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

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

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


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

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


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

O O O O O O O O O O O O O O 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, 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 O 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, 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O

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 .

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 .

O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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


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

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


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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O 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 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 .

O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 .

O O O O O O O O O O O O O O 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 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 ) .

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O 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. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

O O O O O O O O O O O O O O O O O O O O O O O

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


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

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

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


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

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


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

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


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

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


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

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

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


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

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

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


( c ) Quantification of the BrdU positive cells in b . The indicated numbers of cells were counted and the data are given as percentage of BrdU positive cells of this total number .

O O O O O O O O O O O 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) Quantification of the BrdU positive cells in b. The indicated numbers of cells were counted and the data are given as percentage of BrdU positive cells of this total number.


d ) Quantification of cells staining for phosphoH3 . The indicated number of cells were counted and the data are given as percentage of phosphoH3 - positive cells of this total number .

O O O O O O O O O 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) Quantification of cells staining for phosphoH3. The indicated number of cells were counted and the data are given as percentage of phosphoH3-positive cells of this total number.


( e ) Confocal microscopy of control or VHR siRNA - transfected HeLa cells stained for VHR ( green ) and phospho H3 ( red ) to indicate M - phase entry , and DNA ( DAPI , blue ) . The bottom panels represent merges of the images above .

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

(e) Confocal microscopy of control or VHR siRNA-transfected HeLa cells stained for VHR (green) and phospho H3 (red) to indicate M-phase entry, and DNA (DAPI, blue). The bottom panels represent merges of the images above.


( f ) Immunoblots of total lysates of control siRNA transfected cells and VHR siRNA cells with the indicated antibodies . Note that VHR was downregulated by approximately 80 % , wheras actin levels were unchanged .

O O O O O O O O O O O O O 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) Immunoblots of total lysates of control siRNA transfected cells and VHR siRNA cells with the indicated antibodies. Note that VHR was downregulated by approximately 80%, wheras actin levels were unchanged.


( g ) Immunoblot with anti - p21Cip1 - WAF1 of total lysates of cells transfected with control siRNA ( lanes 1 - 2 ) , two different VHR siRNAs cells ( lanes 3 - 6 ) or another control siRNA ( lanes 7 - 8 ) . The scale bars represent 10 μm in b and e .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot with anti-p21Cip1-WAF1 of total lysates of cells transfected with control siRNA (lanes 1-2), two different VHR siRNAs cells (lanes 3-6) or another control siRNA (lanes 7-8). The scale bars represent 10 μm in b and e.


( a ) Anti - VHR immunoblots of the same cell lysates used for assessment of MAP kinase activation in b - e . The cells were stimulated with 10 % serum for 0 , 1 , 5 , 15 , 30 and 60 min , as indicated at the bottom of the figure . Equal amounts of total protein were loaded into each lane . ( b - e ) Immunoblots of the same lysates with the indicated antibodies for assement of MAP kinase activation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Anti-VHR immunoblots of the same cell lysates used for assessment of MAP kinase activation in b-e. The cells were stimulated with 10% serum for 0, 1, 5, 15, 30 and 60 min, as indicated at the bottom of the figure. Equal amounts of total protein were loaded into each lane. (b-e) Immunoblots of the same lysates with the indicated antibodies for assement of MAP kinase activation.


( a ) Proliferation of cells transfected 3 days earlier with a combination of control siRNA or VHR siRNA together with Mek , Jnk or Mek plus Jnk siRNAs . The data are calculated as percentage of control ( control siRNA alone ) and represent the mean ± s . d . from three independent experiments . The starting cell numbers were 150 , 000 or 200 , 000 and the control cells had increased by 5 . 3 , 5 . 0 and 13 - fold in the three experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Proliferation of cells transfected 3 days earlier with a combination of control siRNA or VHR siRNA together with Mek, Jnk or Mek plus Jnk siRNAs. The data are calculated as percentage of control (control siRNA alone) and represent the mean ± s.d. from three independent experiments. The starting cell numbers were 150,000 or 200,000 and the control cells had increased by 5.3, 5.0 and 13-fold in the three experiments.


( b ) Immunoblots of the same cells with the indicated antibodies .

(b) Immunoblots of the same cells with the indicated antibodies.


A . Live - cell imaging assay to detect mitotic perturbations . HeLa cells stably expressing a chromatin marker ( H2B - mCherry ; red ) and a nuclear import substrate ( IBB - eGFP ; green ) were imaged by automated live - cell microscopy and the duration of mitosis from prometaphase to anaphase onset was automatically determined for each dividing cell based on the time from nuclear envelope breakdown ( mitotic entry ) until nuclear reassembly ( mitotic exit ) ( Schmitz et al . , 2010 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Live-cell imaging assay to detect mitotic perturbations. HeLa cells stably expressing a chromatin marker (H2B-mCherry; red) and a nuclear import substrate (IBB-eGFP; green) were imaged by automated live-cell microscopy and the duration of mitosis from prometaphase to anaphase onset was automatically determined for each dividing cell based on the time from nuclear envelope breakdown (mitotic entry) until nuclear reassembly (mitotic exit) (Schmitz et al., 2010).


B . miRNA mimic screen for miRNAs that regulate mitosis genes . miRNA - mimics from a library of all embryonic cortically expressed miRNAs were transfected individually into HeLa cells and prometaphase to anaphase onset duration was determined as in A . Individual points correspond to the mean z - score of the duration of prometaphase to anaphase onset determined in 2 independent experimental replicates for a given miRNA - mimic .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. miRNA mimic screen for miRNAs that regulate mitosis genes. miRNA-mimics from a library of all embryonic cortically expressed miRNAs were transfected individually into HeLa cells and prometaphase to anaphase onset duration was determined as in A. Individual points correspond to the mean z-score of the duration of prometaphase to anaphase onset determined in 2 independent experimental replicates for a given miRNA-mimic.


C . Cumulative histograms of mitotic progression for control cells and for cells transfected with miRNA mimics . Nuclear envelope breakdown is at t = 0 min . ( n ≥ 71 in all conditions ) .

C. Cumulative histograms of mitotic progression for control cells and for cells transfected with miRNA mimics. Nuclear envelope breakdown is at t = 0 min. (n ≥ 71 in all conditions).


D . Confocal time lapse microscopy images of HeLa cells stably expressing H2B - mCherry ( red ) and a microtubule marker ( α - Tub - eGFP ; green ) 48 h after transfection of miR - 449a mimic , or non - targeting control siRNA . Scale bar , 10 μm . E . Quantification of spindle rotation and duration from prometaphase to anaphase onset in time - lapse movies as in D . miR - 449a mimic promotes spindle rotation . Duration from prometaphase to anaphase onset was defined as the time from nuclear envelope breakdown to anaphase onset . Spindle rotation during metaphase was measured as described in methods . Individual data points correspond to single cells ( n ≥ 72 in all conditions ) . Normality was tested with Kolmogorov - Smirnov test . Variance between samples was tested using F test . Significance was tested by Welch ' s t test : p - value = 2 . 598e - 06 comparing spindle rotation of negative control versus miR - 449a mimic - transfected cells ( all data points are compared ) . p - value = 0 . 0005621 ( for cells with prometaphase - anaphase onset duration lower than 60 min .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal time lapse microscopy images of HeLa cells stably expressing H2B-mCherry (red) and a microtubule marker (α-Tub-eGFP; green) 48 h after transfection of miR-449a mimic, or non-targeting control siRNA. Scale bar, 10 μm.E. Quantification of spindle rotation and duration from prometaphase to anaphase onset in time-lapse movies as in D. miR-449a mimic promotes spindle rotation. Duration from prometaphase to anaphase onset was defined as the time from nuclear envelope breakdown to anaphase onset. Spindle rotation during metaphase was measured as described in methods. Individual data points correspond to single cells (n ≥ 72 in all conditions). Normality was tested with Kolmogorov-Smirnov test. Variance between samples was tested using F test. Significance was tested by Welch's t test: p-value = 2.598e-06 comparing spindle rotation of negative control versus miR-449a mimic-transfected cells (all data points are compared). p-value = 0.0005621 (for cells with prometaphase-anaphase onset duration lower than 60 min.


A . The expression levels of endogenous miR - 34 / 449 family members were measured by RT - qPCR in ventricular zone samples derived by laser microdissection of mouse cortices at E14 . The levels of the different miR - 34 / 449 family members and miR - 7a - 1 , a highly expressed miRNA relevant in cortical progenitor biology , were determined . All concentrations were normalized ( norm . ) using miR - 7a - 1 concentration ( n = 8 cortices , 2 different litters ) . Error bars indicate standard error .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The expression levels of endogenous miR-34/449 family members were measured by RT-qPCR in ventricular zone samples derived by laser microdissection of mouse cortices at E14. The levels of the different miR-34/449 family members and miR-7a-1, a highly expressed miRNA relevant in cortical progenitor biology, were determined. All concentrations were normalized (norm.) using miR-7a-1 concentration (n = 8 cortices, 2 different litters). Error bars indicate standard error.


B , C . Expression analysis of miR - 34 / 449 by in situ hybridization using locked nucleic acid ( LNA ) probes in wild type cortices at E14 . Mature miR - 449 , miR - 34b and miR - 34c are preferentially expressed in the subventricular ( SVZ ) and ventricular ( VZ ) zones of the neocortex . Scale bar , 50 μm ( A ) , 10 μm ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C. Expression analysis of miR-34/449 by in situ hybridization using locked nucleic acid (LNA) probes in wild type cortices at E14. Mature miR-449, miR-34b and miR-34c are preferentially expressed in the subventricular (SVZ) and ventricular (VZ) zones of the neocortex. Scale bar, 50 μm (A), 10 μm (B).


B , C . Expression analysis of miR - 34 / 449 by in situ hybridization using locked nucleic acid ( LNA ) probes in wild type cortices at E14 . Mature miR - 449 , miR - 34b and miR - 34c are preferentially expressed in the subventricular ( SVZ ) and ventricular ( VZ ) zones of the neocortex . Scale bar , 50 μm ( B ) , 10 μm ( C ) .

B, C. Expression analysis of miR-34/449 by in situ hybridization using locked nucleic acid (LNA) probes in wild type cortices at E14. Mature miR-449, miR-34b and miR-34c are preferentially expressed in the subventricular (SVZ) and ventricular (VZ) zones of the neocortex. Scale bar, 50 μm (B), 10 μm (C).


D , E . Brains of adult mice ( P23 ) , and quantification of brain weight . Dots indicate individual brains ; red line indicates median . Mice lacking miR - 449abc and miR - 34bc ( DKO ) or miR - 449abc , miR - 34bc , and miR - 34a ( TKO ) have significantly smaller brains compared to littermate controls ( Het ) . Significance was tested by Pairwaise t - test with Bonferroni correction ; Het ( n = 6 brains , 2 different litters ) vs . DKO ( n = 4 brains , 2 different litters ) p - value = 0 . 00215 ; Het ( n = 6 brains , 2 different litters ) vs . TKO ( n = 4 brains , 2 different litters ) p - value = 0 . 00054 . Subfigure G was statistically tested as in E . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E. Brains of adult mice (P23), and quantification of brain weight. Dots indicate individual brains; red line indicates median. Mice lacking miR-449abc and miR-34bc (DKO) or miR-449abc, miR-34bc, and miR-34a (TKO) have significantly smaller brains compared to littermate controls (Het). Significance was tested by Pairwaise t-test with Bonferroni correction; Het (n = 6 brains, 2 different litters) vs. DKO (n = 4 brains, 2 different litters) p-value = 0.00215; Het (n = 6 brains, 2 different litters) vs. TKO (n = 4 brains, 2 different litters) p-value = 0.00054. Subfigure G was statistically tested as in E. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001.


F , G . Confocal images of coronal brain sections ( P23 ) and quantification of cortex width from miR - 34 / 449 DKO and TKO mice and littermate controls ( Het ) . Sections were stained with DAPI . DKO or TKO mice have significantly thinner cortices compared to littermate controls ( Het ) in adult mice ( P23 ) . ( n = 6 brains , 2 different litters ) vs . DKO ( n = 4 brains , 2 different litters ) p - value = 0 . 00084 , Het ( n = 6 brains , 2 different liters ) vs . TKO ( n = 4 brains , 2 different litters ) p - value = 0 . 00211 . Scale bar , 500 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G. Confocal images of coronal brain sections (P23) and quantification of cortex width from miR-34/449 DKO and TKO mice and littermate controls (Het). Sections were stained with DAPI. DKO or TKO mice have significantly thinner cortices compared to littermate controls (Het) in adult mice (P23). (n = 6 brains, 2 different litters) vs. DKO (n = 4 brains, 2 different litters) p-value = 0.00084, Het (n = 6 brains, 2 different liters) vs. TKO (n = 4 brains, 2 different litters) p-value = 0.00211. Scale bar, 500 μm.


A . Confocal images of coronal sections from E16 brains of miR - 34 / 449 KO mice and littermate controls ( Het ) , stained with anti - Satb2 - antibody to label neurons of layers II - IV ( green ) and DAPI to label all cell nuclei ( blue ) . Scale bars : 50 μm . B . Quantification of the number of Satb2 - positive cells per 100 μm ventricular zone surface . Bars indicate mean ± SEM , as for all quantifications shown in this figure ; n = 3 brains per genotype group , from two independent litters . Data was normalized ( norm . ) to the ventricular zone surface analyzed ( 100 µm ) and relativized to the heterozygous control average value . Significance was tested by Welch ' s t test : p - value = 0 . 01146 ( Het vs . KO ) ( n = 4 cortices per genotype group , 2 independent litters ) . All subfigures were statistically tested as in B . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal images of coronal sections from E16 brains of miR-34/449 KO mice and littermate controls (Het), stained with anti-Satb2-antibody to label neurons of layers II-IV (green) and DAPI to label all cell nuclei (blue). Scale bars: 50 μm.B. Quantification of the number of Satb2-positive cells per 100 μm ventricular zone surface. Bars indicate mean ± SEM, as for all quantifications shown in this figure; n = 3 brains per genotype group, from two independent litters. Data was normalized (norm.) to the ventricular zone surface analyzed (100 µm) and relativized to the heterozygous control average value. Significance was tested by Welch's t test: p-value = 0.01146 (Het vs. KO) (n = 4 cortices per genotype group, 2 independent litters). All subfigures were statistically tested as in B. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001.


C - J . Confocal images and cell density quantifications as in A using molecular markers for different cell types : C , D . E16 brains stained with anti - Ctip2 antibody to label layer Vneurons ( n = 4 brains per genotype group , 2 independent litters ) . Scale bars : 50 μm . Images in C were taken from same brain slices as shown in A . p - value = 0 . 03224 ( Het vs . KO ) ( immuno co - staining of Ctip2 ( red , C ) ) .

C-J. Confocal images and cell density quantifications as in A using molecular markers for different cell types:C, D. E16 brains stained with anti-Ctip2 antibody to label layer Vneurons (n = 4 brains per genotype group, 2 independent litters). Scale bars: 50 μm. Images in C were taken from same brain slices as shown in A. p-value = 0.03224 (Het vs. KO) (immuno co-staining of Ctip2(red, C)).


C - J . Confocal images and cell density quantifications as in A using molecular markers for different cell types : E , F . E14 brains stained with anti - Tbr1 antibody to label layer IVneurons p - value = 0 . 01392 ( Het vs . KO ) ( n = 3 and 4 brains per genotype group from 2 independent litters ) . Scale bars : 50 μm .

C-J. Confocal images and cell density quantifications as in A using molecular markers for different cell types:E, F. E14 brains stained with anti-Tbr1 antibody to label layer IVneurons p-value = 0.01392 (Het vs. KO) (n = 3 and 4 brains per genotype group from 2 independent litters). Scale bars: 50 μm.


C - J . Confocal images and cell density quantifications as in A using molecular markers for different cell types : G , H . E16 brains stained with anti - Tbr2 antibody to label intermediate progenitors . p - value = 0 . 01647 ( Het vs . KO ) ( n = 7 brains for each genotype group , 5 independent litters ) . Scale bars : 50 μm .

C-J. Confocal images and cell density quantifications as in A using molecular markers for different cell types:G, H. E16 brains stained with anti-Tbr2 antibody to label intermediate progenitors. p-value = 0.01647 (Het vs. KO) (n = 7 brains for each genotype group, 5 independent litters). Scale bars: 50 μm.


C - J . Confocal images and cell density quantifications as in A using molecular markers for different cell types : I , J . E16 brains stained with anti - Pax6 antibody to label radial glia progenitors . p - value = 0 . 002768 ( n = 5 brains per genotype group , 3 independent litters ) . Scale bars : 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J. Confocal images and cell density quantifications as in A using molecular markers for different cell types:I, J. E16 brains stained with anti-Pax6 antibody to label radial glia progenitors. p-value = 0.002768 (n = 5 brains per genotype group, 3 independent litters). Scale bars: 50 μm.


K . 3D reconstruction of a dividing radial glial progenitor at early anaphase . A coronal section of an E14 brain of a heterozygous control mouse was stained with anti - phospho - Vimentin antibody ( red ) , anti - γ - tubulin antibody ( green / yellow ) , phalloidin ( magenta ) , and DAPI ( blue ) and imaged by 3D confocal microscopy . " s " indicates spindle axis , " α " indicates angle relative to ventricular surface plane , which was determined by a vector path as indicated by thin white lines located on the left side of the image . Yellow dots highlight the centrosomes of the spindle poles from the analyzed dividing cell . Scale bars : 5 μm . L . Quantification of spindle orientation in radial glial cells as in K for E14 brains of miR - 34 / 449 KO and littermate controls ( Het ) . Each dot represents a single dividing cell ; p - value = 3 . 166e - 05 ( Het vs . KO ) ( n = 131 vs . 107 cells , n = 4 brains per genotype group , 2 independent litters ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 3D reconstruction of a dividing radial glial progenitor at early anaphase. A coronal section of an E14 brain of a heterozygous control mouse was stained with anti-phospho-Vimentin antibody (red), anti-γ-tubulin antibody (green/yellow), phalloidin (magenta), and DAPI (blue) and imaged by 3D confocal microscopy. "s" indicates spindle axis, "α" indicates angle relative to ventricular surface plane, which was determined by a vector path as indicated by thin white lines located on the left side of the image. Yellow dots highlight the centrosomes of the spindle poles from the analyzed dividing cell. Scale bars: 5 μm.L. Quantification of spindle orientation in radial glial cells as in K for E14 brains of miR-34/449 KO and littermate controls (Het). Each dot represents a single dividing cell; p-value = 3.166e-05 (Het vs. KO) (n = 131 vs. 107 cells, n = 4 brains per genotype group, 2 independent litters).


A . Quantification of mRNA expression by RT - qPCR of candidate miR - 34 / 449 targets that might regulate spindle orientation in E14 cortex samples from miR - 34 / 449 KO mice compared with littermate controls ( Het ) . Expression was normlaized ( norm . ) to Phosphoglycerate kinase ( PGK ) mRNA levels . Significance was tested by Welch ' s t test : p = 0 . 009756 ( JAM - A ( Het ) vs . JAM - A ( KO ) ) , all the rest Het vs . KO comparisons p = n . s . ( n = 4 cortices per genotype group , 2 independent litters ) . All subfigures were statistically tested as in A . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . Red bar indicates median .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of mRNA expression by RT-qPCR of candidate miR-34/449 targets that might regulate spindle orientation in E14 cortex samples from miR-34/449 KO mice compared with littermate controls (Het). Expression was normlaized (norm.) to Phosphoglycerate kinase (PGK) mRNA levels. Significance was tested by Welch's t test: p = 0.009756 (JAM-A (Het) vs. JAM-A (KO)), all the rest Het vs. KO comparisons p=n.s. (n = 4 cortices per genotype group, 2 independent litters). All subfigures were statistically tested as in A. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001. Red bar indicates median.


B . RT - qPCR quantification of JAM - A mRNA expression in HeLa cells 48 h after transfection of miR - 449a mimic compared to cells transfected with non - targeting negative control siRNA . JAM - A mRNA Expression was normalized ( norm . ) to Glyceraldehyde 3 - phosphate dehydrogenase ( GAPDH ) mRNA levels . p = 0 . 0001218 ( Neg . ctrol . vs . miR - 449a mimic ) ( n = 9 measurements from 3 independent replicates ) .

B. RT-qPCR quantification of JAM-A mRNA expression in HeLa cells 48 h after transfection of miR-449a mimic compared to cells transfected with non-targeting negative control siRNA. JAM-A mRNA Expression was normalized (norm.) to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. p = 0.0001218 (Neg. ctrol. vs. miR-449a mimic) (n = 9 measurements from 3 independent replicates).