( E ) IF of DAXX and ATRX in control ( n = 15 ) and Atrxm - z + ( n = 14 ) PN5 zygotes . Data information : All panels : images of paternal and maternal pronuclei at given stage / condition are from same zygote . Yellow dashed circles represent the contours of the pronuclei . All scale bars , 10µm .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-TISSUE O O O O O O O O O B-SUBCELLULAR O O O O O O O

(E) IF of DAXX and ATRX in control (n=15) and Atrxm-z+ (n=14) PN5 zygotes. Data information: All panels: images of paternal and maternal pronuclei at given stage/condition are from same zygote. Yellow dashed circles represent the contours of the pronuclei. All scale bars, 10µm.


( A ) IF of DAXX and EZH2 in paternal pronuclei of Ezh1m - z + ( n = 5 ) , Ezh2m - z + ( n = 3 ) and Ezh1m - z + ; Ezh2m - z + ( n = 4 ) late zygotes . Yellow dashed circles represent the contours of the pronuclei . Data information : All scale bars , 10µm .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O

(A) IF of DAXX and EZH2 in paternal pronuclei of Ezh1m-z+ (n=5), Ezh2m-z+ (n=3) and Ezh1m-z+; Ezh2m-z+ (n=4) late zygotes. Yellow dashed circles represent the contours of the pronuclei. Data information: All scale bars, 10µm.


( B ) IF of DAXX and RNF2 in PN0 zygotes ( n = 10 ) . Data information : All scale bars , 10µm .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O

(B) IF of DAXX and RNF2 in PN0 zygotes (n=10). Data information: All scale bars, 10µm.


( C ) IF of DAXX in wild type ( n = 30 ) , Ring1m - z + ( n = 12 ) , Rnf2m - z + ( n = 11 ) and Ring1m - z + ; Rnf2m - z + ( n = 12 ) paternal pronuclei of PN5 zygotes . Data information : All scale bars , 10µm .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O O O O O O

(C) IF of DAXX in wild type (n=30), Ring1m-z+ (n=12), Rnf2m-z+ (n=11) and Ring1m-z+; Rnf2m-z+ (n=12) paternal pronuclei of PN5 zygotes. Data information: All scale bars, 10µm.


( D ) Anti - Flag co - IP of DAXXMycHis , RNF2 and H3K27me3 with CBX23 × Flag from extracts of HEK293 cells in presence or absence of benzonase , followed by immuno - blot analyses for indicated antigens .

O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O

(D) Anti-Flag co-IP of DAXXMycHis, RNF2 and H3K27me3 with CBX23×Flag from extracts of HEK293 cells in presence or absence of benzonase, followed by immuno-blot analyses for indicated antigens.


( C ) Occupancy ( % of IP - ed DNA / input DNA ) of GAL4DBDCBX2 , RNF2 , DAXXMycHis , H3 . 3 , and H3 proteins at 5 × UAS site as measured by ChIP - qPCR of chromatin from HEK2935xUAS cells . Data are means ± SEM ( n = 3 ) .

O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-CELL_LINE O O O O O O O O O O O O O

(C) Occupancy (% of IP-ed DNA / input DNA) of GAL4DBDCBX2, RNF2, DAXXMycHis, H3.3, and H3 proteins at 5×UAS site as measured by ChIP-qPCR of chromatin from HEK2935xUAS cells. Data are means ± SEM (n = 3).


( D ) Immunoblots for the indicated proteins in HEK2935xUAS

(D) Immunoblots for the indicated proteins in HEK2935xUAS cells


( 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O

(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 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O

(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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O O O B-GENEPROD 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-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O

(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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O

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

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O

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

O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-CELL_LINE O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

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

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-SUBCELLULAR O B-GENEPROD O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O

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

O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O

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

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-TISSUE O O O O O O O O B-SUBCELLULAR O O O O O O O B-TISSUE O O O O O O

(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 - 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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-SUBCELLULAR O B-TISSUE O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O B-TISSUE O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-TISSUE O O O O O O

(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 + and Daxxm - z - early embryos .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O

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


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

O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O B-SUBCELLULAR O O O O O O O O O

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


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

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


( 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 .

O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-TISSUE O B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O

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

O O O O O B-TISSUE O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-TISSUE O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O

(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 ) 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-GENEPROD O O O B-TISSUE O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O

(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 - and mat - PCH of control Daxxm + z + ( n = 18 ) , Daxxm - z + ( n = 8 ) and Ring1m - z + ; Rnf2m - z + ( n = 12 ) zygotes . Scale bar 10µm .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-TISSUE O O O O O

(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 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 .

O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O B-TISSUE O O O O O O O O O O O O O O

(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 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 .

O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O

(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 ) 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 ) .

O O O O O B-GENEPROD O O O B-TISSUE O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

(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 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 .

O O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O O B-TISSUE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O B-TISSUE O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O B-GENEPROD O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O

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

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE O O O O O O

(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 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 .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 all samples . Dotted lines indicated log2 ( FC ) of 1 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

(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

O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O

(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 all samples . Dotted lines indicated log2 ( FC ) of 1 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O

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 the presence ( + ) and absence ( - ) of 1 mM PMSF . Arrows denote cells that would be scored as positive in the experiment shown in c .

O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

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 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 .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O

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

O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

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


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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O B-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR 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 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 .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O B-TISSUE O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O

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

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O

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 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 .

O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O

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 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 .

O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O 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, 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 , Cell viability of MCF7 clones determined by trypan blue staining . Symbols for each clone are as in

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

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 .

O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O 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, 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 weeks per number of mice injected with each clone .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O

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

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O

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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O

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 cell free lysate batches of E . coli .

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


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 .

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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 primary TSS calls ( in bp from ATG start codon ) of regulatory sequences from in vitro and 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 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 ) .

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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 all 10 bacterial cell - free expression systems .

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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 bacterial species . Example scatter plots with relatively high and low Pearson correlation in the heatmap ( marked i and 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 percent identity ) and pairwise Pearson correlation of transcriptional profiles . Dashed line 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 and 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 and 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 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 .

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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 levels 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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O O

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 ) 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 .

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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 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 .

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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 .

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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 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 .

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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 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 .

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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 analysis of EE purified from P1 . IDO1 . WT cells . Mock - transfected cells 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O

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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

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 . 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 .

O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 + / + 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 .

O O B-TISSUE O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 + / + 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 .

O O O B-TISSUE O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 + / + 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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY B-TISSUE B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-CELL_TYPE O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 ) .

O O B-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 staining ( blue ) of 10 µm testis cryo - sections from neonatal mice ( P5 , P10 and P14 ) CEP250 + / + and CEP250 - / - . Scale bars : 20 µm .

O O B-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE O O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O

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 . 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 ) .

O O O O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 . 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 ) .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O B-TISSUE O O O O O O B-TISSUE O O B-ORGANISM O O O O B-TISSUE O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ) 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 ) .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O B-TISSUE O O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O B-ORGANISM O O O O O B-TISSUE O O B-ORGANISM O O O O B-TISSUE O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O O O B-ORGANISM O O O O O B-TISSUE O O B-ORGANISM O O O O B-TISSUE O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O

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 . 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 ) .

O O O O B-GENEPROD O O O B-TISSUE O B-TISSUE O O O B-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O B-TISSUE O O B-ORGANISM O O O O B-TISSUE O B-GENEPROD O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O B-ORGANISM O O B-TISSUE O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O B-TISSUE O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O O O O

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.


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 ) .

O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O B-TISSUE O O B-ORGANISM O O O O B-TISSUE O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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).


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 ) .

O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O B-TISSUE O O B-ORGANISM O O O O B-TISSUE O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , 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 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 ) .

O O O O O B-ORGANISM B-TISSUE I-TISSUE O B-TISSUE O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE O O O O B-CELL_TYPE O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O B-TISSUE O O B-ORGANISM O O O O B-TISSUE O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O B-TISSUE O O B-ORGANISM O O O O B-TISSUE O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O B-TISSUE O O B-ORGANISM O O O O B-TISSUE O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O B-ORGANISM B-TISSUE I-TISSUE O B-TISSUE O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O B-SUBCELLULAR O O O B-CELL_TYPE O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-SUBCELLULAR O O O B-CELL_TYPE O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM 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.