( H ) Immunofluorescence analysis of γ - tubulin and α - tubulin in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The histogram shows the percentages of control ( n = 55 ) and Mcrs1 - KD ( n = 63 ) oocytes with abnormal γ - tubulin . * P = 0 . 014 . ( I ) Immunofluorescence analysis of acetylated tubulin and α - tubulin in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The scattergram shows the relative fluorescence intensity of acetylated tubulin signals in control ( n = 83 ) and Mcrs1 - KD ( n = 66 ) oocytes . * * P = 0 . 0046 .

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

(H) Immunofluorescence analysis of γ-tubulin and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The histogram shows the percentages of control (n = 55) and Mcrs1-KD (n = 63) oocytes with abnormal γ-tubulin. *P = 0.014. (I) Immunofluorescence analysis of acetylated tubulin and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The scattergram shows the relative fluorescence intensity of acetylated tubulin signals in control (n = 83) and Mcrs1-KD (n = 66) oocytes. **P = 0.0046.


( C ) Levels of p - Aurka and p - Aurkc in control and Mcrs1 - KD MI oocytes . The blots were probed with anti - p - Aurora A / B / C and anti - actin antibodies . ( D ) Co - IP was performed with an anti - Mcrs1 antibody . The immunoblots of protein precipitates were probed with an anti - Aurka antibody .

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

(C) Levels of p-Aurka and p-Aurkc in control and Mcrs1-KD MI oocytes. The blots were probed with anti-p-Aurora A/B/C and anti-actin antibodies. (D) Co-IP was performed with an anti-Mcrs1 antibody. The immunoblots of protein precipitates were probed with an anti-Aurka antibody.


( G ) Percentages of control ( n = 92 ) , Mcrs1 - KD ( n = 73 ) , and Aurka - rescue ( n = 67 ) oocytes that underwent PBE . * P = 0 . 033 ; * P = 0 . 018 .

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

(G) Percentages of control (n = 92), Mcrs1-KD (n = 73), and Aurka-rescue (n = 67) oocytes that underwent PBE. *P = 0.033; *P = 0.018.


( H ) Percentages of control ( n = 69 ) , Mcrs1 - KD ( n = 76 ) , and Aurka - rescue ( n = 43 ) oocytes with abnormal spindles . * P = 0 . 018 ; * P = 0 . 024 .

(H) Percentages of control (n = 69), Mcrs1-KD (n = 76), and Aurka-rescue (n = 43) oocytes with abnormal spindles. *P = 0.018; *P = 0.024.


( K ) Immunofluorescence analysis of Kif2A in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The histogram shows the percentages of control ( n = 50 ) and Mcrs1 - KD ( n = 61 ) oocytes with abnormal Kif2A . * * P = 0 . 003 .

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

(K) Immunofluorescence analysis of Kif2A in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The histogram shows the percentages of control (n = 50) and Mcrs1-KD (n = 61) oocytes with abnormal Kif2A. **P = 0.003.


( L ) Immunofluorescence analysis of TPX2 and α - tubulin in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The scattergram shows the relative fluorescence intensity of TPX2 signals in control ( n = 48 ) and Mcrs1 - KD ( n = 52 ) oocytes .

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

(L) Immunofluorescence analysis of TPX2 and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The scattergram shows the relative fluorescence intensity of TPX2 signals in control (n = 48) and Mcrs1-KD (n = 52) oocytes.


( B ) The active fragment of mTOR or active Akt2 was incubated with Flag - RagC WT purified from HEK - 293E cells . The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Akt inhibitor GDC - 0068 .

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

(B) The active fragment of mTOR or active Akt2 was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Akt inhibitor GDC-0068.


( C ) HEK - 293E Cells were deprived of serum for 2 hr followed by insulin ( 100nM ) stimulation for the indicated time . Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways . Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls .

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

(C) HEK-293E Cells were deprived of serum for 2 hr followed by insulin (100nM) stimulation for the indicated time. Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways. Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls.


( D ) HEK - 293E cells were deprived of total amino acids for 1hr followed by addition of amino acids for the indicated time . Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways . Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls .

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

(D) HEK-293E cells were deprived of total amino acids for 1hr followed by addition of amino acids for the indicated time. Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways. Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls.


( E ) Control or iRapKO MEFs were treated with or without 2 μM 4 - Hydroxytamoxifen ( 4 - OHT ) for 4 days to induce Raptor knockout . Treated cells were deprived of serum for 2 hr , followed by insulin ( 100 nM ) stimulation for 10 min , and samples were analysed by Western blot .

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

(E) Control or iRapKO MEFs were treated with or without 2 μM 4-Hydroxytamoxifen (4-OHT) for 4 days to induce Raptor knockout. Treated cells were deprived of serum for 2 hr, followed by insulin (100 nM) stimulation for 10 min, and samples were analysed by Western blot.


( F ) SIN1 WT was expressed in SIN1 KO MEFs , and cells were selected by FACS . Cell lines were deprived of serum for 2 hr , followed by insulin ( 100 nM ) stimulation for 10 min , and samples were analysed by Western blot .

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

(F) SIN1 WT was expressed in SIN1 KO MEFs, and cells were selected by FACS. Cell lines were deprived of serum for 2 hr, followed by insulin (100 nM) stimulation for 10 min, and samples were analysed by Western blot.


The active fragment of mTOR was incubated with Flag - RagC WT purified from HEK - 293E cells . The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin . Samples were analysed by Mass Spectrum for pRagC

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

The active fragment of mTOR was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin. Samples were analysed by Mass Spectrum for pRagC T394


The active fragment of mTOR was incubated with Flag - RagC WT purified from HEK - 293E cells . The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin . Samples were analysed by Western blot for pRagC

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

The active fragment of mTOR was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin. Samples were analysed by Western blot for pRagC S21


( A ) The HA - RagC HeLa Flp - In T - Rex stable cell lines were created ( See methods for details ) and the expression of RagC WT or mutants was induced by addition of tetracycline overnight . Cells were deprived of serum for 2 hr followed by insulin ( 100nM ) stimulation for 20 min . Lysates were analyzed by Western blot for levels of the specified proteins and the phosphorylation state of S6K1 . Graphs indicate the mean ± S . E . M . of quantitative analyses of the western blots after normalization for total protein ( * p < 0 . 05 , # no significant difference , two tailed Student ' s t test , n = 3 ) . ( B ) Expression of RagC WT , 3A or 3E was induced by addition of tetracycline overnight . Cells were serum starved for 2 hr followed by insulin ( 100nM ) stimulation for 20 min . Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1 . Graphs indicate the mean ± S . E . M . of quantitative analyses of the western blots after normalization for total protein ( * p < 0 . 05 , # no significant difference , two tailed Student ' s t test , n = 3 )

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

(A) The HA-RagC HeLa Flp-In T-Rex stable cell lines were created (See methods for details) and the expression of RagC WT or mutants was induced by addition of tetracycline overnight. Cells were deprived of serum for 2 hr followed by insulin (100nM) stimulation for 20 min. Lysates were analyzed by Western blot for levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, # no significant difference, two tailed Student's t test, n=3). (B) Expression of RagC WT, 3A or 3E was induced by addition of tetracycline overnight. Cells were serum starved for 2 hr followed by insulin (100nM) stimulation for 20 min. Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, # no significant difference, two tailed Student's t test, n=3)


( C ) Expression of RagC WT and 3A was induced by addition of tetracycline overnight . Cells were serum starved for 2 hr followed by insulin ( 100nM ) stimulation for indicated time . Left : Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1 . Right : Graph shows mean ± S . E . M . of quantitative analyses of the Western blots after normalization for total protein , n = 3

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 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 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

(C) Expression of RagC WT and 3A was induced by addition of tetracycline overnight. Cells were serum starved for 2 hr followed by insulin (100nM) stimulation for indicated time. Left: Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Right: Graph shows mean ± S.E.M. of quantitative analyses of the Western blots after normalization for total protein, n=3


( D ) HEK - 293E cells stably expressing RagC WT , 3A or 3E were deprived of total amino acids for 1hr followed by addition of amino acids for 10 min . Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1 . Graphs indicate the mean ± S . E . M . of quantitative analyses of the western blots after normalization for total protein ( * p < 0 . 05 , * * p < 0 . 01 , two tailed Student ' s t test , n = 3 )

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

(D) HEK-293E cells stably expressing RagC WT, 3A or 3E were deprived of total amino acids for 1hr followed by addition of amino acids for 10 min. Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, ** p<0.01, two tailed Student's t test, n=3)


( A ) Expression of RagC WT , 3A , or 3E was induced by overnight addition of tetracycline in HeLa cells followed by cell size analysis using a particle size counter ( Horizontal lines , box limits and whiskers represent the median , the 25th percentile ot the 75th percentile and the minimum to the maximum respectively . * p < 0 . 05 , # no significant difference , One - way ANOVA with Bonferroni multiple comparisons , n = 4 )

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

(A) Expression of RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=4)


( B ) Phospho - RagC positively regulates wing compartment size in Drosophila . Over - expression ( OE ) of dRagC WT , 3A , or 3E in wing posterior compartment using en - Gal4 driver and the posterior compartment sizes were measured in each genotype ( ( Horizontal lines , box limits and whiskers represent the median , the 25th percentile ot the 75th percentile and the minimum to the maximum respectively . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , One - way ANOVA with Bonferroni multiple comparisons , n ≥ 63 flies per condition )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Phospho-RagC positively regulates wing compartment size in Drosophila. Over-expression (OE) of dRagC WT, 3A, or 3E in wing posterior compartment using en-Gal4 driver and the posterior compartment sizes were measured in each genotype ((Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. ** p<0.01, *** p<0.001, **** p<0.0001, One-way ANOVA with Bonferroni multiple comparisons, n ≥ 63 flies per condition)


( C ) Empty vector ( EV ) , RagC WT , 3A or 3E were transiently expressed in stable RagC knockdown HEK - 293E cells followed by cell size analysis using a particle size counter ( Horizontal lines , box limits and whiskers represent the median , the 25th percentile ot the 75th percentile and the minimum to the maximum respectively . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , # no significant difference , One - way ANOVA with Bonferroni multiple comparisons , n = 2 for C2 and n = 4 for other conditions )

O O O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O 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) Empty vector (EV), RagC WT, 3A or 3E were transiently expressed in stable RagC knockdown HEK-293E cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, ** p<0.01, **** p<0.0001, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=2 for C2 and n=4 for other conditions)


( D ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were expressed in HEK - 293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr . Lysates were analyzed by Western blot for phosphorylation of ULK1 and total

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

(D) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Lysates were analyzed by Western blot for phosphorylation of ULK1 and total ULK1


( E ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were expressed in HEK - 293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr . Chloroquine ( 100μM ) was added to media 30 min prior to harvest . Lysates were analyzed by Western blot for levels of LC3 - II and 14 - 3 - 3 . Graphs show mean ± S . E . M . of quantitative analyses of LC3 - II blotting after normalization for 14 - 3 - 3 . * p < 0 . 05 , two tailed Student ' s t test , n = 4

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

(E) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. Lysates were analyzed by Western blot for levels of LC3-II and 14-3-3.Graphs show mean ± S.E.M. of quantitative analyses of LC3-II blotting after normalization for 14-3-3. * p<0.05, two tailed Student's t test, n=4


( F ) GFP - LC3 was co - expressed with empty vector , RagC WT , 3A or 3E in HEK - 293E cells followed 48 hr later by serum deprivation for 4 hr . Chloroquine ( 100μM ) was added to media 30 min prior to harvest . LC3 and RagC were monitored by confocal fluorescence microscopy . Scale bar , 10 µm

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

(F) GFP-LC3 was co-expressed with empty vector, RagC WT, 3A or 3E in HEK-293E cells followed 48 hr later by serum deprivation for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. LC3 and RagC were monitored by confocal fluorescence microscopy. Scale bar, 10 µm


( G ) Quantitative analysis of GFP puncta ( F ) ( * * * * p < 0 . 0001 , two tailed Student ' s t test ; n ≥ 21 ) . Mean value is shown as a horizontal bar

O O O O O O O O O O O O O O O O O O O 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 analysis of GFP puncta (F) (**** p<0.0001, two tailed Student's t test; n ≥ 21). Mean value is shown as a horizontal bar


( A ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were transiently expressed in HEK - 293E cells . RagC and lysosome maker , Lamp1 , were monitored by confocal fluorescence microscopy . Scale bar , 10 μm

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

(A) Empty vector (EV), HA-RagC WT, 3A or 3E were transiently expressed in HEK-293E cells. RagC and lysosome maker, Lamp1, were monitored by confocal fluorescence microscopy. Scale bar, 10 μm


( B ) Empty vector ( EV ) , HA - RagC WT or 3A were transiently expressed with scramble or RagC & RagD shRNA in HEK - 293E cells . mTOR and HA - RagC were monitored by confocal fluorescence microscopy . Scale bar , 10 µm

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

(B) Empty vector (EV), HA-RagC WT or 3A were transiently expressed with scramble or RagC & RagD shRNA in HEK-293E cells. mTOR and HA-RagC were monitored by confocal fluorescence microscopy. Scale bar, 10 µm


( C ) Empty vector ( EV ) , Flag - RagC WT , 3A or 3E were transiently expressed with HA - RagA in HEK - 293E cells . Total cell lysates and amounts of mTOR , Raptor and HA - RagA in the Flag - RagC immunoprecipitates were analyzed by Western blot . Graphs show mean ± S . E . M . of quantitative analyses of Western blots ( * p < 0 . 05 , two tailed Student ' s t test , n ≥ 3 )

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

(C) Empty vector (EV), Flag-RagC WT, 3A or 3E were transiently expressed with HA-RagA in HEK-293E cells. Total cell lysates and amounts of mTOR, Raptor and HA-RagA in the Flag-RagC immunoprecipitates were analyzed by Western blot. Graphs show mean ± S.E.M. of quantitative analyses of Western blots (* p<0.05, two tailed Student's t test, n ≥ 3)


( D ) Expression of HA - RagC WT , 3A , or 3E was induced by overnight addition of tetracycline in HeLa cells . Total cell lysates ( TCL ) and amount of mTOR in the Raptor immunoprecipitates were analyzed by Western blot . Graphs show mean ± S . E . M . of quantitative analyses of Western blots ( * * * p < 0 . 001 , Student ' s t test , n = 3 )

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

(D) Expression of HA-RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells. Total cell lysates (TCL) and amount of mTOR in the Raptor immunoprecipitates were analyzed by Western blot. Graphs show mean ± S.E.M. of quantitative analyses of Western blots (*** p<0.001, Student's t test, n=3)


A , Rb1 protein ( RB ) was expressed in uterine luminal epithelium and stroma during early pregnancy in mice . Scale bar = 100μm ; arrowhead , embryo . B , Phosphorylated RB ( pRB ) protein , the inactive form of RB , was expressed in the luminal epithelium on days 2 and 3 of pregnancy . Scale bar = 100μm .

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

A, Rb1 protein (RB) was expressed in uterine luminal epithelium and stroma during early pregnancy in mice. Scale bar = 100μm; arrowhead, embryo. B, Phosphorylated RB (pRB) protein, the inactive form of RB, was expressed in the luminal epithelium on days 2 and 3 of pregnancy. Scale bar = 100μm.


C , RB was efficiently deleted in the uterus of mice with uterine deletion of Rb1 ( Rb1d / d mice ) on day 4 of pregnancy . Scale bar = 100μm . D , RB expression was normal in the ovary of Rb1d / d mice on day 4 of pregnancy . Scale bar = 100μm .

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

C, RB was efficiently deleted in the uterus of mice with uterine deletion of Rb1 (Rb1d/d mice) on day 4 of pregnancy. Scale bar = 100μm. D, RB expression was normal in the ovary of Rb1d/d mice on day 4 of pregnancy. Scale bar = 100μm.


E , The number of new - born pups was decreased in Rb1d / d dams ( n = 37 different dams ) compared to their littermate controls ( Rb1f / f mice , n = 20 different dams ) ( mean ± SEM , Student ' s t test ) .

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E, The number of new-born pups was decreased in Rb1d/d dams (n=37 different dams) compared to their littermate controls (Rb1f/f mice, n=20 different dams) (mean ± SEM, Student's t test).


A - C , Ovulation , fertilization and development of preimplantation embryos were normal in Rb1d / d mice ( A & C , mean ± SEM , Student ' s t test ; n = 5 mice for each group ; B , Fisher ' s exact test ; n = 5 mice for each group ) .

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A-C, Ovulation, fertilization and development of preimplantation embryos were normal in Rb1d/d mice (A&C, mean ± SEM, Student's t test; n=5 mice for each group; B, Fisher's exact test; n=5 mice for each group).


Number of Ki67 - positive cells on day 4 in uterine epithelium of Rb1d / d mice was higher than that of Rb1f / f mice ( mean ± SEM , Student ' s t test ; n = 5 mice for each group ) . Scale bar , 100μm ; le , luminal epithelium ; s , stroma .

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Number of Ki67-positive cells on day 4 in uterine epithelium of Rb1d/d mice was higher than that of Rb1f/f mice (mean ± SEM, Student's t test; n=5 mice for each group). Scale bar, 100μm; le, luminal epithelium; s, stroma.


F - G , Embryo attachment occurred normally in Rb1d / d mice at 0900h on day 5 ( mean ± SEM , Student ' s t test ; n = 5 mice for each group ) . Arrow , implantation site ; scale bar = 1 cm .

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F-G, Embryo attachment occurred normally in Rb1d/d mice at 0900h on day 5 (mean ± SEM, Student's t test; n=5 mice for each group). Arrow, implantation site; scale bar = 1 cm.


H - I , Rb1d / d mice showed higher rate of embryo resorption on day 8 ( * P < 0 . 05 ; Fisher ' s exact test ; n = 45 implantation sites for Rb1f / f mice and n = 46 implantation sites for Rb1d / d mice ) . Arrow , implantation site ; scale bar = 1 cm .

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H-I, Rb1d/d mice showed higher rate of embryo resorption on day 8 (*P<0.05; Fisher's exact test; n=45 implantation sites for Rb1f/f mice and n=46 implantation sites for Rb1d/d mice). Arrow, implantation site; scale bar = 1 cm.


J , H & E staining showed embryo resorption in Rb1d / d mice on day 8 of pregnancy . Scale bar = 200 μm ; arrowhead , embryo ; arrow , broken embryo with blood cell infiltration .

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J, H&E staining showed embryo resorption in Rb1d/d mice on day 8 of pregnancy. Scale bar = 200 μm; arrowhead, embryo; arrow, broken embryo with blood cell infiltration.


K , Weight of implantation site was comparable between Rb1f / f and Rb1d / d mice on day 6 , but reduced on day 8 in Rb1d / d mice compared to Rb1f / f mice ( mean ± SEM , Student ' s t test ; n = 6 mice for each group ) .

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K, Weight of implantation site was comparable between Rb1f/f and Rb1d/d mice on day 6, but reduced on day 8 in Rb1d/d mice compared to Rb1f/f mice (mean ± SEM, Student's t test; n=6 mice for each group).


L - M , Expression of Cox2 and Bmp2 , markers of decidualization , was comparable in between Rb1d / d and Rb1f / f mice at 0900h on day 6 in implantation sites ( mean ± SEM , Student ' s t test ; n = 4 mice for each group ) .

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L-M, Expression of Cox2 and Bmp2, markers of decidualization, was comparable in between Rb1d/d and Rb1f/f mice at 0900h on day 6 in implantation sites (mean ± SEM, Student's t test; n=4 mice for each group).


A , Immunostaining of cytokeratin 8 ( CK8 ) , a marker of epithelial cell lineage , showed that epithelial alignment is collapsed and the trophoblast invades the stroma at implantation sites in Rb1f / f mice , while epithelial alignment is persistent and trophoblast invasion into the stroma is compromised in Rb1d / d mice . Scale bar = 200μm ; arrowhead , an embryo .

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A, Immunostaining of cytokeratin 8 (CK8), a marker of epithelial cell lineage, showed that epithelial alignment is collapsed and the trophoblast invades the stroma at implantation sites in Rb1f/f mice, while epithelial alignment is persistent and trophoblast invasion into the stroma is compromised in Rb1d/d mice. Scale bar = 200μm; arrowhead, an embryo.


B , Transmission electron microscopic analyses ( TEM ) showed that the uterine epithelium surrounding the embryo disappears and the trophoblast contacts directly with the stroma at 2000h on day 5 in Rb1f / f mice , while the uterine epithelium is persistent and the embryo does not attach to the stroma in Rb1d / d mice . Scale bar = 10µm ; red dotted line , stroma ; green dotted line , luminal epithelium ; blue dotted line , trophoblast .

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B, Transmission electron microscopic analyses (TEM) showed that the uterine epithelium surrounding the embryo disappears and the trophoblast contacts directly with the stroma at 2000h on day 5 in Rb1f/f mice, while the uterine epithelium is persistent and the embryo does not attach to the stroma in Rb1d/d mice. Scale bar = 10µm; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.


C , TEM showed that fragmented luminal epithelial cells with cytoplasmic lipid droplets are engulfed by the trophoblast in the control mice ( Rb1f / f mice ) . Scale bar = 2µm . Arrowhead , cytoplasmic fragments engulfed by trophoblast ; yellow dotted line circle , cytoplasmic lipid droplets in the luminal epithelium ; red dotted line , stroma ; green dotted line , luminal epithelium ; blue dotted line , trophoblast .

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C, TEM showed that fragmented luminal epithelial cells with cytoplasmic lipid droplets are engulfed by the trophoblast in the control mice (Rb1f/f mice). Scale bar = 2µm. Arrowhead, cytoplasmic fragments engulfed by trophoblast; yellow dotted line circle, cytoplasmic lipid droplets in the luminal epithelium; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.


D , TEM showed that the microvilli , markers of uterine receptivity , were observed at the surface of intact luminal epithelium on day 4 of pregnancy and epithelial cells with cytoplasmic lipid droplets were observed at 2000h on day 5 of pregnancy in the control mice ( Rb1f / f mice ) . Scale bar = 2µm ; red dotted line , stroma ; green dotted line , luminal epithelium ; blue dotted line , trophoblast .

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D, TEM showed that the microvilli, markers of uterine receptivity, were observed at the surface of intact luminal epithelium on day 4 of pregnancy and epithelial cells with cytoplasmic lipid droplets were observed at 2000h on day 5 of pregnancy in the control mice (Rb1f/f mice). Scale bar = 2µm; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.


E , Immunofluorescence of MFG - E8 , a marker of programmed cell death , showed that MFG - E8 is not expressed at the implantation sites of Rb1f / f mice at 0900h on day 5 and Rb1d / d mice at 0900h and 2000h on day 5 , but is expressed at the implantation sites of Rb1f / f mice at 2000h on day 5 . Scale bar = 100μm ; blue signal , nuclei stained by DAPI ; yellow signal , MFG - E8 ; arrowhead , embryo .

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E, Immunofluorescence of MFG-E8, a marker of programmed cell death, showed that MFG-E8 is not expressed at the implantation sites of Rb1f/f mice at 0900h on day 5 and Rb1d/d mice at 0900h and 2000h on day 5, but is expressed at the implantation sites of Rb1f/f mice at 2000h on day 5. Scale bar = 100μm; blue signal, nuclei stained by DAPI; yellow signal, MFG-E8; arrowhead, embryo.


A , Serum estradiol - 17β ( E2 ) levels on days 4 and 6 of pregnancy were comparable between Rb1f / f and Rb1d / d mice ( mean ± SEM , Student ' s t test ; n = 4 mice for each group ) .

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A, Serum estradiol-17β (E2) levels on days 4 and 6 of pregnancy were comparable between Rb1f/f and Rb1d/d mice (mean ± SEM, Student's t test; n=4 mice for each group).


B , Serum P4 levels on days 4 and 6 of pregnancy were comparable between Rb1f / f and Rb1d / d mice . In Rb1d / d mice , pre - implantation P4 treatment on days 2 and 3 increased serum P4 levels on day 4 but did not affect those on day 6 ( mean ± SEM , Student ' s t test ; n = 4 mice for each group ) .

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B, Serum P4 levels on days 4 and 6 of pregnancy were comparable between Rb1f/f and Rb1d/d mice. In Rb1d/d mice, pre-implantation P4 treatment on days 2 and 3 increased serum P4 levels on day 4 but did not affect those on day 6 (mean ± SEM, Student's t test; n=4 mice for each group).


D , Persistent Ki67 expression in the epithelium of Rb1d / d mice on day 4 was recovered by pre - implantation P4 treatment on days 2 and 3 . Scale bar = 100μm ; le , luminal epithelium ; s , stroma . E , Ratio of Ki67 - positive epithelial cells in Rb1d / d mice was completely suppressed by pre - implantation P4 supplementation on days 2 and 3 . The same data of Rb1f / f and Rb1d / d mice without P4 treatment in Fig 2D were used ( mean ± SEM , Student ' s t test ; n = 5 different mice for each group ) . Three different high - powered fields per mouse were analyzed and each of the Ki67 - posivie ratios was demonstrated .

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D, Persistent Ki67 expression in the epithelium of Rb1d/d mice on day 4 was recovered by pre-implantation P4 treatment on days 2 and 3. Scale bar = 100μm; le, luminal epithelium; s, stroma. E, Ratio of Ki67-positive epithelial cells in Rb1d/d mice was completely suppressed by pre-implantation P4 supplementation on days 2 and 3. The same data of Rb1f/f and Rb1d/d mice without P4 treatment in Fig 2D were used (mean ± SEM, Student's t test; n=5 different mice for each group). Three different high-powered fields per mouse were analyzed and each of the Ki67-posivie ratios was demonstrated.


G , Resorption rate was reduced on day 8 in Groups 1 ( n = 41 different implantation sites ) and 2 ( n = 30 different implantation sites ) , but not in Group 3 ( n = 40 different implantation sites ) . The same data of Rb1f / f and Rb1d / d mice without P4 treatment in Fig 2I were used . H & E staining was performed using all sections that shows the implantation sites , and resorption rate was evaluated . * P < 0 . 05 vs Rb1f / f mice ; * * P < 0 . 05 vs Rb1d / d mice ; Fisher ' s exact test .

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G, Resorption rate was reduced on day 8 in Groups 1 (n=41 different implantation sites) and 2 (n=30 different implantation sites), but not in Group 3 (n=40 different implantation sites). The same data of Rb1f/f and Rb1d/d mice without P4 treatment in Fig 2I were used. H&E staining was performed using all sections that shows the implantation sites, and resorption rate was evaluated. *P<0.05 vs Rb1f/f mice; **P<0.05 vs Rb1d/d mice; Fisher's exact test.


H , Number of pups delivered by Rb1d / d dams was normalized by pre - implantation P4 treatment on days 2 and 3 ( Group 2 ) ( mean ± SEM , Student ' s t test ) . The same data of Rb1f / f and Rb1d / d dams without P4 treatment in Fig 1E were used . The numbers of pups in 20 Rb1f / f and 37 Rb1d / d dams without P4 treatment and 7 Rb1d / d dams with P4 treatment were evaluated .

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H, Number of pups delivered by Rb1d/d dams was normalized by pre-implantation P4 treatment on days 2 and 3 (Group 2)(mean ± SEM, Student's t test). The same data of Rb1f/f and Rb1d/d dams without P4 treatment in Fig 1E were used. The numbers of pups in 20 Rb1f/f and 37 Rb1d/d dams without P4 treatment and 7 Rb1d/d dams with P4 treatment were evaluated.


A , pRIP3 , a central mediator of necroptosis , was not expressed at the implantation sites of Rb1d / d mice but was expressed at those of Rb1f / f mice at 1500h on day 5 . Scale bar = 100μm ; blue signal , nuclei stained by DAPI ; purple signal , pRIP3 ; le , luminal epithelium ; s , stroma ; arrowhead , embryo ; green dotted line , luminal epithelium ; red dotted line , stroma .

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A, pRIP3, a central mediator of necroptosis, was not expressed at the implantation sites of Rb1d/d mice but was expressed at those of Rb1f/f mice at 1500h on day 5. Scale bar = 100μm; blue signal, nuclei stained by DAPI; purple signal, pRIP3; le, luminal epithelium; s, stroma; arrowhead, embryo; green dotted line, luminal epithelium; red dotted line, stroma.


B , TNFα stimulated the expression of pMLKL , a critical mediator of necroptosis , in the primary mouse uterine epithelial cells . Scale bar = 50μm ; blue signal , nuclei stained by DAPI ; red signal , pMLKL .

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B, TNFα stimulated the expression of pMLKL, a critical mediator of necroptosis, in the primary mouse uterine epithelial cells. Scale bar = 50μm; blue signal, nuclei stained by DAPI; red signal, pMLKL.


C , Uterine expression of Tnfα was elevated in the control mice ( Rb1f / f mice ) on days 5 and 6 compared to on day 4 ( mean ± SEM , Student ' s t test ; n = 3 mice for each group ) . As for the uterine samples on days 5 and 6 , those with implantation sites were examined .

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C, Uterine expression of Tnfα was elevated in the control mice (Rb1f/f mice) on days 5 and 6 compared to on day 4 (mean ± SEM, Student's t test; n=3 mice for each group). As for the uterine samples on days 5 and 6, those with implantation sites were examined.


D , The implanting embryo and the uterus produced TNFα at the implantation site of both Rb1f / f and Rb1d / d mice . Immunostaining of TNFα at the implantation sites of Rb1f / f and Rb1d / d mice were performed . Scale bar = 100μm ; arrowhead , embryo .

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D, The implanting embryo and the uterus produced TNFα at the implantation site of both Rb1f/f and Rb1d/d mice. Immunostaining of TNFα at the implantation sites of Rb1f/f and Rb1d/d mice were performed. Scale bar = 100μm; arrowhead, embryo.


A - B , In vitro analyses using primary mouse uterine epithelial cells demonstrated that TNFα induces the expression of annexin V , a marker of PS presentation at the outer membrane of Rb1f / f epithelial cells but does not in Rb1d / d ones , and P4 restores TNFα - primed annexin V expression in Rb1d / d uterine epithelial cells ( mean ± SEM , Student ' s t test ) . Using three lines of primary mouse epithelial cells obtained from both Rb1f / fand Rb1d / d mice , the in vivo experiments were performed three times . Each of the annexin V - positive ratios was demonstrated . Scale bar = 100μm ; green signal , annexin V .

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A-B, In vitro analyses using primary mouse uterine epithelial cells demonstrated that TNFα induces the expression of annexin V, a marker of PS presentation at the outer membrane of Rb1f/f epithelial cells but does not in Rb1d/d ones, and P4 restores TNFα-primed annexin V expression in Rb1d/d uterine epithelial cells (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d mice, the in vivo experiments were performed three times. Each of the annexin V-positive ratios was demonstrated. Scale bar = 100μm; green signal, annexin V.


C - D , Annexin V assay using primary mouse uterine epithelial cells showed that TNFα administration increases the expression of annexin V in growth - arrested mouse epithelial cells with thymidine treatment , but does not in the control cells ( mean ± SEM , Student ' s t test ) . Using three lines of primary mouse epithelial cells obtained from both Rb1f / fand Rb1d / d mice , the in vivo experiments were performed three times . Each of the annexin V - positive ratios was demonstrated . Scale bar = 100μm ; green signal , annexin V .

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C-D, Annexin V assay using primary mouse uterine epithelial cells showed that TNFα administration increases the expression of annexin V in growth-arrested mouse epithelial cells with thymidine treatment, but does not in the control cells (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d mice, the in vivo experiments were performed three times. Each of the annexin V-positive ratios was demonstrated. Scale bar = 100μm; green signal, annexin V.


E , The expression of TNF receptor type 2 ( TNFR2 ) was upregulated in growth - arrested epithelial cells with thymidine and P4 supplementation ( mean ± SEM , Student ' s t test ) . Using three lines of primary mouse epithelial cells obtained from both Rb1f / fand Rb1d / d groups , qPCR was performed in duplicate .

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E, The expression of TNF receptor type 2 (TNFR2) was upregulated in growth-arrested epithelial cells with thymidine and P4 supplementation (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d groups, qPCR was performed in duplicate.


A , In Rb1d / d mice with pre - implantation P4 treatment , the fragmented uterine epithelial cells with the cytoplasmic lipid droplets were engulfed by the trophoblast cells . Scale bar = 2µm . Arrowhead , cytoplasmic fragments engulfed by trophoblast ; dotted line circle , lipid droplets in the cytoplasm ; tr , trophoblast ; le , luminal epithelium ; s , stroma ; red dotted line , stroma ; green dotted line , luminal epithelium ; blue dotted line , trophoblast .

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A, In Rb1d/d mice with pre-implantation P4 treatment, the fragmented uterine epithelial cells with the cytoplasmic lipid droplets were engulfed by the trophoblast cells. Scale bar = 2µm. Arrowhead, cytoplasmic fragments engulfed by trophoblast; dotted line circle, lipid droplets in the cytoplasm; tr, trophoblast; le, luminal epithelium; s, stroma; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.


B , Pre - implantation P4 treatment rescued the expression of MFG - E8 on day 5 of pregnancy in Rb1d / d uteri . Scale bar = 100μm . Blue signal , nuclei stained by DAPI ; yellow signal , MFG - E8 ; le , luminal epithelium ; s , stroma ; arrowhead , embryo . C , Pre - implantation P4 treatment recovered the expression of pRIP3 , a mediator of necroptosis , on day 5 of pregnancy in Rb1d / d uteri . Scale bar = 100μm ; blue signal , nuclei stained by DAPI ; purple signal , pRIP3 ; le , luminal epithelium ; s , stroma ; arrowhead , embryo .

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B, Pre-implantation P4 treatment rescued the expression of MFG-E8 on day 5 of pregnancy in Rb1d/d uteri. Scale bar = 100μm. Blue signal, nuclei stained by DAPI; yellow signal, MFG-E8; le, luminal epithelium; s, stroma; arrowhead, embryo. C, Pre-implantation P4 treatment recovered the expression of pRIP3, a mediator of necroptosis, on day 5 of pregnancy in Rb1d/d uteri. Scale bar = 100μm; blue signal, nuclei stained by DAPI; purple signal, pRIP3; le, luminal epithelium; s, stroma; arrowhead, embryo.


A , RB was efficiently deleted in the uterine epithelium of uterine epithelium - specific conditional knockout mice ( Rb1 eKO mice ) . Scale bar = 100μm . Rb1 eKO mice , Rb1loxP / loxPLtfCre / + mice ; Rb1 eControl mice ( littermate controls of Rb1 eKO mice ) , Rb1loxP / loxP mice ; le , luminal epithelium ; s , stroma .

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A, RB was efficiently deleted in the uterine epithelium of uterine epithelium-specific conditional knockout mice (Rb1 eKO mice). Scale bar = 100μm. Rb1 eKO mice, Rb1loxP/loxPLtfCre/+ mice; Rb1 eControl mice (littermate controls of Rb1 eKO mice), Rb1loxP/loxP mice; le, luminal epithelium; s, stroma.


B , Number of new - born pups was comparable between Rb1 eKO and eControl dams ( mean ± SEM , Student ' s t test ; n = 10 different dams for each group ) .

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B, Number of new-born pups was comparable between Rb1 eKO and eControl dams (mean ± SEM, Student's t test; n=10 different dams for each group).


C , The expression patterns of Ki67 on day 4 in uterine epithelium were comparable between Rb1 eKO and Rb1 eControl mice . Scale bar , 100μm ; le , luminal epithelium ; s , stroma .

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C, The expression patterns of Ki67 on day 4 in uterine epithelium were comparable between Rb1 eKO and Rb1 eControl mice. Scale bar, 100μm; le, luminal epithelium; s, stroma.


A , Representative pictures of Ki67 staining in the human endometria obtained from women at the time of embryo implantation . Samples were divided into two groups according to the outcome of clinical pregnancy following endometrial biopsy . Scale bar = 100μm ; le , luminal epithelium ; s , stroma . B , Ratio of Ki67 - positive cells in the human uterine luminal epithelium during the implantation period was lower in the group with pregnancy than the group without pregnancy ( mean ± SEM , Student ' s t test ; n = 10 different individuals for each group ) . Three different high - powered fields per sample were analyzed and each of the Ki67 - posivie ratios was demonstrated .

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A, Representative pictures of Ki67 staining in the human endometria obtained from women at the time of embryo implantation. Samples were divided into two groups according to the outcome of clinical pregnancy following endometrial biopsy. Scale bar = 100μm; le, luminal epithelium; s, stroma. B, Ratio of Ki67-positive cells in the human uterine luminal epithelium during the implantation period was lower in the group with pregnancy than the group without pregnancy (mean ± SEM, Student's t test; n=10 different individuals for each group). Three different high-powered fields per sample were analyzed and each of the Ki67-posivie ratios was demonstrated.


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 ) .

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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 .

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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 ) .

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

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 .

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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 ) .

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

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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 .

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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 ) ) .

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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 .

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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 .

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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 ) .

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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 .

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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 ) .

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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).


C , D . mouse J110 cells were transfected with either miR - 449a mimic or a negative control mimic . Whole cell extracts were subjected to immunoblot analysis to detect endogenous JAM - A and ACTIN . ACTIN was used to normalize JAM - A expression ( norm . ) as loading control . p = 0 . 04454 ( Neg . ctrol . vs . miR - 449a mimic ) ( n = 4 independent wells , 2 independent transfections ) .

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

C, D. mouse J110 cells were transfected with either miR-449a mimic or a negative control mimic. Whole cell extracts were subjected to immunoblot analysis to detect endogenous JAM-A and ACTIN. ACTIN was used to normalize JAM-A expression (norm.) as loading control. p = 0.04454 (Neg. ctrol. vs. miR-449a mimic) (n = 4 independent wells, 2 independent transfections).


E , F . HeLa cells were cotransfected with either miR - 449a mimic or a negative control mimic , together with JAM - A - GFP fusion protein and GFP . Whole cell extracts were subjected to immunoblot analysis to detect GFP . GFP was used to normalize ( norm . ) by transfection efficiency as loading control . p = 0 . 001176 ( JAM - A WT Neg . ctrol . vs . miR - 449a mimic ) ( n = 9 independent wells , 4 independent transfections ) .

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 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

E, F. HeLa cells were cotransfected with either miR-449a mimic or a negative control mimic, together with JAM-A-GFP fusion protein and GFP. Whole cell extracts were subjected to immunoblot analysis to detect GFP. GFP was used to normalize (norm.) by transfection efficiency as loading control. p = 0.001176 (JAM-A WT Neg. ctrol. vs. miR-449a mimic) (n = 9 independent wells, 4 independent transfections).


G , H . Confocal images of coronal sections from E14 brains of wild type mice stained with anti - JAM - A antibody ( green ) , phalloidin to label actin ( gray ) and DAPI to label cell nuclei ( blue ) . Scale bars : 10 μm ( G ) , 5 μm ( H ) . I . Quantification of JAM - A protein expression in the ventricular zone of E14 brain coronal sections from miR - 34 / 449 KO mice compared with littermate controls ( Het ) by immunofluorescence . ACTIN ( phalloidin signal ) was used to normalize ( norm . ) the expresion levels of JAM - A . p = 0 . 04842 ( n = 5 ( KO ) or 7 ( het ) cortices per genotype group , 3 independent litters ) .

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

G, H. Confocal images of coronal sections from E14 brains of wild type mice stained with anti-JAM-A antibody (green), phalloidin to label actin (gray) and DAPI to label cell nuclei (blue). Scale bars: 10 μm (G), 5 μm (H).I. Quantification of JAM-A protein expression in the ventricular zone of E14 brain coronal sections from miR-34/449 KO mice compared with littermate controls (Het) by immunofluorescence. ACTIN (phalloidin signal) was used to normalize (norm.) the expresion levels of JAM-A. p = 0.04842 (n = 5 (KO) or 7 (het) cortices per genotype group, 3 independent litters).


A , D . Confocal time lapse images of metaphase HeLa cells stably expressing a centriole marker ( Centrin2 - GFP ; green ) , the microtubule marker ( α - tubulin - mRFP ; red ) and no extra protein ( A ) or mouse JAM - A ( D ) , 48 h after transfection of miR - 449a mimic , JAM - A siRNA , or non - targeting negative control siRNA . Scale bar , 10 μm . B , E . Quantification of spindle rotation for cells as shown in A and D . Each dot represents a single cell . Red bar indicates median . Significance was tested by Pairwise t - test with Bonferroni correction . p - value = 2e - 11 ( Neg . control versus miR449 mimic ) , p - value = 0 . 01928 ( Neg . control versus JAM - A siRNA ) , p - value = 0 . 17492 ( Neg . control versus miR449 mimic , under JAM - A overexpression ) . * indicates p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; p * * * p ≤ 0 . 001 for all panels . C , F . Cumulative histograms of mitotic duration from nuclear envelope breakdown until anaphase onset , measured for cells as shown in A and D ; n ≥ 20 cells for all conditions , 2 independent replicates .

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

A, D. Confocal time lapse images of metaphase HeLa cells stably expressing a centriole marker (Centrin2-GFP; green), the microtubule marker (α-tubulin-mRFP; red) and no extra protein (A) or mouse JAM-A (D), 48 h after transfection of miR-449a mimic, JAM-A siRNA, or non-targeting negative control siRNA. Scale bar, 10 μm.B, E. Quantification of spindle rotation for cells as shown in A and D. Each dot represents a single cell. Red bar indicates median. Significance was tested by Pairwise t-test with Bonferroni correction. p-value = 2e-11 (Neg. control versus miR449 mimic), p-value = 0.01928 (Neg. control versus JAM-A siRNA), p-value = 0.17492 (Neg. control versus miR449 mimic, under JAM-A overexpression). * indicates p ≤ 0.05; ** p ≤ 0.01; p*** p ≤ 0.001 for all panels.C, F. Cumulative histograms of mitotic duration from nuclear envelope breakdown until anaphase onset, measured for cells as shown in A and D; n ≥ 20 cells for all conditions, 2 independent replicates.


A - Distribution of Casp3 + cells in rostral and ventral domains of an E11 . 5 Rx - Dicer mutant embryo . NCx , neocortex ; OB , olfactory bulb ; Spt , septum .

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

A - Distribution of Casp3+ cells in rostral and ventral domains of an E11.5 Rx-Dicer mutant embryo. NCx, neocortex; OB, olfactory bulb; Spt, septum.


B , C - Marker analysis of Casp3 + cells in the rostral telencephalon of E12 . 5 Rx - Dicer mutant embryos . Most apoptotic cells are Pax6 + RGCs ( solid arrowheads ) and not Tbr1 + neurons ( open arrowheads ) . N = 3 replicates per marker .

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 B-MEASURED_VAR 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,C - Marker analysis of Casp3+ cells in the rostral telencephalon of E12.5 Rx-Dicer mutant embryos. Most apoptotic cells are Pax6+ RGCs (solid arrowheads) and not Tbr1+ neurons (open arrowheads). N = 3 replicates per marker.


D , E - Distribution and abundance of apoptotic cells ( Casp3 + ) in the rostral telencephalic primordium of control and Rx - Dicer mutant embryos at the indicated ages . Dotted line indicates basal surface , dashed line apical surface . N = 3 replicates per genotype and age .

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 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

D,E - Distribution and abundance of apoptotic cells (Casp3+) in the rostral telencephalic primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted line indicates basal surface, dashed line apical surface. N = 3 replicates per genotype and age.


F , G - Distribution and abundance of mitoses ( PH3 + ) and neurons ( Tuj1 ) in the rostral telencephalic primordium of control and Rx - Dicer mutant embryos at the indicated ages . Dotted line indicates basal surface , dashed line apical surface . N = 3 - 4 replicates per genotype and age . No significant differences were found between control and mutant embryos in apical nor basal mitoses .

O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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 - Distribution and abundance of mitoses (PH3+) and neurons (Tuj1) in the rostral telencephalic primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted line indicates basal surface, dashed line apical surface. N = 3-4 replicates per genotype and age. No significant differences were found between control and mutant embryos in apical nor basal mitoses.


A - DAPI stain of sagittal sections through the rostral telencephalon of E17 . 5 control and Rx - Dicer mutant littermates showing the neocortex ( NCx ) and olfactory bulb ( OB ; dashed line ) .

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

A - DAPI stain of sagittal sections through the rostral telencephalon of E17.5 control and Rx-Dicer mutant littermates showing the neocortex (NCx) and olfactory bulb (OB; dashed line).


B - Expression pattern of Grm1 mRNA in OB ( dotted line ) of control and Rx - Dicer mutants . C - Quantification of OB perimeter ( mean ± SEM ; symbols indicate values for individual embryos ) ; t - test , * * * P < 0 . 001 . N = 14 replicates per genotype .

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

B - Expression pattern of Grm1 mRNA in OB (dotted line) of control and Rx-Dicer mutants. C - Quantification of OB perimeter (mean ± SEM; symbols indicate values for individual embryos); t-test, ***P < 0.001. N = 14 replicates per genotype.


D - Immunostains of E17 . 5 control and Rx - Dicer mutant brains showing the distribution of progenitor cells ( Ki67 , red ) and neurons ( Tuj1 , green ) . Arrowheads indicate rosettes . Sp , septum .

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

D - Immunostains of E17.5 control and Rx-Dicer mutant brains showing the distribution of progenitor cells (Ki67, red) and neurons (Tuj1, green). Arrowheads indicate rosettes. Sp, septum.


E - Detail of a rosette from an E17 . 5 Rx - Dicer mutant embryo displaying typical features : closed apical surface ( dotted line ) with PH3 + apical mitoses ( white arrowhead ) and basal mitoses , surrounded by Tuj1 + neurons ( green ) . Dashed line indicates the basal border of the rosette .

O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O

E - Detail of a rosette from an E17.5 Rx-Dicer mutant embryo displaying typical features: closed apical surface (dotted line) with PH3+ apical mitoses (white arrowhead) and basal mitoses, surrounded by Tuj1+ neurons (green). Dashed line indicates the basal border of the rosette.


F - Rostral half of an Rx - Dicer mutant E17 . 5 brain immunostained for Pax6 , clarified and segmented to reveal rosettes ( yellow ) . C , caudal ; R , rostral .

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

F - Rostral half of an Rx-Dicer mutant E17.5 brain immunostained for Pax6, clarified and segmented to reveal rosettes (yellow). C, caudal; R, rostral.


G , H - BrdU incorporation and cell cycle re - entry analysis with the progenitor cell marker Ki67 at E17 . 5 , in the rostral cortex of control embryos compared to rostral rosettes of Rx - Dicer mutants . Dotted lines indicate apical surface , dashed lines indicate basal border of VZ . Arrowheads indicate double - positive cells . Data in histograms are mean ± SEM , symbols indicate values for individual embryos ; n ≥ 3 embryos ; X2 - test , * P < 0 . 05 , * * * P < 0 . 001 .

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

G,H - BrdU incorporation and cell cycle re-entry analysis with the progenitor cell marker Ki67 at E17.5, in the rostral cortex of control embryos compared to rostral rosettes of Rx-Dicer mutants. Dotted lines indicate apical surface, dashed lines indicate basal border of VZ. Arrowheads indicate double-positive cells. Data in histograms are mean ± SEM, symbols indicate values for individual embryos; n ≥ 3 embryos; X2-test, *P < 0.05, ***P < 0.001.


I - Apical lumen of rosettes immunostained against apical complex proteins ( Par3 , β - Catenin ) , primary cilia ( Arl13b ) , apical mitoses ( PhVim ) and neurons ( Tuj1 ) .

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

I - Apical lumen of rosettes immunostained against apical complex proteins (Par3, β-Catenin), primary cilia (Arl13b), apical mitoses (PhVim) and neurons (Tuj1).


J - Coronal section through the rostral telencephalon of an E14 . 5 Rx - Dicer mutant embryo illustrating the high abundance and location of rosettes ( arrows ) , as revealed by the distribution of mitoses ( PH3 ) and neurons ( Tuj1 ) . K - S - Analysis of the regional identity of rosettes in E14 . 5 Rx - Dicer mutants . ( K ) Schema of normal transcription factor expression patterns defining telencephalic regional identity . Expression of Ngn2 , Tbr2 and Pax6 identifies rosettes in the rostro - dorsal telencephalon as having dorsal identity ( L - N , P ) ; Gsx2 , Dlx2 and Dlx5 identify rosettes in the ventral telencephalon as having ventral identity ( O , Q ) ; Nkx2 . 1 identifies MGE rosettes as being normotopic ( R ) ; absence of Gsx2 in LGE rosette cells identifies them as ectopic ( S ) . Tuj1 labels neurons . In ( L , M ) , arrowheads indicate the border between dorsal and ventral territories , and dotted line indicates the outer border of the telencephalon . LGE , lateral ganglionic eminence ; MGE , medial ganglionic eminence ; P , pallium ; SP , subpallium .

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

J - Coronal section through the rostral telencephalon of an E14.5 Rx-Dicer mutant embryo illustrating the high abundance and location of rosettes (arrows), as revealed by the distribution of mitoses (PH3) and neurons (Tuj1). K-S - Analysis of the regional identity of rosettes in E14.5 Rx-Dicer mutants. (K) Schema of normal transcription factor expression patterns defining telencephalic regional identity. Expression of Ngn2, Tbr2 and Pax6 identifies rosettes in the rostro-dorsal telencephalon as having dorsal identity (L-N,P); Gsx2, Dlx2 and Dlx5 identify rosettes in the ventral telencephalon as having ventral identity (O,Q); Nkx2.1 identifies MGE rosettes as being normotopic (R); absence of Gsx2 in LGE rosette cells identifies them as ectopic (S). Tuj1 labels neurons. In (L,M), arrowheads indicate the border between dorsal and ventral territories, and dotted line indicates the outer border of the telencephalon. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; P, pallium; SP, subpallium.


A - Coronal section through the rostral telencephalon of an E14 . 5 Rx - Dicer mutant embryo displaying the three types of rosettes .

O 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 - Coronal section through the rostral telencephalon of an E14.5 Rx-Dicer mutant embryo displaying the three types of rosettes.


B - High magnification of rosettes in Rx - Dicer mutants at E12 . 5 ( Type 1 ) , E14 . 5 ( Type 2 ) and E17 . 5 ( Type 3 ) , immunostained for PH3 and Tuj1 . Dashed line indicates ventricular surface ; arrowheads indicate basal mitoses ; arrows indicate a stream of apical mitoses connecting the lumen of the rosette with the lumen of the telencephalic ventricle .

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

B - High magnification of rosettes in Rx-Dicer mutants at E12.5 (Type 1), E14.5 (Type 2) and E17.5 (Type 3), immunostained for PH3 and Tuj1. Dashed line indicates ventricular surface; arrowheads indicate basal mitoses; arrows indicate a stream of apical mitoses connecting the lumen of the rosette with the lumen of the telencephalic ventricle.


C , D - Abundance of rosette types at the indicated ages ( C ) , and rostro - caudal distribution of total rosette abundance per age , independent of type ( D ) . n ≥ 2 brains per age .

O O O O O O O O O O O O O O O O O O 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,D - Abundance of rosette types at the indicated ages (C), and rostro-caudal distribution of total rosette abundance per age, independent of type (D). n ≥ 2 brains per age.


F , G - Distribution of neurons ( Tuj1 + cells ) and apical adherens junction protein Par3 in the OB primordium of control and Rx - Dicer mutant embryos at the indicated ages . Dotted lines indicate basal border , dashed lines indicate apical border . White arrowheads indicate ectopic neurons and / or absence of Par3 . Arrow in ( G ) indicates accumulation of Par3 at the lumen of a nascent rosette .

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

F,G - Distribution of neurons (Tuj1+ cells) and apical adherens junction protein Par3 in the OB primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted lines indicate basal border, dashed lines indicate apical border. White arrowheads indicate ectopic neurons and/or absence of Par3. Arrow in (G) indicates accumulation of Par3 at the lumen of a nascent rosette.


A , B - Changes in expression levels of miRNAs between control and Rx - Dicer mutants at E11 . 5 in the rostral telencephalon . Red dots indicate statistically differentially expressed miRNAs according to FDR correction of P values based on the Wald statistic ( DESeq2 analysis ) ( B ) . Dashes lines indicate stringency limits in evaluation of results .

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

A,B - Changes in expression levels of miRNAs between control and Rx-Dicer mutants at E11.5 in the rostral telencephalon. Red dots indicate statistically differentially expressed miRNAs according to FDR correction of P values based on the Wald statistic (DESeq2 analysis) (B). Dashes lines indicate stringency limits in evaluation of results.


D , E - Changes in mRNA expression levels between control and Rx - Dicer mutants at E11 . 5 in the rostral telencephalon . Statistically differentially expressed mRNAs according to FDR correction of P values based on the Wald statistic ( DESeq2 analysis ; Adj . P < 0 . 1 and FC > 1 . 25 ) are highlighted in red , and the three with the highest Adj . P value are identified by name . Blue dot indicates Irs2 : FC = 1 . 748 , Adj . P = 5 . 95e - 08 .

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

D,E - Changes in mRNA expression levels between control and Rx-Dicer mutants at E11.5 in the rostral telencephalon. Statistically differentially expressed mRNAs according to FDR correction of P values based on the Wald statistic (DESeq2 analysis; Adj. P < 0.1 and FC > 1.25) are highlighted in red, and the three with the highest Adj. P value are identified by name. Blue dot indicates Irs2: FC=1.748, Adj. P = 5.95e-08.


G - Heatmaps of relative expression levels of DEGs related to apoptosis ( top ) and proliferation ( bottom ) . Rows correspond to independent biological replicates . Indicated are some example genes .

O O O O O O O O O O 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 - Heatmaps of relative expression levels of DEGs related to apoptosis (top) and proliferation (bottom). Rows correspond to independent biological replicates. Indicated are some example genes.


H - Enrichment plots from GSEA for MSigDB Hallmark Neurogenesis ( NES = 2 . 98 ; p = 0 ; Adj . P = 0 . 00045 ) , Regulation of cell proliferation ( NES = 2 . 40 ; p = 0 ; Adj . P = 0 . 0033 ) , Positive regulation of cell adhesion ( NES = 2 . 06 ; p = 0 . 0058 ; Adj . P = 0 . 013 ) , Regulation of response to stress ( NES = 2 . 50 ; p = 0 ; Adj . P = 0 . 0023 ) , Positive regulation of cell death ( NES = 1 . 84 ; p = 0 . 0179 ; Adj . P = 0 . 0326 ) , Apoptotic signaling pathway ( NES = 1 . 59 ; p = 0 . 047 ; Adj . P = 0 . 084 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - Enrichment plots from GSEA for MSigDB Hallmark Neurogenesis (NES = 2.98; p = 0; Adj. P = 0.00045), Regulation of cell proliferation (NES = 2.40; p=0; Adj. P = 0.0033), Positive regulation of cell adhesion (NES = 2.06; p = 0.0058; Adj. P = 0.013), Regulation of response to stress (NES = 2.50; p = 0; Adj. P = 0.0023), Positive regulation of cell death (NES = 1.84; p = 0.0179; Adj. P = 0.0326), Apoptotic signaling pathway (NES = 1.59; p= 0.047; Adj. P = 0.084).