated samples as a function of averaged expression across samples . Dotted lines indicated log2 ( FC ) of 1 .

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(F) MA-plot showing FC (in log2) in gene expression between indicated samples as a function of averaged expression across all samples. Dotted lines indicated log2(FC) of 1.


( G - I ) Boxplots showing FC ( log2 ) in gene expression between indicated samples for 3 groups of genes classified according to H3K27me3 enrichments ( as shown in E ) . Lower hinge , central upper hinge represent 25th , 50th ( median ) 75th percentiles respectively . Upper / lower whiskers extend to largest / smallest values no further than 1 . 5 * IQR from upper / lower hinge , where IQR is inter - quartile range or between 25th 75th percentiles . Outliers are not displayed . notches extend $ 1 . 5 * IQR / \ sqrt { n } $ where n is a number of genes in each H3K27me3 class . Number of genes ( n ) in " Low " , " Medium " " High " H3K27me3 class used for boxplots are 9824 , 2311 , 2647 respectively . Expression changes were estimated using 2 or 3 biological replicates for each condition . Statistical significance was estimated using two - sided 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G - I) Boxplots showing FC (log2) in gene expression between indicated samples for 3 groups of genes classified according to H3K27me3 enrichments (as shown in E). Lower hinge, central line and upper hinge represent 25th, 50th (median) and 75th percentiles respectively. Upper/lower whiskers extend to the largest/smallest values no further than 1.5 * IQR from the upper/lower hinge, where IQR is inter-quartile range or distance between 25th and 75th percentiles. Outliers are not displayed. The notches extend $1.5*IQR/\sqrt{n}$ where n is a number of genes in each H3K27me3 class. Number of genes (n) in "Low", "Medium" and "High" H3K27me3 class used for boxplots are 9824, 2311, 2647 respectively. Expression changes were estimated using 2 or 3 biological replicates for each condition. Statistical significance was estimated using two-sided t-tests.


( J ) Boxplot showing FC ( log2 ) in gene expression between Daxx deficient control MII oocytes for five groups of genes classified according to FC ( log2 ) in gene expression between Ring1 Rnf2 deficient control GV oocytes . Boxplots were created number of genes used for each boxplot is displayed below . Expression changes were estimated using 3 biological replicates for each condition . Statistical significance was estimated using two - sided t - .

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(J) Boxplot showing FC (log2) in gene expression between Daxx deficient and control MII oocytes for five groups of genes classified according to FC (log2) in gene expression between Ring1 Rnf2 deficient and control GV oocytes. Boxplots were created and number of genes used for each boxplot is displayed below. Expression changes were estimated using 3 biological replicates for each condition. Statistical significance was estimated using two-sided t-tests.


a , Western blot of yeast lysates with polyclonal anti - flag antibody to detect flag - tagged human Beclin 1 expression . Two - hundred micrograms of per lane was loaded . SEY6210 , wild - type yeast ; JCY3000 , SEY6210 disrupted in apg6 / vps30 ; ( + ) control , lysates of mammalian transfected with flag - beclin 1 .

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a, Western blot analysis of yeast cell lysates with polyclonal anti-flag antibody to detect flag-tagged human Beclin 1 expression. Two-hundred micrograms of protein per lane was loaded. SEY6210, wild-type yeast; JCY3000, SEY6210 disrupted in apg6/vps30; (+) control, lysates of mammalian cells transfected with flag-beclin 1.


b , Autophagicbody formation in yeast deprived of nitrogen in presence ( + ) absence ( - ) of 1 mM PMSF . Arrows denote that would be scored as positive in experiment shown in c .

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b, Autophagicbody formation in yeast deprived of nitrogen in the presence (+) and absence (-) of 1 mM PMSF. Arrows denote cells that would be scored as positive in the experiment shown in c.


c , Quantitative effects of apg6 / vps30 beclin 1 transformation on autophagicbody formation in apg6 / vps30 - disrupted yeast in presence of PMSF . with one or more autophagic bodies within vacuole were scored as positive ( see arrows in b ) . A minimum of 100 was counted for each sample . Results represent mean ( ± s . e . m . ) of with autophagic bodies within vacuole for triplicate samples . Similar results were obtained in five independent experiments .

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c, Quantitative effects of apg6/vps30 and beclin 1 transformation on autophagicbody formation in apg6/vps30-disrupted yeast in the presence of PMSF. Cells with one or more autophagic bodies within the vacuole were scored as positive (see arrows in b). A minimum of 100 cells was counted for each sample. Results represent the mean (±s.e.m.) percentage of cells with autophagic bodies within the vacuole for triplicate samples. Similar results were obtained in five independent experiments.


d , Sorting of vacuolar carboxypeptidase Y , ( CPY ) . p2CPY , precursor form ; mCPY , sorted mature form . Mr , relative molecular mass .

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d, Sorting of vacuolar protein carboxypeptidase Y, (CPY). p2CPY, precursor form; mCPY, sorted mature form. Mr, relative molecular mass.


a - f , Electron of MCF7 . control ( clone 38 ) ( a , b ) MCF7 . beclin1 ( clone 17 ) ( c - f ) grown in nutrient - rich media ( a , c ) or subjected to 4 h of serum - acid deprivation ( b , d - f ) . Asterisks in e f denote autophagic vacuoles that would be counted in experiment shown in g . Autophagic vacuoles were defined as double - vacuolar structures containing recognizable contents . Scale bars , 1 µm .

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a-f, Electron micrographs of MCF7.control (clone 38) (a, b) and MCF7.beclin1 (clone 17) cells (c- f) grown in nutrient-rich media (a, c) or subjected to 4 h of serum and amino-acid deprivation (b, d-f). Asterisks in e and f denote autophagic vacuoles that would be counted in the experiment shown in g. Autophagic vacuoles were defined as double-membrane vacuolar structures containing recognizable cytoplasmic contents. Scale bars, 1 µm.


g , Quantitative effects of beclin 1 on basal nutrient - deprivation - induced autophagy of MCF7 . Bars indicate mean ( ± s . e . m . ) number of autophagic vacuoles per for growing in normal media ( black ) , for subjected to 4 h of serum - acid deprivation ( grey ) , for pre - treated for 30 min with 10 mM 3 - MA subjected to 4 h of serum - acid deprivation in presence of 3 - MA ( open ) . Mean was determined by counting total number of autophagic vacuoles in each for 100 per clone per treatment .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

g, Quantitative effects of beclin 1 on basal and nutrient-deprivation-induced autophagy of MCF7 cells. Bars indicate mean (±s.e.m.) number of autophagic vacuoles per cell for cells growing in normal media (black), for cells subjected to 4 h of serum and amino-acid deprivation (grey), and for cells pre-treated for 30 min with 10 mM 3-MA and subjected to 4 h of serum and amino-acid deprivation in the presence of 3-MA (open). Mean was determined by counting the total number of autophagic vacuoles in each cell for 100 cells per clone per treatment.


h , Comparison of rates of degradation of long - lived in MCF7 . control ( squares , clone 38 ) , MCF7 . beclin1 ( circles , clone 17 ) MCF7 . beclin1stop ( triangles , clone 70 ) , in EBSS + 10 % serum complete ( open symbols , solid lines ) in EBSS alone ( closed symbols , solid lines ) , in EBSS + 10 mM 3 - MA ( closed symbols , dotted lines ) . Results are mean ( ± s . e . m . ) of triplicate wells . Similar results were obtained in three independent experiments . Results shown with these clones are representative of results obtained with other clones analysed by electron microscopy in g .

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h, Comparison of the rates of degradation of long-lived proteins in MCF7.control cells (squares, clone 38), MCF7.beclin1 cells (circles, clone 17) and MCF7.beclin1stop cells (triangles, clone 70), in EBSS + 10% serum and complete amino acids (open symbols, solid lines) in EBSS alone (closed symbols, solid lines), and in EBSS + 10 mM 3-MA (closed symbols, dotted lines). Results are mean (±s.e.m.) of triplicate wells. Similar results were obtained in three independent experiments. Results shown with these clones are representative of results obtained with other clones analysed by electron microscopy in g.


a , Western blot of flag - tagged beclin - 1 - transfected MCF7 clones . C , MCF7 . control ( clone 38 ) ; St , MCF7 . beclin1stop ( clone 70 ) . Mr , relative molecular mass .

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a, Western blot analysis of flag-tagged beclin-1-transfected MCF7 clones. C, MCF7.control cells (clone 38); St, MCF7.beclin1stop cells (clone 70). Mr, relative molecular mass.


b , Western blot of flag - tagged beclin1stop - transfected MCF7 clones . C , MCF . control ( clone 38 ) ; B , MCF7 . beclin1 ( clone 1 ) .

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b, Western blot analysis of flag-tagged beclin1stop-transfected MCF7 clones. C, MCF.control cells (clone 38); B, MCF7.beclin1 cells (clone 1).


c , Photomicrographs of MCF7 . control ( left panel , clone 38 ) , MCF7 . beclin1 ( middle panel , clone 17 ) MCF7 . beclin1stop ( right panel , clone 70 ) 48 h after seeding at similar densities .

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c, Photomicrographs of MCF7.control cells (left panel, clone 38), MCF7. beclin1 cells (middle panel, clone 17) and MCF7.beclin1stop cells (right panel, clone 70) 48 h after seeding at similar densities.


d , Proliferation of MCF7 . control ( clones 37 38 ; closed squares circles , respectively , solid lines ) , MCF7 . beclin1 ( clones 1 , 6 , 17 ; open squares , circles , triangles , respectively , solid lines ) MCF7 . beclin1stop ( clone 70 , closed triangles , dotted ) . Results represent mean Absorbance ( A ) ( ± s . e . m . ) for six wells ; similar results were obtained in three independent experiments .

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d, Proliferation of MCF7.control cells (clones 37 and 38; closed squares and circles, respectively, solid lines), MCF7.beclin1 cells (clones 1, 6, 17; open squares, circles, triangles, respectively, solid lines) and MCF7.beclin1stop cells (clone 70, closed triangles, dotted line). Results represent mean Absorbance (A) (±s.e.m.) for six wells; similar results were obtained in three independent experiments.


e , viability of MCF7 clones determined by trypan blue . Symbols for each clone are as in

e, Cell viability of MCF7 clones determined by trypan blue staining. Symbols for each clone are as in d


f , Clonigenicity in semisolid medium ( soft agar ) of MCF7 clones . Results are mean ( ± s . e . m . ) for pooled triplicate wells from 3 - 4 independent experiments .

f, Clonigenicity in semisolid medium (soft agar) of MCF7 clones. Results are mean (±s.e.m.) for pooled triplicate wells from 3-4 independent experiments.


g , Tumour formation in NCR nude mice injected subcutaneously with MCF7 clones . Numbers on top of bar indicate number of autopsy - confirmed tumours at 8 per number of mice injected with each clone .

g, Tumour formation in NCR nude mice injected subcutaneously with MCF7 clones. Numbers on top of bar indicate number of autopsy-confirmed tumours at 8 weeks per number of mice injected with each clone.


a , Western blot of human breast carcinoma lines . Results are from 8 representative lines out of a total of 11 analysed .

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a, Western blot analysis of human breast carcinoma cell lines. Results are from 8 representative cell lines out of a total of 11 analysed.


b , Western blot of matched normal breast breast carcinoma tissue from with sporadic invasive breast carcinoma . Results are from 7 representative cases out of a total of 17 analysed . N , normal breast tissue ; T , tumour tissue ; Mr , relative molecular mass .

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b, Western blot analysis of matched normal breast and breast carcinoma tissue from patients with sporadic invasive breast carcinoma. Results are from 7 representative cases out of a total of 17 analysed. N, normal breast tissue; T, tumour tissue; Mr, relative molecular mass.


c , Immunohistochemical of paraffin - embedded sections of breast tissue from 441 shown in b . Adjacent sections are stained with pre - immune 843 rabbit serum ( left column ) , 843 anti - Beclin 1serum ( middle column ) anti - cytokeratin ( right column ) . Top row shows low power view of region of breast containing tumour ( T ) normal mammary epithelial ( N ) . Middle row shows higher power view of normal mammary epithelial . Bottom row shows higher power view of tumour . Scale bars , 1 mm .

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c, Immunohistochemical analysis of paraffin-embedded sections of breast tissue from patient 441 shown in b. Adjacent sections are stained with pre-immune 843 rabbit serum (left column), 843 anti-Beclin 1serum (middle column) and anti-cytokeratin (right column). Top row shows low power view of region of breast containing tumour (T) and normal mammary epithelial duct (N). Middle row shows higher power view of normal mammary epithelial duct. Bottom row shows higher power view of tumour. Scale bars, 1 mm.


Biological replicates of transcriptional profiles ( Tx ) measured in separately prepared free lysate batches of E . coli .

Biological replicates of transcriptional profiles (Tx) measured in separately prepared cell free lysate batches of E. coli.


Correlation between transcriptional profiles ( Tx ) for regulatory libraries from in vitro in vivo measurements in E . coli . color scale indicates density of data points in a given of plot .

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Correlation between transcriptional profiles (Tx) for regulatory sequence libraries from in vitro and in vivo measurements in E. coli. The color scale indicates the density of data points in a given area of the plot.


Correlation between TSS calls ( in bp from ATG start codon ) of regulatory sequences from in vitro in vivo measurements in E . coli .

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Correlation between primary TSS calls (in bp from ATG start codon) of regulatory sequences from in vitro and in vivo measurements in E. coli.


Optimization of transcriptional output using different concentrations of Mg - glutamate K - glutamate in 10 - free expression systems with Broccoli as a reporter ( solid lines , optimal buffer composition shown in red ) . No template controls are shown as grey dashed lines . 12 . 5 nM of template was used in systems , except L . lactis ( 50 nM used ) .

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Optimization of transcriptional output using different concentrations of Mg-glutamate and K-glutamate in 10 bacterial cell-free expression systems with Broccoli as a reporter (solid lines, optimal buffer composition shown in red). No DNA template controls are shown as grey dashed lines. 12.5 nM of DNA template was used in all systems, except L. lactis (50 nM used).


Transcriptional activities ( Tx ) of 421 regulatory sequences that were active in 10 - free expression systems .

Transcriptional activities (Tx) of 421 regulatory sequences that were active in all 10 bacterial cell-free expression systems.


Pairwise comparison of transcriptional profiles of 421 universally - active regulatory sequences between species . Example scatter plots with relatively high low Pearson correlation in ( marked i ii ) are shown on left .

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Pairwise comparison of transcriptional profiles of 421 universally-active regulatory sequences between bacterial species. Example scatter plots with relatively high and low Pearson correlation in the heatmap (marked i and ii) are shown on left.


Correlation between evolutionary divergences ( 16S rRNA identity ) pairwise Pearson correlation of transcriptional profiles . Dashed represents linear regression .

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Correlation between evolutionary divergences (16S rRNA percent identity) and pairwise Pearson correlation of transcriptional profiles. Dashed line represents linear regression.


Pairwise correlation of RS7003 transcriptional profiles between hybrid lysate single constituent species lysates for ( d ) E . coli + B .

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Pairwise correlation of RS7003 transcriptional profiles between hybrid lysate and single constituent species lysates for (d) E. coli + B. subtilis


Pairwise correlation of RS7003 transcriptional profiles between hybrid lysate single constituent species lysates for ( e ) E . coli + C . glutamicum hybrids with different mixing ratios .

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Pairwise correlation of RS7003 transcriptional profiles between hybrid lysate and single constituent species lysates for (e) E. coli + C. glutamicum hybrids with different mixing ratios.


A Expression of transcripts coding for PI3K subunit genes in pDCs ( n = 12 biological replicates ) . In boxplot , middle , bottom , top of box represent median , 1st quantile 3rd quantile , respectively ; height of box is given by difference between 3rd 1st quantiles ( IQR , interquartile range ) ; whiskers is 1 . 5 times interquartile range . Data points beyond whiskers represent values more extreme than 1 . 5 times interquartile range above or below 3rd or 1st quantile , respectively .

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A Expression of transcripts coding for PI3K subunit genes in pDCs (n =12 biological replicates). In the boxplot, the middle, the bottom, and the top of the box represent the median, the 1st quantile and the 3rd quantile, respectively; the height of the box is given by the difference between the 3rd and the 1st quantiles (IQR, interquartile range); the whiskers length is 1.5 times the interquartile range. Data points beyond the whiskers represent values more extreme than 1.5 times the interquartile range above or below the 3rd or the 1st quantile, respectively.


C Kyn in culture supernatants of pDCs incubated for 24 h with medium alone ( Ctrl ) or IFN - γ after 1 − h pretreatment with PI3K inhibitors .

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C Kyn levels in culture supernatants of pDCs incubated for 24 h with medium alone (Ctrl) or IFN-γ after 1−h pretreatment with PI3K inhibitors.


D Real - time PCR of Ido1 Tgfb1 transcripts in pDCs stimulated for 18 h with TGF - β , in presence or absence ( Ctrl , control ) of catalytic PI3K inhibitors , normalized to expression of Gapdh presented relative to results in untreated ( dotted , 1 - fold ) .

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D Real-time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs stimulated for 18 h with TGF-β, in the presence or absence (Ctrl, control) of catalytic PI3K inhibitors, normalized to the expression of Gapdh and presented relative to results in untreated cells (dotted line, 1-fold).


E In vivo suppression of activity of HY - pulsed CD8 − DCs in combination with a minority fraction ( 5 % ) of pDCs conditioned in vitro with TGF - β ( or medium ) for 24 h in presence or absence ( Ctrl ) of catalytic inhibitors as in C . of skin reactivity of recipient mice to eliciting peptide at 15 days is presented as change in footpad ( experimental versus control footpads ) .

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E In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs conditioned in vitro with TGF-β (or medium) for 24 h in the presence or absence (Ctrl) of catalytic inhibitors as in C. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads).


F Real - time PCR of Ido1 Tgfb1 transcripts in pDCs transfected with p110α - , β - , δ - specific , or negative control ( nc ) siRNA stimulated for 18 h with TGF - β , normalized to expression of Gapdh presented relative to results in unstimulated ( dotted , 1 - fold ) .

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F Real-time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs transfected with p110α-, β-, δ-specific, or negative control (nc) siRNA and stimulated for 18 h with TGF-β, normalized to the expression of Gapdh and presented relative to results in unstimulated cells (dotted line, 1-fold).


G In vivo suppression of activity of HY - pulsed CD8 − DCs in combination with a minority fraction ( 5 % ) of pDCs transfected with siRNA as in ( F ) conditioned in vitro with TGF - β ( or medium ) for 24 h . of skin reactivity of recipient mice to eliciting peptide at 15 days is presented as change in footpad ( experimental versus control footpads ) .

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G In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs transfected with siRNA as in (F) and conditioned in vitro with TGF-β (or medium) for 24 h. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads).


C Pull - down ( PD ) of lysates from P1 . HTR with unphosphorylated ( YENM ) tyrosine - phosphorylated IDO1 peptides ( pYENM , pITIM1 , pITIM2 ) , analyzed by sequential immunoblotting with anti − p85 , − SHP - 1 , - SHP - 2 antibodies .

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C Pull-down (PD) assay of lysates from P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated IDO1 peptides (pYENM, pITIM1, and pITIM2), analyzed by sequential immunoblotting with anti−p85, −SHP-1, and -SHP-2 antibodies.


D Immunoblotting with anti - IDO1 - p85 antibodies of IDO1 immunoprecipitates obtained from P1 . HTR transfected with IDO1 . WT or IDO1 . Y149F . Mock - transfected P1 . HTR were used as a control .

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D Immunoblotting with anti-IDO1 and -p85 antibodies of IDO1 immunoprecipitates obtained from P1.HTR cells transfected with IDO1.WT or IDO1.Y149F. Mock-transfected P1.HTR cells were used as a control.


E Tyrosine phosphatase activity in anti - IDO1 immunoprecipitates from lysates of P1 . HTR transfectants as in D .

E Tyrosine phosphatase activity in anti-IDO1 immunoprecipitates from lysates of P1.HTR transfectants as in D.


F Kyn release over 24 h by P1 . HTR transfectants as in D .

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F Kyn release over 24 h by P1.HTR transfectants as in D.


A Confocal immunofluorescence microscopy of P1 . HTR either untransfected ( Untr ) or transfected with IDO1 . WT ( WT ) or vector alone ( Mock ) stained with antibodies recognizing IDO1 ( green ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 20 µm .

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A Confocal immunofluorescence microscopy images of P1.HTR cells either untransfected (Untr) or transfected with IDO1.WT (WT) or vector alone (Mock) and stained with antibodies recognizing IDO1 (green). Nuclei were stained with DAPI (blue). Scale bar, 20 µm.


B Confocal immunofluorescence microscopy of P1 . HTR transfected with IDO1 . WT , co - stained with antibodies recognizing IDO1 ( green ) markers of intracellular organelles structures ( as indicated ; red ) . Nuclei were stained with DAPI ( blue ) . merge imagine is shown for stainings or without DAPI ( no DAPI ) . Scale bar , 10 µm .

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B Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT, co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (as indicated; red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.


C Immunoblotting of EE purified from P1 . IDO1 . WT . Mock - transfected as in A were used as control .

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C Immunoblotting analysis of EE purified from P1.IDO1.WT cells. Mock-transfected cells as in A were used as control.


D Distribution of IDO1 . WT expressed by P1 . HTR transfected in isopycnic sucrose gradients . Fractions were immunoblotted using antibodies specific for IDO1 intracellular organelles or structures as indicated .

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D Distribution of the IDO1.WT protein expressed by P1.HTR transfected cells in isopycnic sucrose gradients. Fractions were immunoblotted using antibodies specific for IDO1 and intracellular organelles or structures as indicated.


A Confocal immunofluorescence microscopy of P1 . HTR transfected with IDO1 . WT or IDO1 . Y149F co - stained with antibodies recognizing IDO1 ( green ) EEA1 ( red ) . Nuclei were stained with DAPI ( blue ) . merge imagine is shown for stainings or without DAPI ( no DAPI ) . Scale bar , 10 µm .

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A Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT or IDO1.Y149F and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.


B Pearson ' s coefficient of IDO1 colocalization with EEA1 in P1 . HTR transfected as in A , with each point representing colocalization within an image stack . Data represent mean ± SD ( n > 24 from three independent experiments ) . Statistical was performed using two - unpaired Student ' s t - . * * * P < 0 . 001 .

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B Pearson's coefficient of IDO1 colocalization with EEA1 in P1.HTR cells transfected as in A, with each point representing colocalization within an image stack. Data represent mean ± SD (n > 24 cells from three independent experiments). Statistical analysis was performed using two-tailed unpaired Student's t-test. ***P < 0.001.


C Distribution of IDO1 . WT IDO1 . Y149F mutants in homogenates of P1 . HTR along isopycnic sucrose gradient as in Fig 3D . Fractions were immunoblotted using antibodies specific for IDO1 .

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C Distribution of IDO1.WT and the IDO1.Y149F mutants in homogenates of P1.HTR cells along the isopycnic sucrose gradient as in Fig 3D. Fractions were immunoblotted using antibodies specific for IDO1.


D distribution of IDO1 . WT IDO1 . Y149F along isopycnic sucrose gradient is shown in term of densitometric in each fraction ( mean ± SD of three experiments ) .

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D The distribution of IDO1.WT and IDO1.Y149F proteins along the isopycnic sucrose gradient is shown in term of densitometric analysis in each fraction (mean ± SD of three experiments).


A Pull - down of lysates from untransfected P1 . HTR with unphosphorylated ( YENM ) tyrosine - phosphorylated ( pYENM ) IDO1 peptides , analyzed by immunoblotting with anti - p85 , - p110α , - p110β , - p110γ , - p110δ antibodies . Whole lysates ( WCL ) were used as control of lysate amount .

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A Pull-down assay of lysates from untransfected P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated (pYENM) IDO1 peptides, analyzed by immunoblotting with anti-p85, -p110α, -p110β, -p110γ, and -p110δ antibodies. Whole cell lysates (WCL) were used as control of lysate amount.


B Confocal immunofluorescence microscopy of P1 . HTR transfected with IDO1 . WT incubated with class IA PI3K p110 isoform inhibitors ( indicated ) or medium alone ( Ctrl ) for 24 h co - stained with antibodies recognizing IDO1 ( green ) EEA1 ( red ) . Nuclei were stained with DAPI ( blue ) . merge imagine is shown for stainings or without DAPI ( no DAPI ) . Scale bar , 10 µm .

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B Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT incubated with class IA PI3K p110 isoform inhibitors (indicated) or medium alone (Ctrl) for 24 h and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.


C Pearson ' s coefficient of IDO1 colocalization with EEA1 in P1 . HTR transfected treated as in ( B ) , with each point representing colocalization within an image stack . Data represent mean ± SD ( n > 20 from two independent experiments ) .

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C Pearson's coefficient of IDO1 colocalization with EEA1 in P1.HTR cells transfected and treated as in (B), with each point representing colocalization within an image stack. Data represent mean ± SD (n > 20 cells from two independent experiments).


D Tyrosine phosphatase activity in anti - IDO1 immunoprecipitates from P1 . HTR transfected with IDO1 . WT treated with PI3K catalytic inhibitors ( indicated ) or medium alone ( ctrl ) . Data represent mean ± SD ( n = 3 technical replicates ) .

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D Tyrosine phosphatase activity in anti-IDO1 immunoprecipitates from P1.HTR cells transfected with IDO1.WT cells and treated with PI3K catalytic inhibitors (indicated) or medium alone (ctrl). Data represent the mean ± SD (n = 3 technical replicates).


A Confocal immunofluorescence microscopy of freshly purified pDCs co - stained with antibodies recognizing IDO1 ( green ) markers of intracellular organelles structures ( indicated ; red ) . Nuclei were stained with DAPI ( blue ) . merge imagine is shown for stainings or without DAPI ( no DAPI ) . Scale bar , 5 µm .

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A Confocal immunofluorescence microscopy images of freshly purified pDCs co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (indicated; red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 5 µm.


B Confocal immunofluorescence microscopy of pDCs incubated for 24 h with TGF - β or IFN - γ co - stained with antibodies recognizing IDO1 ( green ) EEA1 ( red ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 5 µm . C Pearson ' s coefficient of IDO1 colocalization with EEA1 in pDCs either untreated or stimulated with TGF - β or IFN - γ ( as in B ) , with each point representing colocalization within an image stack . Data represent mean ± SD ( n > 20 from three independent experiments ) .

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B Confocal immunofluorescence microscopy images of pDCs incubated for 24 h with TGF-β or IFN-γ and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). Scale bar, 5 µm. C Pearson's coefficient of IDO1 colocalization with EEA1 in pDCs either untreated or stimulated with TGF-β or IFN-γ (as in B), with each point representing colocalization within an image stack. Data represent mean ± SD (n > 20 cells from three independent experiments).


A . of adult ( 9 - 14 week ) CEP250 + / + CEP250 - / - mice . B . of CEP250 + / + CEP250 - / - mice ( 9 - 14 week ) . CEP250 + / + n = 5 mice , CEP250 - / - n = 5 mice . Data information : In ( B , data are presented as mean ± . n . s . , not significant , * * P < 0 . 01 , * * * * P < 0 . 0001 ( unpaired Student ' s t - ) . In ( B whiskers represent range of data , with bottom top represent min max value of dataset respectively .

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A. Size of adult (9-14 week) CEP250+/+ and CEP250-/- mice. B. Body weight of CEP250+/+ and CEP250-/- mice (9-14 week). CEP250+/+ n=5 mice, CEP250-/- n=5 mice. Data information: In (B , data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test). In (B whiskers represent the range of the data, with bottom and top represent min and max value of the dataset respectively.


C . Brains from adult CEP250 + / + CEP250 - / - mice ( 9 - 14 week ) . Scale bars : 1000 µm . D . Brain of 9 - 14 - week - old mice . CEP250 + / + n = 5 mice , CEP250 - / - n = 5 mice . Data information : In data are presented as mean ± . n . s . , not significant , * * P < 0 . 01 , * * * * P < 0 . 0001 ( unpaired Student ' s t - ) . In whiskers represent range of data , with bottom top represent min max value of dataset respectively .

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C. Brains from adult CEP250+/+ and CEP250-/- mice (9-14 week). Scale bars: 1000 µm. D. Brain weight of 9-14-week-old mice. CEP250+/+ n=5 mice, CEP250-/- n=5 mice. Data information: In data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test). In whiskers represent the range of the data, with bottom and top represent min and max value of the dataset respectively.


F . Adult testis ( 9 - 14 week ) of CEP250 + / + CEP250 - / - mice . Scale bars : 1000 µm . G . Adult testis ( 9 - 14 week ) of CEP250 + / + CEP250 - / - mice . CEP250 + / + n = 10 mice , CEP250 - / - n = 10 mice . Data information : In data are presented as mean ± . n . s . , not significant , * * P < 0 . 01 , * * * * P < 0 . 0001 ( unpaired Student ' s t - ) . In G ) whiskers represent range of data , with bottom top represent min max value of dataset respectively .

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F. Adult testis (9-14 week) of CEP250+/+ and CEP250-/- mice. Scale bars: 1000 µm. G. Adult testis (9-14 week) weight of CEP250+/+ and CEP250-/- mice. CEP250+/+ n=10 mice, CEP250-/- n=10 mice. Data information: In data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test). In G) whiskers represent the range of the data, with bottom and top represent min and max value of the dataset respectively.


K . Bright - field image of live epididymal sperm from CEP250 + / + CEP250 - / - mice ( 9 - 14 week ) . Scale bars : 10 µm . Arrow heads highlight sperm heads . L . of ( K ) , sperm number per mouse . CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . Data information : In data are presented as mean ± . n . s . , not significant , * * P < 0 . 01 , * * * * P < 0 . 0001 ( unpaired Student ' s t - ) .

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K. Bright-field image of live epididymal sperm from CEP250+/+ and CEP250-/- mice (9-14 week). Scale bars: 10 µm. Arrow heads highlight the sperm heads. L. Quantification of (K), sperm number per mouse. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. Data information: In data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test).


N . Immunofluorescence for GATA4 ( green ) with DAPI ( blue ) of 10 µm testis cryo - sections from adult CEP250 + / + CEP250 - / - mice ( 9 - 14 week ) . Scale bars : 100 µm . Dashed lines highlight STs . O . of ( N ) , CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . 50 STs were analyzed for each mouse . See Appendix Fig S2 for ST number per section ST diameter . Data information : In O ) , data are presented as mean ± . n . s . , not significant , * * P < 0 . 01 , * * * * P < 0 . 0001 ( unpaired Student ' s t - ) .

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N. Immunofluorescence for GATA4 (green) with DAPI staining (blue) of 10 µm testis cryo-sections from adult CEP250+/+ and CEP250-/- mice (9-14 week). Scale bars: 100 µm. Dashed lines highlight the STs. O. Quantification of (N), CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 50 STs were analyzed for each mouse. See Appendix Fig S2 for ST number per section and ST diameter. Data information: In O), data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test).


D . Immunofluorescence for MVH ( green ) with DAPI ( blue ) of 10 µm testis cryo - sections from neonatal mice ( P5 , P10 P14 ) CEP250 + / + CEP250 - / - . Scale bars : 20 µm .

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D. Immunofluorescence for MVH (green) with DAPI staining (blue) of 10 µm testis cryo-sections from neonatal mice (P5, P10 and P14) CEP250+/+ and CEP250-/-. Scale bars: 20 µm.


E . of CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . Data information : E ) , data are presented as mean ± . n . s . , not significant , * * P < 0 . 01 , * * * * P < 0 . 0001 ( unpaired Student ' s t - ) .

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E. Quantification of CEP250+/+ n=3 mice, CEP250-/- n=3 mice. Data information: E), data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test).


F . of C - CASP3 positive normalized to total number per seminiferous tubule ( ST ) . Dots ( colors reflect STs from different mice ) represent value per ST , CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . 30 STs were analyzed for each mouse . Data information : In ( F data are presented as mean ± . n . s . , not significant , * * * P < 0 . 005 , * * * * P < 0 . 0001 ( Mann - Whitney ) .

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F. Quantification of C-CASP3 positive cells normalized to total cell number per seminiferous tubule (ST). Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 30 STs were analyzed for each mouse. Data information: In (F data are presented as mean ± SEM. n.s., not significant, ***P < 0.005 , ****P < 0.0001 (the Mann-Whitney test).


G . C - CASP3 ( green ) MVH ( magenta ) with DAPI ( blue ) of 10 µm testis cryo - sections from neonatal mice ( P5 , P10 P14 ) CEP250 + / + CEP250 - / - , scale bars : 20 µm . H . of ( G ) . of C - CASP3 MVH double positive in relation to total C - CASP3 positive per ST from P5 , P10 P14 mice . Dots ( colors reflect STs from different mice ) represent value per ST , CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . 30 STs were analyzed for each mouse . I . of ( G ) . of C - CASP3 MVH double positive in relation to total MVH positive ( germ ) per ST from P5 , P10 P14 mice . Dots ( colors reflect STs from different mice ) represent value per ST , CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . 30 STs were analyzed for each mouse . Data information In H , I ) , data are presented as mean ± . n . s . , not significant , * * * P < 0 . 005 , * * * * P < 0 . 0001 ( Mann - Whitney ) .

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G. C-CASP3 (green) and MVH (magenta) staining with DAPI (blue) of 10 µm testis cryo-sections from neonatal mice (P5, P10 and P14) CEP250+/+ and CEP250-/-, scale bars: 20 µm. H. Quantification of (G). Percentage of C-CASP3 and MVH double positive cells in relation to total C-CASP3 positive cells per ST from P5, P10 and P14 mice. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 30 STs were analyzed for each mouse. I. Quantification of (G). Percentage of C-CASP3 and MVH double positive cells in relation to total MVH positive cells (germ cells) per ST from P5, P10 and P14 mice. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 30 STs were analyzed for each mouse. Data information In H, I), data are presented as mean ± SEM. n.s., not significant, ***P < 0.005 , ****P < 0.0001 (the Mann-Whitney test).


A . Immunofluorescence for PLZF ( red ) with DAPI ( blue ) of 10 µm cryo - sections of testes from CEP250 + / + CEP250 - / - mice at P5 , P10 P14 . Scale bars : 20 μm .

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A. Immunofluorescence staining for PLZF (red) with DAPI staining (blue) of 10 µm cryo-sections of testes from CEP250+/+ and CEP250-/- mice at P5, P10 and P14. Scale bars: 20 μm.


B . of PLZF + per seminiferous ( ST ) section of CEP250 + / + CEP250 - / - , testes at P5 , P10 P14 . Dots ( colors reflect STs from different mice ) represent value per ST , CEP250 + / + ( P5 ) n = 3 mice , CEP250 - / - ( P5 ) n = 3 mice , 50 STs were analyzed for each mouse ; CEP250 + / + ( P10 ) n = 3 mice , CEP250 - / - ( P10 ) n = 3 mice , at least 30 STs were analyzed for each mouse ; CEP250 + / + ( P14 ) n = 3 mice , CEP250 - / - ( P14 ) n = 3 mice , at least 50 ST were analyzed for each mouse . Data information : In ( B data are presented as mean ± . n . s . , not significant , * P < 0 . 05 , * * * P < 0 . 005 , * * * * P < 0 . 0001 ( Mann - Whitney ) .

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B. Quantification of PLZF+ cells per seminiferous (ST) section of CEP250+/+ and CEP250-/-, testes at P5, P10 and P14. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ (P5) n=3 mice, CEP250-/- (P5) n=3 mice, 50 STs were analyzed for each mouse; CEP250+/+ (P10) n=3 mice, CEP250-/- (P10) n=3 mice, at least 30 STs were analyzed for each mouse; CEP250+/+ (P14) n=3 mice, CEP250-/- (P14) n=3 mice, at least 50 ST were analyzed for each mouse. Data information: In (B data are presented as mean ± SEM. n.s., not significant, *P<0.05, ***P < 0.005, ****P < 0.0001 (the Mann-Whitney test).


C . Immunofluorescence for SCP1 ( green ) , PLZF ( red ) DAPI ( blue ) of 10 µm cryo - sections of testes from CEP250 + / + CEP250 - / - mice at P2 , P3 , P5 P7 . Arrowheads highlight PLZF - SCP1 + ( green ) , which represent differentiated spermatogonia . Scale bars : 20 μm .

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C. Immunofluorescence staining for SCP1 (green), PLZF (red) and DAPI (blue) of 10 µm cryo-sections of testes from CEP250+/+ and CEP250-/- mice at P2, P3, P5 and P7. Arrowheads highlight PLZF- SCP1+ (green) cells, which represent differentiated spermatogonia. Scale bars: 20 μm.


of number of PLZF + SCP1 - , PLZF + SCP1 + , PLZF - SCP1 + normalized to total number per ST section in CEP250 + / + CEP250 - / - testes at P2 , P3 respectively . Dots ( colors reflect STs from different mice ) represent value per ST , CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice , at least 40 STs were analyzed for each mouse . Data information : data are presented as mean ± . n . s . , not significant , * P < 0 . 05 , * * * P < 0 . 005 , * * * * P < 0 . 0001 ( Mann - Whitney ) .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of The number of PLZF+ SCP1-, PLZF+ SCP1+, PLZF- SCP1+ cells normalized to total cell number per ST section in CEP250+/+ and CEP250-/- testes at P2, P3 respectively. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 40 STs were analyzed for each mouse. Data information: data are presented as mean ± SEM. n.s., not significant, *P<0.05, ***P < 0.005, ****P < 0.0001 (the Mann-Whitney test).


of number of PLZF + SCP1 - , PLZF + SCP1 + , PLZF - SCP1 + normalized to total number per ST section in CEP250 + / + CEP250 - / - testes at , P5 P7 respectively . Dots ( colors reflect STs from different mice ) represent value per ST , CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice , at least 40 STs were analyzed for each mouse . Data information : data are presented as mean ± . n . s . , not significant , * P < 0 . 05 , * * * P < 0 . 005 , * * * * P < 0 . 0001 ( Mann - Whitney ) .

Quantification of The number of PLZF+ SCP1-, PLZF+ SCP1+, PLZF- SCP1+ cells normalized to total cell number per ST section in CEP250+/+ and CEP250-/- testes at , P5 and P7 respectively. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 40 STs were analyzed for each mouse. Data information: data are presented as mean ± SEM. n.s., not significant, *P<0.05, ***P < 0.005, ****P < 0.0001 (the Mann-Whitney test).


B . Representative of mouse seminiferous tubule ( ST ) sections stained for PLZF ( red ) , SCP1 ( green ) DAPI ( blue ) at P4 , P6 P7 . Scale bars : 20 μm . Arrowheads highlight PLZF - SCP1 + ( green ) localize to middle of ST lumen , which represent differentiated spermatogonia that translocated to middle of ST lumen . C . of ( B ) . Shown is number of PLZF - SCP1 + which translocated inwards seminiferous tubule lumen per seminiferous tubule section , as normalized to total number per ST section . Dots ( colors reflect STs from different mice ) represent value per ST , CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice , at least 30 STs were analyzed for each mouse . D . of ( B ) . Shown is total number of SCP1 + which translocated inwards seminiferous tubule lumen per ST section , as normalized to total number per seminiferous tubule section . Dots ( colors reflect STs from different mice ) represent value per ST , CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice , at least 30 STs were analyzed for each mouse . E . of ( B ) . Shown is number of PLZF + SCP1 + which translocated inwards seminiferous tubule lumen per seminiferous tubule section , as normalized to total number per seminiferous tubule section . Dots ( colors reflect STs from different mice ) represent value per ST , CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice , at least 30 STs were analyzed for each mouse . Data information : In ( C - E ) , data are presented as mean ± . n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 ( Mann - Whitney ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O 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-MEASURED_VAR O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of mouse seminiferous tubule (ST) sections stained for PLZF (red), SCP1 (green) and DAPI (blue) at P4, P6 and P7. Scale bars: 20 μm. Arrowheads highlight PLZF- SCP1+ (green) cells localize to the middle of the ST lumen, which represent differentiated spermatogonia that translocated to the middle of the ST lumen. C. Quantification of (B). Shown is the number of PLZF- SCP1+ cells which translocated inwards the seminiferous tubule lumen per seminiferous tubule section, as normalized to the total cell number per ST section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse. D. Quantification of (B). Shown is the total number of SCP1+ cells which translocated inwards the seminiferous tubule lumen per ST section, as normalized to the total cell number per seminiferous tubule section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse. E. Quantification of (B). Shown is the number of PLZF+ SCP1+ cells which translocated inwards the seminiferous tubule lumen per seminiferous tubule section, as normalized to the total cell number per seminiferous tubule section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse. Data information: In (C-E), data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the Mann-Whitney test).


A . Representative of P4 mouse seminiferous tubule ( ST ) 10 µm cryo - sections stained for PLZF ( red ) , γ - Tubulin ( green ) , PH3 ( magenta ) DAPI ( blue ) . Arrowheads highlight centrosomes . Dashed yellow highlights basement . Semi - transparent , which bisects both spindle poles was used to determine spindle angle Ø orientation in relation to basement . Anaphase were analyzed . Scale bars : 5 µm . B . Representative of P5 mouse seminiferous tubule 10 µm cryo - sections stained for PLZF ( red ) , γ - Tubulin ( green ) , PH3 ( magenta ) DAPI ( blue ) . Arrowheads highlight centrosomes . Dashed yellow highlights basement . Semi - transparent , which bisects both spindle poles was used to determine spindle angle Ø orientation in relation to basement . Anaphase were analyzed . Scale bars : 5 µm . ( C Quantitation of spindle angle of anaphase GSCs in P4 ST sections ( from A ) . Spindle angle relative to basement for CEP250 + / + CEP250 - / - ) . In ( C ) , dots ( colors reflect different mice ) represent spindle angle of each GSC . CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . At least 10 were analyzed for each mouse . ( D Quantitation of spindle angle of anaphase GSCs in P5 ST sections ( from B ) . for P5 . In ( D ) , dots ( colors reflect different mice ) represent spindle angle of each GSC . CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . At least 10 were analyzed in each mouse .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

A. Representative images of P4 mouse seminiferous tubule (ST) 10 µm cryo-sections stained for PLZF (red), γ-Tubulin (green), PH3 (magenta) and DAPI (blue). Arrowheads highlight the centrosomes. Dashed yellow line highlights the basement membrane. Semi-transparent white line, which bisects both spindle poles was used to determine spindle angle Ø orientation in relation to the basement membrane. Anaphase cells were analyzed. Scale bars: 5 µm. B. Representative images of P5 mouse seminiferous tubule 10 µm cryo-sections stained for PLZF (red), γ-Tubulin (green), PH3 (magenta) and DAPI (blue). Arrowheads highlight the centrosomes. Dashed yellow line highlights the basement membrane. Semi-transparent white line, which bisects both spindle poles was used to determine spindle angle Ø orientation in relation to the basement membrane. Anaphase cells were analyzed. Scale bars: 5 µm. (C Quantitation of spindle angle of anaphase GSCs in P4 ST sections (from A). Spindle angle relative to basement membrane for CEP250+/+ and CEP250-/-). In (C), dots (colors reflect different mice) represent spindle angle of each GSC. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed for each mouse. (D Quantitation of spindle angle of anaphase GSCs in P5 ST sections (from B). for P5. In (D), dots (colors reflect different mice) represent spindle angle of each GSC. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed in each mouse.


E ) Quantitation of spindle angle of anaphase GSCs in P4 ST sections Spindle angle relative to basement for CEP250 + / + CEP250 - / - ) . In ( E ) , three dots represent averaged spindle angle per mouse . CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . At least 10 were analyzed for each mouse . F ) Quantitation of spindle angle of anaphase GSCs in P5 ST sections for P5 . In ( F ) , three dots represent averaged spindle angle per mouse . CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . At least 10 were analyzed in each mouse . Data information : In ( E , F ) , data are presented as mean ± . n . s . , not significant , * P < 0 . 05 , * * * P < 0 . 005 ( unpaired Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Quantitation of spindle angle of anaphase GSCs in P4 ST sections Spindle angle relative to basement membrane for CEP250+/+ and CEP250-/-). In (E), the three dots represent averaged spindle angle per mouse. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed for each mouse. F) Quantitation of spindle angle of anaphase GSCs in P5 ST sections for P5. In (F), the three dots represent averaged spindle angle per mouse. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed in each mouse. Data information: In (E, F), data are presented as mean ± SEM. n.s., not significant, *P<0.05, ***P < 0.005 (the unpaired Student's t-test).


B . Representative of P5 mouse ST . 10 µm cryo - sections stained for PLZF ( red ) , E - cadherin ( green ) DAPI ( blue ) showing GSCs in interphase . Scale bars : 5 μm . C . of ( B ) . of GSCs displaying different polarity status are indicated . CEP250 + / + n = 5 mice , CEP250 - / - n = 4 mice . At least 100 were analyzed in each mouse average was calculated for each mouse . Data information : In arrowheads highlight basement ; yellow arrowhead highlights cortical enrichment of E - cadherin towards basement . In ( C data are presented as mean ± . n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 ( unpaired Student ' s t - ) .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of P5 mouse ST. 10 µm cryo-sections stained for PLZF (red), E-cadherin (green) and DAPI (blue) showing GSCs in interphase. Scale bars: 5 μm. C. Quantification of (B). Percentage of GSCs displaying different polarity status are indicated. CEP250+/+ n=5 mice, CEP250-/- n=4 mice. At least 100 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In white arrowheads highlight basement membrane; yellow arrowhead highlights cortical enrichment of E-cadherin towards the basement membrane. In (C data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the unpaired Student's t-test).


D . Representative of P5 mouse ST . 10 µm cryo - sections stained for PLZF ( red ) , E - cadherin ( green ) PH3 ( magenta ) showing GSCs in prophase . PH3 marks mitotic . Scale bars : 5 μm . E . of ( D ) . of GSCs displaying different polarity status are indicated . CEP250 + / + n = 5 mice , CEP250 - / - n = 4 mice . At least 10 were analyzed in each mouse average was calculated for each mouse . Data information : In arrowheads highlight basement ; yellow arrowhead highlights cortical enrichment of E - cadherin towards basement . In data are presented as mean ± . n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 ( unpaired Student ' s t - ) .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Representative images of P5 mouse ST. 10 µm cryo-sections stained for PLZF (red), E-cadherin (green) and PH3 (magenta) showing GSCs in prophase. PH3 staining marks the mitotic chromatin. Scale bars: 5 μm. E. Quantification of (D). Percentage of GSCs displaying different polarity status are indicated. CEP250+/+ n=5 mice, CEP250-/- n=4 mice. At least 10 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In white arrowheads highlight basement membrane; yellow arrowhead highlights cortical enrichment of E-cadherin towards the basement membrane. In data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the unpaired Student's t-test).


F . Representative of P5 mouse ST 10 µm cryo - sections stained for PLZF ( red ) , E - cadherin ( green ) PH3 ( magenta ) showing GSCs in prometaphase . PH3 marks mitotic chromosome condensation . Scale bars : 5 μm . G . of ( F ) , of GSCs displaying different polarity status are indicated . CEP250 + / + n = 5 mice , CEP250 - / - n = 4 mice . At least 30 were analyzed in each mouse average was calculated for each mouse . Data information : In F , arrowheads highlight basement ; yellow arrowhead highlights cortical enrichment of E - cadherin towards basement . In G ) , data are presented as mean ± . n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 ( unpaired Student ' s t - ) .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Representative images of P5 mouse ST 10 µm cryo-sections stained for PLZF (red), E-cadherin (green) and PH3 (magenta) showing GSCs in prometaphase. PH3 staining marks the mitotic chromosome condensation. Scale bars: 5 μm. G. Quantification of (F), percentage of GSCs displaying different polarity status are indicated. CEP250+/+ n=5 mice, CEP250-/- n=4 mice. At least 30 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In F, white arrowheads highlight basement membrane; yellow arrowhead highlights cortical enrichment of E-cadherin towards the basement membrane. In G), data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the unpaired Student's t-test).


A . Representative of P5 mouse seminiferous tubule ( ST ) 10 µm cryo - sections stained for PLZF ( red ) , ODF2 ( green ) , NIN ( magenta ) PH3 ( blue ) showing GSCs in prophase . Scale bars : 2 μm . Yellow arrowheads highlight two centrosomes .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative images of P5 mouse seminiferous tubule (ST) 10 µm cryo-sections stained for PLZF (red), ODF2 (green), NIN (magenta) and PH3 (blue) showing GSCs in prophase. Scale bars: 2 μm. Yellow arrowheads highlight the two centrosomes.


B . of Centrosome in GSCs in prophase at P5 . CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . At least 50 were analyzed in each mouse ( color of dot indicates mouse ) . Dashed indicates 4 μm as threshold of well separated centrosomes . Data information : In ( B ) , data are presented as mean ± . * * * * P < 0 . 001 ( Mann - Whitney ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of Centrosome distance in GSCs in prophase at P5. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 50 cells were analyzed in each mouse (color of dot indicates mouse). Dashed line indicates 4 μm as the threshold of well separated centrosomes. Data information: In (B), data are presented as mean ± SEM. ****P < 0.001 (the Mann-Whitney test).


D . Representative of P5 mouse seminiferous tubule 10 µm cryo - sections stained for PLZF ( red ) , ODF2 ( green ) , NIN ( magenta ) PH3 ( blue ) , focusing on PLZF + germ ( GSCs ) in prometaphase . Scale bars : 2 μm . Yellow lines highlight basement . Yellow arrowheads highlight mother centrosomes arrowheads highlight daughter centrosomes .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Representative images of P5 mouse seminiferous tubule 10 µm cryo-sections stained for PLZF (red), ODF2 (green), NIN (magenta) and PH3 (blue), focusing on PLZF+ germ stem cells (GSCs) in prometaphase. Scale bars: 2 μm. Yellow lines highlight basement membrane. Yellow arrowheads highlight the mother centrosomes and white arrowheads highlight the daughter centrosomes.


E . of of prometaphase GSCs which preferentially position their mother centrosome close to basement in CEP250 + / + CEP250 - / - testis at P5 . CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . At least 50 were analyzed in each mouse average was calculated for each mouse . Data information : In ( E ) , data are presented as mean ± . * * * * P < 0 . 001 ( unpaired Student ' s t - ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Quantification of Percentage of prometaphase GSCs which preferentially position their mother centrosome close to the basement membrane in CEP250+/+ and CEP250-/- testis at P5. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 50 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In (E), data are presented as mean ± SEM. ****P < 0.001 (the unpaired Student's t-test).


( a ) Confocal microscopy of synchronized HeLa stained for VHR ( green ) BrdU ( red ) to indicate S - phase entry , ( DAPI ; blue ) . were synchronized using a double thymidine block then washed placed in culture for indicated times . shown are representative of majority of in each sample .

O O O O O O O O O O B-MEASURED_VAR O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(a) Confocal microscopy of synchronized HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI; blue). The cells were synchronized using a double thymidine block and then washed and placed in culture for the indicated times. The cells shown are representative of the majority of cells in each sample.


b ) Anti - VHR , anti - actin anti - Prc1immunoblots of lysates of same .

O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O

b) Anti-VHR, anti-actin and anti-Prc1immunoblots of lysates of the same cells.


( c ) histograms of same at indicated time points .

(c) DNA histograms of the same cells at the indicated time points.


( d ) Confocal microscopy of HeLa at various stages of mitosis stained for tubulin ( green ) , VHR ( red ) ( DAPI ; blue ) .

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(d) Confocal microscopy of HeLa cells at various stages of mitosis stained for tubulin (green), VHR (red) and DNA (DAPI; blue).


( e ) Anti - VHR immunoblot of synchronized from G1 , S , G2 M phase treated for 30 min with 50 μg ml − 1 of synthesis inhibitor cycloheximide . Note that only in G1 showed a rapid loss of VHR . scale bars represent 5 μm in a 2 μm in b .

O O O O O B-MEASURED_VAR 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-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

(e) Anti-VHR immunoblot of synchronized cells from G1, S, G2 and M phase treated for 30 min with 50 μg ml−1 of the protein synthesis inhibitor cycloheximide. Note that only cells in G1 showed a rapid loss of VHR protein. The scale bars represent 5 μm in a and 2 μm in b.


( a ) Anti - VHR anti - actin immunoblots of lysates of HeLa transfected with a control luciferase siRNA or VHR siRNA cultured for indicated number of days .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

(a) Anti-VHR and anti-actin immunoblots of lysates of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for the indicated number of days.


( b ) Number of at start of experiment ( 200 , 000 ) on day 3 for HeLa transfected with a control luciferase siRNA ( black bars ) or with VHR siRNA ( bars ) .

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(b) Number of cells at the start of the experiment (200,000) and on day 3 for HeLa cells transfected with a control luciferase siRNA (black bars) or with VHR siRNA (white bars).


( c ) microscopy of HeLa transfected with a control luciferase siRNA or VHR siRNA cultured for three days .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

(c) Light microscopy of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days.


( d ) Thymidine incorporation by HeLa transfected with a control luciferase siRNA or with VHR siRNA . data represent mean ± sd . ( n = 3 ) .

O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

(d) Thymidine incorporation by HeLa cells transfected with a control luciferase siRNA or with VHR siRNA. The data represent the mean ± sd. (n = 3).


( e ) β - galactosidase of HeLa transfected with a control luciferase siRNA or VHR siRNA cultured for three days . Note increased number of blue - granules in many . (

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(e) β-galactosidase staining of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days. Note the increased number of blue-staining granules in many cells. (


( f ) Telomerase activity of treated with indicated concentrations of VHR siRNA . Each sample was analysed in triplicate , with RNAse added to a fourth sample .

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(f) Telomerase activity of cells treated with the indicated concentrations of VHR siRNA. Each sample was analysed in triplicate, with RNAse added to a fourth sample.


( g ) Anti - VHR immunoblot of same as in f . ( h ) Anti - actin control immunoblot of same as in f . scale bars represent 10 μm in c 25 μm in e .

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(g) Anti-VHR immunoblot of the same cells as in f. (h) Anti-actin control immunoblot of the same cells as in f. The scale bars represent 10 μm in c and 25 μm in e.


( a ) - cycle distribution number of transfected with control or VHR siRNA observed for 4 - 5 days . were stained with propidium iodide for content analysed by FACS after indicated number of days in culture .

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(a) Cell-cycle distribution and cell number count of cells transfected with control or VHR siRNA and observed for 4-5 days. The cells were stained with propidium iodide for DNA content and analysed by FACS after the indicated number of days in culture.


( b ) Confocal microscopy of same HeLa stained for VHR ( green ) BrdU ( red ) to indicate S - phase entry , ( DAPI , blue ) . Control siRNA - treated treated with VHR are shown . bottom panels represent merges of 3

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(b) Confocal microscopy of the same HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI, blue). Control siRNA-treated cells and cells treated with VHR are shown. The bottom panels represent merges of the 3 images above