( e ) The indicated HA - Ulk1 proteins were immunopurified from transfected HEK293 cells grown in high - glucose medium , and then incubated with AMPK in the presence of cold ATP for 15 minin vitro . After the reaction , AMPK was removed by extensive washing , the resulting Ulk1 immuno - complexes were assayed for kinase activity in the presence of 32P - ATP .

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(e) The indicated HA-Ulk1 proteins were immunopurified from transfected HEK293 cells grown in high-glucose medium, and then incubated with AMPK in the presence of cold ATP for 15 minin vitro. After the reaction, AMPK was removed by extensive washing, the resulting Ulk1 immuno-complexes were assayed for kinase activity in the presence of 32P-ATP.


( f ) HA - Ulk1 proteins ( wild type or S317 / 777A mutant ) were immunoprecipitated from the transfected HEK293 cells , which were incubated with or without glucose ( 4 h ) before lysis . An in vitro kinase reaction was performed in the presence of GST - ATG13 and FIP200 . Uncropped images of blots are shown in Supplementary Fig . S5 .

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(f) HA-Ulk1 proteins (wild type or S317/777A mutant) were immunoprecipitated from the transfected HEK293 cells, which were incubated with or without glucose (4 h) before lysis. An in vitro kinase reaction was performed in the presence of GST-ATG13 and FIP200. Uncropped images of blots are shown in Supplementary Fig. S5.


( a ) AMPK interacts with Ulk1 . HEK293 cells were transfected with the various Flag - Ulk1 deletion mutants together with AMPK α / β / γ , Atg13 and FIP200 . Flag - Ulk1 protein ( indicated by white arrows ) was immunoprecipitated and co - immunoprecipitation of AMPK α / β / γ , Atg13 and FIP200 were examined by western blots .

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(a) AMPK interacts with Ulk1. HEK293 cells were transfected with the various Flag-Ulk1 deletion mutants together with AMPK α/β/γ, Atg13 and FIP200. Flag-Ulk1 protein (indicated by white arrows) was immunoprecipitated and co-immunoprecipitation of AMPK α/β/γ, Atg13 and FIP200 were examined by western blots.


( b ) Deletion analysis of Ulk1 regions responsible for AMPK interaction . The indicated Flag - Ulk1 truncation mutants were immunoprecipitated from transfected HEK293 cells co - expressing AMPK complex ( α / β / γ ) . Co - immunoprecipitation of AMPK subunits was determined by western blots .

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(b) Deletion analysis of Ulk1 regions responsible for AMPK interaction. The indicated Flag-Ulk1 truncation mutants were immunoprecipitated from transfected HEK293 cells co-expressing AMPK complex (α/β/γ). Co-immunoprecipitation of AMPK subunits was determined by western blots.


( c ) Rheb inhibits the Ulk1 - AMPK interaction . HA - AMPKα , Flag - Ulk1 and Myc - Rheb were co - transfected into HEK293 cells as indicated . Cells were treated with or without rapamycin ( 50 nM Rapa ) for 1 h before lysis . Flag - Ulk1 was immunoprecipitated and co - immunoprecipitates of AMPKα were determined by western blot .

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(c) Rheb inhibits the Ulk1-AMPK interaction. HA-AMPKα, Flag-Ulk1 and Myc-Rheb were co-transfected into HEK293 cells as indicated. Cells were treated with or without rapamycin (50 nM Rapa) for 1 h before lysis. Flag-Ulk1 was immunoprecipitated and co-immunoprecipitates of AMPKα were determined by western blot.


( d ) Rapamycin treatment enhances the interaction of endogenous Ulk1 and AMPK . Endogenous Ulk1 proteins were immunoprecipitated from either Ulk1 or AMPK wild - type and knockout ( single - knockout ; KO or double - knockout ; DKO ) MEFs . Treatment with 50 nM rapamycin for 1 h is indicated ( Rapa ) . Co - immunoprecipitation of endogenous AMPKα protein was determined by western blot . The arrow indicates AMPKα protein .

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(d) Rapamycin treatment enhances the interaction of endogenous Ulk1 and AMPK. Endogenous Ulk1 proteins were immunoprecipitated from either Ulk1 or AMPK wild-type and knockout (single-knockout; KO or double-knockout; DKO) MEFs. Treatment with 50 nM rapamycin for 1 h is indicated (Rapa). Co-immunoprecipitation of endogenous AMPKα protein was determined by western blot. The arrow indicates AMPKα protein.


( e ) Phosphorylation by mTORC1 inhibits the ability of Ulk1 to bind AMPK in vitro . CBP / SBP - Ulk1 was purified from transfected HEK293 cells by streptavidin beads and the Ulk1 - bead complex was incubated with mTORC1 , which was prepared by Raptor immunoprecipitation , in the presence of cold ATP , as indicated . The resulting Ulk1 complex was incubated with the cell lysates containing AMPK , then extensively washed . The Ulk1 and associated AMPKα were detected by western blot . Uncropped images of blots are shown in Supplementary Fig . S5 .

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(e) Phosphorylation by mTORC1 inhibits the ability of Ulk1 to bind AMPK in vitro. CBP/SBP-Ulk1 was purified from transfected HEK293 cells by streptavidin beads and the Ulk1-bead complex was incubated with mTORC1, which was prepared by Raptor immunoprecipitation, in the presence of cold ATP, as indicated. The resulting Ulk1 complex was incubated with the cell lysates containing AMPK, then extensively washed. The Ulk1 and associated AMPKα were detected by western blot. Uncropped images of blots are shown in Supplementary Fig. S5.


( a ) mTORC1 phosphorylates the Ulk1 S / T domain . Ulk1 deletion mutants were prepared from the transfected HEK293 cells and used for in vitro mTORC1 assay . Phosphorylation was examined by 32P - autoradiogram ( top ) and protein level was determined by western blot ( bottom ) .

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(a) mTORC1 phosphorylates the Ulk1 S/T domain. Ulk1 deletion mutants were prepared from the transfected HEK293 cells and used for in vitro mTORC1 assay. Phosphorylation was examined by 32P-autoradiogram (top) and protein level was determined by western blot (bottom).


( b ) Ser 757 is phosphorylated by mTORC1 . Left : the indicated recombinant GST - mUlk1 mutants were purified from E . coli and used for in vitro mTORC1 assay as substrates . Deletion analyses isolated the fragment ( 753 - 771 ) as a target for mTORC1 . The Ulk1 ( 753 - 771 ) fragment contains five conserved serine / threonine residues , Thr 754 , Ser 757 , Ser 760 , Thr 763 and Thr 770 . Right : mutation of Ser 757 abolished Ulk1 phosphorylation by mTORC1 in vitro . GST was used as negative control for mTORC1 phosphorylation reaction . Phosphorylation was determined by 32P - autoradiograph ( top ) , whereas protein levels were detected by Coomassie staining ( bottom ) .

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(b) Ser 757 is phosphorylated by mTORC1. Left: the indicated recombinant GST-mUlk1 mutants were purified from E. coli and used for in vitro mTORC1 assay as substrates. Deletion analyses isolated the fragment (753-771) as a target for mTORC1. The Ulk1 (753-771) fragment contains five conserved serine/threonine residues, Thr 754, Ser 757, Ser 760, Thr 763 and Thr 770. Right: mutation of Ser 757 abolished Ulk1 phosphorylation by mTORC1 in vitro. GST was used as negative control for mTORC1 phosphorylation reaction. Phosphorylation was determined by 32P-autoradiograph (top), whereas protein levels were detected by Coomassie staining (bottom).


( c ) Rheb increases Ulk1 Ser 757 phosphorylation . HA - Ulk1 wild type and the S757A mutant were immunoprecipitated from transfected HEK293 cells . Co - transfection with Rheb and rapamycin ( Rapa ) treatment are indicated . Ulk1 Ser 757 phosphorylation was determined by western blot .

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(c) Rheb increases Ulk1 Ser 757 phosphorylation. HA-Ulk1 wild type and the S757A mutant were immunoprecipitated from transfected HEK293 cells. Co-transfection with Rheb and rapamycin (Rapa) treatment are indicated. Ulk1 Ser 757 phosphorylation was determined by western blot.


( d ) Rheb induces a mobility shift in wild - type Ulk1 , but not the Ulk1S757A mutant . HA - Ulk1 was transfected with or without Rheb into HEK293 cells . HA - Ulk1 was immunoprecipitated from the cells under nutrient - rich medium and Ulk1 mobility was examined by western blot .

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(d) Rheb induces a mobility shift in wild-type Ulk1, but not the Ulk1S757A mutant. HA-Ulk1 was transfected with or without Rheb into HEK293 cells. HA-Ulk1 was immunoprecipitated from the cells under nutrient-rich medium and Ulk1 mobility was examined by western blot.


( e ) Endogenous Ulk1 Ser 757 phosphorylation is elevated in Tsc1 − / − Tsc1 . Tsc1 + / + ( WT ) and Tsc1 − / − ( KO ) MEFs were starved of glucose ( 4 h ) , or treated with 50 nM rapamycin ( Rapa , 1 h ) . Ser 757 phosphorylation of endogenous Ulk1 was detected by a phospho - Ulk1 Ser 757 antibody . Uncropped images of blots are shown in Supplementary Fig . S5 .

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(e) Endogenous Ulk1 Ser 757 phosphorylation is elevated in Tsc1−/− Tsc1. Tsc1+/+ (WT) and Tsc1−/− (KO) MEFs were starved of glucose (4 h), or treated with 50 nM rapamycin (Rapa, 1 h). Ser 757 phosphorylation of endogenous Ulk1 was detected by a phospho-Ulk1 Ser 757 antibody. Uncropped images of blots are shown in Supplementary Fig. S5.


( a ) Ulk1 Ser 757 is required for mTORC1 to regulate the interaction of Ulk1 with AMPK in vivo . CBP / SBP tagged Ulk1 ( wild type or S757C ) was co - transfected with HA - AMPKα and Rheb into HEK293 cells as indicated . Ulk1 was purified by streptavidin beads and the co - precipitatedHA - AMPKα was examined by western blot ( Rapa , 50 nM rapamycin treatment for 1 h before cell lysis ) .

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(a) Ulk1 Ser 757 is required for mTORC1 to regulate the interaction of Ulk1 with AMPK in vivo. CBP/SBP tagged Ulk1 (wild type or S757C) was co-transfected with HA-AMPKα and Rheb into HEK293 cells as indicated. Ulk1 was purified by streptavidin beads and the co-precipitatedHA- AMPKα was examined by western blot (Rapa, 50 nM rapamycin treatment for 1 h before cell lysis).


( b ) Ulk1 Ser 757 is required for rapamycin to enhance the Ulk1 - AMPK interaction in vitro . CBP / SBP Ulk1 proteins ( wild type or S757C ) were prepared from transfected HEK293 cells , which were pre - incubated with 50 nM rapamycin ( Rapa , 1h ) as indicated . The Ulk1 proteins were purified by streptavidin beads and the resulting Ulk1 - bead was incubated with the bacterial purified AMPKα / β / γ complex . AMPKα protein levels in the in vitro pulldown assays were examined by western blot using AMPKα antibody . L . E . ; long exposure .

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(b) Ulk1 Ser 757 is required for rapamycin to enhance the Ulk1-AMPK interaction in vitro. CBP/SBP Ulk1 proteins (wild type or S757C) were prepared from transfected HEK293 cells, which were pre-incubated with 50 nM rapamycin (Rapa, 1h) as indicated. The Ulk1 proteins were purified by streptavidin beads and the resulting Ulk1-bead was incubated with the bacterial purified AMPKα/β/γ complex. AMPKα protein levels in the in vitro pulldown assays were examined by western blot using AMPKα antibody. L.E.; long exposure.


( c ) Phosphorylation of AMPK sites Ser 317 and Ser 777 in Ulk1 are decreased in Tsc1 − / − Tsc1 . Tsc1 + / + ( WT ) and Tsc1 − / − ( KO ) MEFs were starved of glucose ( 4 h ) , or treated with 50 nM rapamycin ( Rapa , 1 h ) . Ser 317 and Ser 777 phosphorylation of endogenous Ulk1 was examined by western blotting with antibodies against Ulk1 phosphorylated at Ser 317 or Ser 777 .

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(c) Phosphorylation of AMPK sites Ser 317 and Ser 777 in Ulk1 are decreased in Tsc1−/− Tsc1. Tsc1+/+ (WT) and Tsc1−/− (KO) MEFs were starved of glucose (4 h), or treated with 50 nM rapamycin (Rapa, 1 h). Ser 317 and Ser 777 phosphorylation of endogenous Ulk1 was examined by western blotting with antibodies against Ulk1 phosphorylated at Ser 317 or Ser 777.


( d ) Rheb suppresses Ulk1 Ser 317 and Ser 777 phosphorylation in a manner dependent on mTORC1 . HA - Ulk1 , AMPKα kinase - dead mutant ( DN ) , and Myc - Rheb were co - transfected into HEK293 cells as indicated . The cells were incubated with glucose - free medium ( - Glu , glucose ) , in which either 20 μM compound C ( C . C . ) or 50 nM Rapamycin ( Rapa ) was added . Total cell lysates were probed with antibodies against Ulk1 phosphorylated at Ser 317 , Ser 777 , Ser 757 , and HA , as indicated .

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(d) Rheb suppresses Ulk1 Ser 317 and Ser 777 phosphorylation in a manner dependent on mTORC1. HA-Ulk1, AMPKα kinase-dead mutant (DN), and Myc-Rheb were co-transfected into HEK293 cells as indicated. The cells were incubated with glucose-free medium (-Glu, glucose), in which either 20 μM compound C (C.C.) or 50 nM Rapamycin (Rapa) was added. Total cell lysates were probed with antibodies against Ulk1 phosphorylated at Ser 317, Ser 777, Ser 757, and HA, as indicated.


( e ) Rheb inhibits glucose starvation - induced Ulk1 activation . HA - Ulk1 and Myc - Rheb was transfected into HEK293 cells , which were incubated with glucose - free ( - Glu ) , amino - acid - free ( - A . A ) medium , or 50 nM rapamycin ( Rapa ) for 4 h before lysis . HA - Ulk1 was immunoprecipitated and kinase assays were performed . Ulk1 activity was measured by 32P - autoradiogram and the protein level of HA - Ulk1 and GST - Atg13 used in the assay was determined by western blot and by Coomassie staining , respectively . Uncropped images of blots are shown in Supplementary Fig . S5 .

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(e) Rheb inhibits glucose starvation-induced Ulk1 activation. HA-Ulk1 and Myc-Rheb was transfected into HEK293 cells, which were incubated with glucose-free (-Glu), amino-acid-free (-A.A) medium, or 50 nM rapamycin (Rapa) for 4 h before lysis. HA-Ulk1 was immunoprecipitated and kinase assays were performed. Ulk1 activity was measured by 32P-autoradiogram and the protein level of HA-Ulk1 and GST-Atg13 used in the assay was determined by western blot and by Coomassie staining, respectively. Uncropped images of blots are shown in Supplementary Fig. S5.


( a ) Ser 317 / Ser 777 is required for Ulk1 to protect cells from glucose starvation . Viability ( 24 h , mean ± s . d . , n = 4 ; top ) and PARP cleavage ( 8 h ; western blot , middle ; quantification , n = 2 , bottom ) was examined in Ulk1 + / + ( WT ) , Ulk1 − / − ( KO ) , Ulk1 − / − re - expressing wild - type Ulk1 ( KO - WT ) , and Ulk1 − / − re - expressing Ulk1 S317 / 777A mutant ( KO - S317 / 777A ) MEFs . Arrows in western blots indicate non - cleaved and cleaved PARP .

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(a) Ser 317/Ser 777 is required for Ulk1 to protect cells from glucose starvation. Viability (24 h, mean ± s.d., n = 4; top) and PARP cleavage (8 h; western blot, middle; quantification, n = 2, bottom) was examined in Ulk1+/+ (WT), Ulk1−/− (KO), Ulk1−/− re-expressing wild-type Ulk1 (KO-WT), and Ulk1−/− re-expressing Ulk1 S317/777A mutant (KO-S317/777A) MEFs. Arrows in western blots indicate non-cleaved and cleaved PARP.


( b ) The Ulk1 S317 / 777A mutant is compromised in LC3 lipidation in response to glucose starvation . ULK1MEFs were cultured in MEFs - free medium for the indicated times . LC3 - II level was determined by western blotting and the LC3 - II accumulation was normalized by α - tubulin and quantified ( bottom , n = 3 , mean ± s . d . ) . A representative western blot was shown . The LC3 antibody used in this experiment seemed to preferentially recognise the lipid - modified form of LC3 - II , which migrated faster on the gel .

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(b) The Ulk1 S317/777A mutant is compromised in LC3 lipidation in response to glucose starvation. ULK1MEFs were cultured in MEFs-free medium for the indicated times. LC3-II level was determined by western blotting and the LC3-II accumulation was normalized by α-tubulin and quantified (bottom, n = 3, mean ± s.d.). A representative western blot was shown. The LC3 antibody used in this experiment seemed to preferentially recognise the lipid-modified form of LC3-II, which migrated faster on the gel.


( c ) The Ulk1 S317 / 777A mutant is defective in autophagosome formation . The indicated MEFs were starved of glucose ( 4 h ) and the formation of GFP - LC3 - positive autophagosomes was examined by confocal microscopy . GFP - LC3 ; green and DAPI ; blue . Scale bar , 20 μm .

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(c) The Ulk1 S317/777A mutant is defective in autophagosome formation. The indicated MEFs were starved of glucose (4 h) and the formation of GFP-LC3-positive autophagosomes was examined by confocal microscopy. GFP-LC3; green and DAPI; blue. Scale bar, 20 μm.


( d ) Autophagy vacuole analysis by electron microscopy . Low - magnification images of Ulk1 − / − ( KO , upper left panel ) , Ulk1 − / − reconstituted with wild - type Ulk1 ( KO - WT , two middle panels with accompanying higher magnification images ) , and Ulk1 − / − reconstituted with Ulk1 S317 / 777A ( KO - S317 / 777A , lower left panel ) are shown . High - magnification images of autophagosomes from KO - WT are shown in upper right and lower right panels . Autophagosome / autolysosome - like structures indicated by arrowheads on the lower - magnification images and arrows in higher - magnification images . Scale bars ; lower - magnification , 1 μm ; higher - magnification , 200 nm .

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(d) Autophagy vacuole analysis by electron microscopy. Low-magnification images of Ulk1−/− (KO, upper left panel), Ulk1−/− reconstituted with wild-type Ulk1 (KO-WT, two middle panels with accompanying higher magnification images), and Ulk1−/− reconstituted with Ulk1 S317/777A (KO-S317/777A, lower left panel) are shown. High-magnification images of autophagosomes from KO-WT are shown in upper right and lower right panels. Autophagosome/autolysosome-like structures indicated by arrowheads on the lower-magnification images and arrows in higher-magnification images. Scale bars; lower-magnification, 1 μm; higher-magnification, 200 nm.


A cdc14 ‐ 3 temperature ‐ sensitive cells reconstituted with CDC14 or CDC14 ‐ NLS were released from a G1 arrest at the restrictive temperature and stained for Cdc14 and the spindle ( Tub1 ) once cells had reached late anaphase .

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A cdc14‐3 temperature‐sensitive cells reconstituted with CDC14 or CDC14‐NLS were released from a G1 arrest at the restrictive temperature and stained for Cdc14 and the spindle (Tub1) once cells had reached late anaphase.


B Wild ‐ type , cdc14 ‐ 3 , + CDC14 and + CDC14 ‐ NLS cells were released from an α ‐ factor ‐ induced G1 arrest and re ‐ arrested in the next G1 . Samples were harvested at the indicated time points and stained with DAPI and for the nucleolar protein Nop1 ( n > 100 ) . Photographs of examples of cells without ( top ) and with ( bottom ) rDNA segregation defects at 80 min after release are shown together with quantification over time .

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B Wild‐type, cdc14‐3, +CDC14 and +CDC14‐NLS cells were released from an α‐factor‐induced G1 arrest and re‐arrested in the next G1. Samples were harvested at the indicated time points and stained with DAPI and for the nucleolar protein Nop1 (n > 100). Photographs of examples of cells without (top) and with (bottom) rDNA segregation defects at 80 min after release are shown together with quantification over time.


C Cells from the experiment in ( B ) were stained for the spindle ( n > 100 ) . Cells without ( top ) and with ( bottom ) anaphase spindles at 100 min after release are shown together with their quantification over time .

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C Cells from the experiment in (B) were stained for the spindle (n > 100). Cells without (top) and with (bottom) anaphase spindles at 100 min after release are shown together with their quantification over time.


D FACS analysis of DNA content and Western blot analysis of cell cycle markers of the samples from ( B ) . Tub1 served as a loading control .

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D FACS analysis of DNA content and Western blot analysis of cell cycle markers of the samples from (B). Tub1 served as a loading control.


A + CDC14 and + CDC14 ‐ NLS cells were grown at 35 . 5 ° C for 5 h and sonicated , and differential interference contrast ( DIC ) images were acquired .

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A +CDC14 and +CDC14‐NLS cells were grown at 35.5°C for 5 h and sonicated, and differential interference contrast (DIC) images were acquired.


B Cells from a synchronous culture ( as in Fig B ) were treated with zymolyase to remove the cell wall . The fraction of remaining budded cells is shown over time ( n = 100 ) .

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B Cells from a synchronous culture (as in Fig B) were treated with zymolyase to remove the cell wall. The fraction of remaining budded cells is shown over time (n = 100).


C Cells expressing the plasma membrane marker GFP ‐ Spo2051 - 91 , exemplifying the indicated bud neck phenotypes and stages of cytokinesis . D Bud neck phenotypes of synchronized GFP ‐ Spo2051 - 91 ‐ expressing + CDC14 or + CDC14 ‐ NLS cells during the progression of a cell cycle ( n > 75 ) .

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C Cells expressing the plasma membrane marker GFP‐Spo2051-91, exemplifying the indicated bud neck phenotypes and stages of cytokinesis.D Bud neck phenotypes of synchronized GFP‐Spo2051-91‐expressing +CDC14 or +CDC14‐NLS cells during the progression of a cell cycle (n > 75).


E + CDC14 or + CDC14 ‐ NLS cells expressing Spc42 ‐ YFP and GFP fusions with the indicated cytokinetic protein were synchronized in G1 using α ‐ factor . Upon release at 35 . 5 ° C , cells were imaged every 2 min and the time from anaphase onset to the indicated events was recorded . Error bars represent SD ; Student ' s t ‐ test was applied to analyze differences between the indicated conditions ; * * P ≤ 0 . 01 ; * * * * P ≤ 0 . 0001 . For representative image sequences , see Supplementary Fig S2C .

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E +CDC14 or +CDC14‐NLS cells expressing Spc42‐YFP and GFP fusions with the indicated cytokinetic protein were synchronized in G1 using α‐factor. Upon release at 35.5°C, cells were imaged every 2 min and the time from anaphase onset to the indicated events was recorded. Error bars represent SD; Student's t‐test was applied to analyze differences between the indicated conditions; **P ≤ 0.01; ****P ≤ 0.0001. For representative image sequences, see Supplementary Fig S2C.


A MET3pr ‐ CDC20cdc15 ‐ as1 cells were arrested in metaphase by Cdc20 depletion and subsequently treated with 1NM ‐ PP1 to inhibit Cdc15 ‐ as1 . Cells were released from the metaphase arrest by Cdc20 re ‐ induction ( left graph ) , or Cdc14 and / or Sic1 expression were induced from the GAL1 promoter while cells remained arrested ( right graph ) . The fraction of large ‐ budded cells after spheroplastation ( n = 100 ) was quantified . Representative photographs 120 min after galactose addition are shown . Scale bar , 10 μm .

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A MET3pr‐CDC20cdc15‐as1 cells were arrested in metaphase by Cdc20 depletion and subsequently treated with 1NM‐PP1 to inhibit Cdc15‐as1. Cells were released from the metaphase arrest by Cdc20 re‐induction (left graph), or Cdc14 and/or Sic1 expression were induced from the GAL1 promoter while cells remained arrested (right graph). The fraction of large‐budded cells after spheroplastation (n = 100) was quantified. Representative photographs 120 min after galactose addition are shown. Scale bar, 10 μm.


B As ( A ) , the plasma membrane marker GFP ‐ Spo2051 - 91 was visualized following Cdc14 induction ( n = 100 ) .

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B As (A), the plasma membrane marker GFP‐Spo2051-91 was visualized following Cdc14 induction (n = 100).


C Samples from the experiment in ( B ) were processed for Western blot analysis of the indicated cell cycle proteins . Arrowheads indicate endogenous and exogenous forms of Cdc14 . Tub1 served as a loading control . A schematic of downstream processing for the phosphoproteome analysis is shown on the right .

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C Samples from the experiment in (B) were processed for Western blot analysis of the indicated cell cycle proteins. Arrowheads indicate endogenous and exogenous forms of Cdc14. Tub1 served as a loading control. A schematic of downstream processing for the phosphoproteome analysis is shown on the right.


A Phospho ‐ peptide abundance during Cdc14 ‐ induced mitotic exit . Examples of phospho ‐ peptides that disappear with early ( red ) , intermediate ( orange ) and late ( green ) timing or that remain stable ( black ) are shown . This color coding is used throughout all panels of this figure . B Numbers of unique peptides and corresponding proteins that do or do not contain a minimal Cdk consensus motif are shown , categorized according to timing of disappearance as described in ( A ) . C Protein stability data for proteins corresponding to disappearing phospho ‐ peptides ( blue ) and for all other proteins ( black ) . D The fractions of peptides that contain a minimal Cdk consensus motif ( [ S / T ] ‐ P ) within the timing categories shown in ( B ) were calculated . The ratio and P ‐ value of enrichment of each category relative to the combined stable / rest category ( gray ) are shown . E Similar to ( D ) , the enrichment for reported physical Cdc14 interactors is assessed within [ S / T ] ‐ P ‐ containing phospho ‐ peptides . F Peptides that contain the indicated Cdk consensus sequence motifs and that have disappeared over time are represented as a fraction of all phospho ‐ peptides containing that motif .

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A Phospho‐peptide abundance during Cdc14‐induced mitotic exit. Examples of phospho‐peptides that disappear with early (red), intermediate (orange) and late (green) timing or that remain stable (black) are shown. This color coding is used throughout all panels of this figure.B Numbers of unique peptides and corresponding proteins that do or do not contain a minimal Cdk consensus motif are shown, categorized according to timing of disappearance as described in (A).C Protein stability data for proteins corresponding to disappearing phospho‐peptides (blue) and for all other proteins (black).D The fractions of peptides that contain a minimal Cdk consensus motif ([S/T]‐P) within the timing categories shown in (B) were calculated. The ratio and P‐value of enrichment of each category relative to the combined stable/rest category (gray) are shown.E Similar to (D), the enrichment for reported physical Cdc14 interactors is assessed within [S/T]‐P‐containing phospho‐peptides.F Peptides that contain the indicated Cdk consensus sequence motifs and that have disappeared over time are represented as a fraction of all phospho‐peptides containing that motif.


G Unique phospho ‐ peptides of Spa2 , as an example , are categorized based on their dephosphorylation timing ( graph ) . Enrichment of clustered timing for all proteins covered by phospho ‐ peptides was compared to random for Cdk or non ‐ Cdk consensus site ‐ containing peptides ( table ) . Fisher ' s exact tests were used to determine significance .

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G Unique phospho‐peptides of Spa2, as an example, are categorized based on their dephosphorylation timing (graph). Enrichment of clustered timing for all proteins covered by phospho‐peptides was compared to random for Cdk or non‐Cdk consensus site‐containing peptides (table). Fisher's exact tests were used to determine significance.


B Western blot analysis of cells expressing conditional Clb2m ( + ) or Clb2mΔCdk ( Δ ) fusions to the indicated proteins , before ( − ) and 5 h after ( + ) β ‐ estradiol treatment . Tub1 served as a loading control . c , control cells .

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B Western blot analysis of cells expressing conditional Clb2m (+) or Clb2mΔCdk (Δ) fusions to the indicated proteins, before (−) and 5 h after (+) β‐estradiol treatment. Tub1 served as a loading control. c, control cells.


C FACS analysis of DNA content after conditional Clb2m or Clb2mΔCdk fusion to the indicated proteins . Quantification of the fraction of cells with > 2C DNA content is shown in the bar graph for two independent biological replicates , with red dots representing individual and bars representing average values . Bnr1 fusions were included as controls for cytokinetic defects . Student ' s t ‐ test was applied to compare matched Clb2m versus Clb2mΔCdk fusion strains . ‐ P > 0 . 05 ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 .

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C FACS analysis of DNA content after conditional Clb2m or Clb2mΔCdk fusion to the indicated proteins. Quantification of the fraction of cells with > 2C DNA content is shown in the bar graph for two independent biological replicates, with red dots representing individual and bars representing average values. Bnr1 fusions were included as controls for cytokinetic defects. Student's t‐test was applied to compare matched Clb2m versus Clb2mΔCdk fusion strains. ‐P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.


D Serial dilutions of cells from ( C ) were plated 6 h following β ‐ estradiol treatment and grown for 1 day at 36 ° C .

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D Serial dilutions of cells from (C) were plated 6 h following β‐estradiol treatment and grown for 1 day at 36°C.


E The length of cell chains containing a common cytoplasm was scored 6 h after β ‐ estradiol treatment using the GFP ‐ Spo2051 - 91 plasma membrane marker ( n > 150 ) . c , control cells ; scale bars , 10 μm .

E The length of cell chains containing a common cytoplasm was scored 6 h after β‐estradiol treatment using the GFP‐Spo2051-91 plasma membrane marker (n > 150). c, control cells; scale bars, 10 μm.


A Genes corresponding to the indicated proteins were replaced by wild ‐ type ( wt ) or Cdk phospho ‐ site mutant ( xA ) sequences and then conditionally fused to Clb2m or Clb2mΔCdk . Cytokinetic defects were scored by FACS analysis of DNA content as in Fig C . The individual values from two independent clones ( red dots ) as well as their average ( bars ) are shown . Student ' s t ‐ test was applied to compare matched wt and xA strains . c , control cells .

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A Genes corresponding to the indicated proteins were replaced by wild‐type (wt) or Cdk phospho‐site mutant (xA) sequences and then conditionally fused to Clb2m or Clb2mΔCdk. Cytokinetic defects were scored by FACS analysis of DNA content as in Fig C. The individual values from two independent clones (red dots) as well as their average (bars) are shown. Student's t‐test was applied to compare matched wt and xA strains. c, control cells.


B Cell chain formation in the samples from ( A ) . The fraction of cells with > 2 cell bodies for each clone are represented with red dots and their averages by the black bar ( n > 150 ) . Significance is determined by Student ' s t ‐ tests for the indicated conditions . Scale bars , 5 μm .

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B Cell chain formation in the samples from (A). The fraction of cells with > 2 cell bodies for each clone are represented with red dots and their averages by the black bar (n > 150). Significance is determined by Student's t‐tests for the indicated conditions. Scale bars, 5 μm.


C The indicated strains were spotted in serial dilutions onto auxin ‐ containing plates and grown for 2 days .

C The indicated strains were spotted in serial dilutions onto auxin‐containing plates and grown for 2 days.


D Auxin was added to cultures of the indicated strains , and after 6 h , cells were sonicated and the number of attached cell bodies was counted ( n > 200 ) . Significance was calculated using a Wilcoxon rank ‐ sum test .

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D Auxin was added to cultures of the indicated strains, and after 6 h, cells were sonicated and the number of attached cell bodies was counted (n > 200). Significance was calculated using a Wilcoxon rank‐sum test.


E The fraction of cells in asynchronously proliferating cultures of the indicated strains with a DNA content of > 2C was determined by FACS analysis ( n = 3 ) . Error bars represent SD .

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E The fraction of cells in asynchronously proliferating cultures of the indicated strains with a DNA content of > 2C was determined by FACS analysis (n = 3). Error bars represent SD.


A Cyk3 ‐ GFP signal intensity at the GFP relative to the onset of actomyosin ring contraction ( t = 0 ) . The mean and SEM from at least 20 cells are shown .

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A Cyk3‐GFP signal intensity at the GFP relative to the onset of actomyosin ring contraction (t = 0). The mean and SEM from at least 20 cells are shown.


B Inn1 ‐ Clb2m , Inn1 ‐ Clb2mΔCdk and control cells before Clb2m fusion were arrested in G1 using α ‐ factor , released and harvested 90 - 100 min later at the time of cytokinesis . Indirect immunofluorescence microscopy was used to determine Inn1bud neck staining and the spindle status . Representative images of full spindles and partially and fully disassembled spindles are shown . nr , non ‐ recombined cells . Scale bars , 5 μm .

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B Inn1‐Clb2m, Inn1‐Clb2mΔCdk and control cells before Clb2m fusion were arrested in G1 using α‐factor, released and harvested 90-100 min later at the time of cytokinesis. Indirect immunofluorescence microscopy was used to determine Inn1bud neck staining and the spindle status. Representative images of full spindles and partially and fully disassembled spindles are shown. nr, non‐recombined cells. Scale bars, 5 μm.


C The time from anaphase onset until the GFP appearance of Inn1 was determined by filming synchronized Inn1 ‐ Clb2m ‐ GFP and Inn1 ‐ Clb2mΔCdk ‐ GFP cells containing Spc42 ‐ YFP ‐ labeled spindle pole bodies . Error bars represent SD .

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C The time from anaphase onset until the GFP appearance of Inn1 was determined by filming synchronized Inn1‐Clb2m‐GFP and Inn1‐Clb2mΔCdk‐GFP cells containing Spc42‐YFP‐labeled spindle pole bodies. Error bars represent SD.


D As in ( C ) , now the persistence of a full actomyosin ring was measured in cells expressing Inn1 ‐ Clb2m , Inn1 ‐ Clb2mΔCdk or their respective non ‐ phosphorylatable mutants together with Myo1 ‐ GFP and Spc42 ‐ YFP . Error bars represent SD .

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D As in (C), now the persistence of a full actomyosin ring was measured in cells expressing Inn1‐Clb2m, Inn1‐Clb2mΔCdk or their respective non‐phosphorylatable mutants together with Myo1‐GFP and Spc42‐YFP. Error bars represent SD.


E Montage of medial view projections of actomyosin rings in Inn1 ‐ Clb2m and Inn1 ‐ Clb2mΔCdk cells . Frames are shown in 2 ‐ min intervals . F Actomyosin ring contraction phenotypes from at least 21 cells filmed as in ( E ) were blindly scored as having a normal ( " constriction " ) or aberrant ( " disintegration " ) cytokinetic phenotype .

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E Montage of medial view projections of actomyosin rings in Inn1‐Clb2m and Inn1‐Clb2mΔCdk cells. Frames are shown in 2‐min intervals.F Actomyosin ring contraction phenotypes from at least 21 cells filmed as in (E) were blindly scored as having a normal ("constriction") or aberrant ("disintegration") cytokinetic phenotype.


G Cytokinetic defect and cell viability of Inn1 ‐ Clb2m or Inn1 ‐ Clb2mΔCdk cells in the absence or presence of Chs2V377I . In the bar graph , red dots represent values of duplicate biological replicates with bars representing the average . Significance is calculated using Student ' s t ‐ test . 10 ‐ fold spot dilution assays are represented , together with the Western blotting analysis for Inn1 before and after its fusion to Clb2m . Tub1 served as a loading control .

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G Cytokinetic defect and cell viability of Inn1‐Clb2m or Inn1‐Clb2mΔCdk cells in the absence or presence of Chs2V377I. In the bar graph, red dots represent values of duplicate biological replicates with bars representing the average. Significance is calculated using Student's t‐test. 10‐fold spot dilution assays are represented, together with the Western blotting analysis for Inn1 before and after its fusion to Clb2m. Tub1 served as a loading control.


B . Typical examples of stage 1 ( day 0 ) , stage 2 ( day 7 ) and stage 3 ( day 12 ) human iPSC - derived NSCs / neurons immunostained for γ - Tubulin and Centrin , marked by arrows . Cells were co - immunostained with Ki67 and DAPI ( day 0 ) or Trim46 and MAP2 ( day 7 and 12 ) to define their neuronal stages . Arrowheads mark the AIS . Scale bar = 10µm in overview , 2µm in zooms .

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B. Typical examples of stage 1 (day 0), stage 2 (day 7) and stage 3 (day 12) human iPSC-derived NSCs/neurons immunostained for γ-Tubulin and Centrin, marked by arrows. Cells were co-immunostained with Ki67 and DAPI (day 0) or Trim46 and MAP2 (day 7 and 12) to define their neuronal stages. Arrowheads mark the AIS. Scale bar = 10µm in overview, 2µm in zooms.


D . 3D STED imaging of αTubulin in stage 1 ( day 0 ) , stage 2 ( day 7 ) and stage 3 ( day 13 ) hiPSC - derived neurons . Scale bar = 5µm in overview , 1µm in zoom .

D. 3D STED imaging of αTubulin in stage 1 (day 0), stage 2 (day 7) and stage 3 (day 13) hiPSC-derived neurons. Scale bar = 5µm in overview, 1µm in zoom.


F . Typical examples of centrosomes of human iPSC - derived NSCs ( day 0 ) with STED imaging of Trim46 and Centrin immunostaining , and confocal imaging of γ - Tubulin immunostaining . Scale bar = 1µm .

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F. Typical examples of centrosomes of human iPSC-derived NSCs (day 0) with STED imaging of Trim46 and Centrin immunostaining, and confocal imaging of γ-Tubulin immunostaining. Scale bar = 1µm.


G . Typical examples of stage 2 and stage 3 human iPSC - derived neurons immunostained for Trim46 and MAP2 . Inserts represent centrosomes . Zooms on the right represent a non - polarized neurite or a developing axon in stage 2 or stage 3 neurons , respectively . Scale bar = 20µm in overview , 1µm in insert , 10µm in zoom .

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G. Typical examples of stage 2 and stage 3 human iPSC-derived neurons immunostained for Trim46 and MAP2. Inserts represent centrosomes. Zooms on the right represent a non-polarized neurite or a developing axon in stage 2 or stage 3 neurons, respectively. Scale bar = 20µm in overview, 1µm in insert, 10µm in zoom.


A . Typical examples of Centrinone - B treated or control human iPSC - derived NSCs immunostained for Pericentrin and Centrin . Inserts represent centriole ( s ) . Scale bar = 5µm in overview , 2µm in inserts . B . Quantifications of the percentage of cells with 0 , 1 or 2 centrioles per cell after 0 ( control ) , 2 or 5 days Centrinone - B treatment and prior to neuronal induction . n = 48 - 51 cells in 2 independent experiments . C Data information : Data represents mean ± SEM . Chi - square - test including post - hoc analysis with Bonferroni correction * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns p > 0 .

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A. Typical examples of Centrinone-B treated or control human iPSC-derived NSCs immunostained for Pericentrin and Centrin. Inserts represent centriole(s). Scale bar = 5µm in overview, 2µm in inserts. B. Quantifications of the percentage of cells with 0, 1 or 2 centrioles per cell after 0 (control), 2 or 5 days Centrinone-B treatment and prior to neuronal induction. n = 48-51 cells in 2 independent experiments. C Data information: Data represents mean ± SEM. Chi-square-test including post-hoc analysis with Bonferroni correction *** p<0.001, ** p<0.01, * p < 0.05, ns p>0.05


C . Typical examples of Centrinone - B treated or control human iPSC - derived neurons ( 12 - 15 days ) immunostained for AnkG , Trim46 , MAP2 and Centrin . Arrowheads mark AIS structures , arrows mark centrosomes . Inserts represent centrosomes , zooms on the right represent AIS structures . Scale bar = 20µm in overview , 2µm in insert , 10µm in zooms .

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C. Typical examples of Centrinone-B treated or control human iPSC-derived neurons (12-15 days) immunostained for AnkG, Trim46, MAP2 and Centrin. Arrowheads mark AIS structures, arrows mark centrosomes. Inserts represent centrosomes, zooms on the right represent AIS structures. Scale bar = 20µm in overview, 2µm in insert, 10µm in zooms.


M . AP threshold , amplitude and after - hyperpolarization recorded in Centrinone - B treated ( n = 43 cells in 3 independent experiments ) and control human iPSC - derived neuron cultures ( n = 59 cells in 4 independent experiments ) . Data information : Data represents mean ± SEM . Chi - square - test including post - hoc analysis with Mann Whitney test after - hyperpolarization Student ' s t test ( M : threshold , AP amplitude ) . * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns p > 0 .

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M. AP threshold, amplitude and after-hyperpolarization recorded in Centrinone-B treated (n = 43 cells in 3 independent experiments) and control human iPSC-derived neuron cultures (n = 59 cells in 4 independent experiments). Data information: Data represents mean ± SEM. Chi-square-test including post-hoc analysis with Mann Whitney test after-hyperpolarization Student's t test (M: threshold, AP amplitude). *** p<0.001, ** p<0.01, * p < 0.05, ns p>0.05


D . Representative images of fan - like growth cones at day 5 of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions . Growth cone visualized by immunostaining for phalloidin . Scale bar = 5µm . E . Quantifications of the average area ( µm² ) of growth cones of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions at different time points . n = 25 - 83 growth cones in 3 independent experiments . F Data information : Data represent mean ± SEM . One - way ANOVA including Tukey ' s post - hoc analysis * * * p < 0 . 001 , * * p < 0 . 005 , * p < 0 . 05 , ns p ≥ 0 .

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D. Representative images of fan-like growth cones at day 5 of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions. Growth cone visualized by immunostaining for phalloidin. Scale bar = 5µm. E. Quantifications of the average area (µm²) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. n = 25-83 growth cones in 3 independent experiments. F Data information: Data represent mean ± SEM. One-way ANOVA including Tukey's post-hoc analysis *** p<0.001, ** p<0.005, * p < 0.05, ns p≥0.05


F . Representative images of different growth cone morphological categories : fan - like , torpedo - like and bulb - like . Scale bar = 5µm . G . Quantifications of the ratios of different subtypes ( fan - like , torpedo - like , bulb - like ) of growth cones of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions at different time points . H . Quantifications of the average area ( µm² ) of different subtypes ( fan - like , torpedo - like , bulb - like ) of growth cones of control and Centrinone - B treated neurons in untreated , or nocodazole - treated conditions at different time points . n = 5 - 55 growth cones in 3 independent experiments . Da Data information : Chi - square - test including post - hoc analysis with Bonferroni correction ( G ) , One - way ANOVA including Sidak ' s post - hoc analysis ( H ) . * * * p < 0 . 001 , * * p < 0 . 005 , * p < 0 . 05 , ns p ≥ 0 .

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F. Representative images of different growth cone morphological categories: fan-like, torpedo-like and bulb-like. Scale bar = 5µm. G. Quantifications of the ratios of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. H. Quantifications of the average area (µm²) of different subtypes (fan-like, torpedo-like, bulb-like) of growth cones of control and Centrinone-B treated neurons in untreated, or nocodazole-treated conditions at different time points. n = 5-55 growth cones in 3 independent experiments. Da Data information: Chi-square-test including post-hoc analysis with Bonferroni correction (G), One-way ANOVA including Sidak's post-hoc analysis (H). *** p<0.001, ** p<0.005, * p < 0.05, ns p≥0.05


A . Example stills from a spinning - disk time - lapse recording of a neurite transfected with mRFP and GFP - MT + TIP . The top panel is a still of a neurite in mRFP , showing neurite morphology . The other panels show moving GFP - MT + TIP comets pointing in either an anterograde direction ( green arrowheads ) or retrograde direction ( blue arrowheads ) . P indicates the proximal direction and D the distal direction of the neurite . Timestamp in minutes : seconds given on bottom right . Scale bar = 5 µm . B . Kymographs and schematic representations of time - lapse recordings of the distal axon as shown in ( A ) , for different time points ( day 7 ; day 13 ) and conditions ( control ; Centrinone - B ) . Scale bar = 5 µm . C

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

A. Example stills from a spinning-disk time-lapse recording of a neurite transfected with mRFP and GFP-MT+TIP. The top panel is a still of a neurite in mRFP, showing neurite morphology. The other panels show moving GFP-MT+TIP comets pointing in either an anterograde direction (green arrowheads) or retrograde direction (blue arrowheads). P indicates the proximal direction and D the distal direction of the neurite. Timestamp in minutes:seconds given on bottom right. Scale bar = 5 µm. B. Kymographs and schematic representations of time-lapse recordings of the distal axon as shown in (A), for different time points (day 7; day 13) and conditions (control; Centrinone-B). Scale bar = 5 µm. C


J . Example stills from a spinning - disk time - lapse recording of a neurite transfected with mRFP and GFP - MT + TIP . Red line denotes the location of LS . Scale bar = 5 µm . K . Kymographs and schematic representations of time - lapse recordings of the distal axon as shown in ( J ) following LS , for different time points ( day 7 ; day 13 ) and conditions ( control ; Centrinone - B ) . Red line and red arrowhead denote location and time of LS . Scale bar = 5 µm . L

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

J. Example stills from a spinning-disk time-lapse recording of a neurite transfected with mRFP and GFP-MT+TIP. Red line denotes the location of LS. Scale bar = 5 µm. K. Kymographs and schematic representations of time-lapse recordings of the distal axon as shown in (J) following LS, for different time points (day 7; day 13) and conditions (control; Centrinone-B). Red line and red arrowhead denote location and time of LS. Scale bar = 5 µm. L


( C ) Period length decreases with continual differentiation of PSCs into HIOs . Maturation of HIOs into kcHIEs leads to 24 - hour rhythms with greater reproducibility . ( D ) The amplitude of oscillations are significantly higher in kcHIEs / bHIEs compared to HIOs . Bmal1 - luc traces in B were generated by plotting the standard deviation of n ≥ 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Period length decreases with continual differentiation of PSCs into HIOs. Maturation of HIOs into kcHIEs leads to 24-hour rhythms with greater reproducibility. (D) The amplitude of oscillations are significantly higher in kcHIEs/bHIEs compared to HIOs. Bmal1-luc traces in B were generated by plotting the standard deviation of n≥3 biological replicates.


( A - C ) mRNA expression of Bmal1 ( black ) , Rev - erbα ( grey ) and Per2 ( red ) in HIOs ( A ) , kcHIEs ( B ) and bHIEs ( C ) .

O O O O O O O O O O O O O O O O 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-C) mRNA expression of Bmal1 (black), Rev-erbα (grey) and Per2 (red) in HIOs (A), kcHIEs (B) and bHIEs (C).


( A ) Representative images of necrotic cell death ( SYTOX orange - red fluorescence ) at 2 - , 24 - and 48 - hours post exposure to 10ng / mL TcdB in control Bmal1f / f - EsrCRE enteroids . Scale bar = 250μm . ( B ) Quantitative analysis of fluorescent intensity from SYTOX orange in control Bmal1f / f - EsrCRE enteroids without tamoxifen treatment . ( C ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 10ng / mL TcdB in tamoxifen - treated circadian arrhythmic Bmal1f / f - EsrCRE enteroids . Scale bar = 250μm . ( D ) Quantitative analysis of fluorescent intensity from SYTOX orange in tamoxifen - treated circadian arrhythmic Bmal1f / f - EsrCRE enteroids .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 necrotic cell death (SYTOX orange - red fluorescence) at 2-, 24- and 48-hours post exposure to 10ng/mL TcdB in control Bmal1f/f-EsrCRE enteroids. Scale bar = 250μm. (B) Quantitative analysis of fluorescent intensity from SYTOX orange in control Bmal1f/f-EsrCRE enteroids without tamoxifen treatment. (C) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 10ng/mL TcdB in tamoxifen-treated circadian arrhythmic Bmal1f/f-EsrCRE enteroids. Scale bar = 250μm. (D) Quantitative analysis of fluorescent intensity from SYTOX orange in tamoxifen-treated circadian arrhythmic Bmal1f/f-EsrCRE enteroids.


( A ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 0 . 1μg / mL TcdB in HIOs . Scale bar = 250μm . ( B ) Quantification of necrotic cell death in HIOs . ( C ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 0 . 5μg / mL TcdB in kcHIEs . Scale bar = 250μm . ( D ) Quantification of necrotic cell death in kcHIEs . ( E ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 0 . 5μg / mL TcdB in bHIEs . Scale bar = 250μm . ( F ) Quantification of necrotic cell death in bHIEs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.1μg/mL TcdB in HIOs. Scale bar = 250μm. (B) Quantification of necrotic cell death in HIOs. (C) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.5μg/mL TcdB in kcHIEs. Scale bar = 250μm. (D) Quantification of necrotic cell death in kcHIEs. (E) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.5μg/mL TcdB in bHIEs. Scale bar = 250μm. (F) Quantification of necrotic cell death in bHIEs.


( A ) Heatmap of circadian transcripts over 48 hours in mouse and human enteroids .

(A) Heatmap of circadian transcripts over 48 hours in mouse and human enteroids.


( B ) Spearman ' s rho for circadian clock and clock - associated genes .

(B) Spearman's rho for circadian clock and clock-associated genes.


( A ) Validation of anti - phasic Rac1 expression in mouse ( mRac1 , red ) and human enteroids ( hRac1 , black ) by qRT - PCR . ( B ) Quantitative analysis of fluorescent intensity from SYTOX orange in Rac1 - KD bHIEs .

O O O O O O O O O O O O O O O O O O O O O O O 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) Validation of anti-phasic Rac1 expression in mouse (mRac1, red) and human enteroids (hRac1, black) by qRT-PCR. (B) Quantitative analysis of fluorescent intensity from SYTOX orange in Rac1-KD bHIEs.


( C - D ) Quantitative analysis of fluorescent intensity from SYTOX orange in control Rac1 f / f ; CAGGCre - ERTM without tamoxifen ( C ) or Rac1 f / f ; CAGGCre - ERTM with tamoxifen - treated ( D ) enteroids .

O O O O O O O O O O O O O O O O O O O O O O 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) Quantitative analysis of fluorescent intensity from SYTOX orange in control Rac1 f/f;CAGGCre-ERTM without tamoxifen (C) or Rac1 f/f;CAGGCre-ERTM with tamoxifen-treated (D) enteroids.


B . FPKM of the 7 LncRNAs identified in the RNA - seq with high expression in PGCs . The solid lines at each end of the box plot represent the maximum and minimum values , respectively . The three dotted lines in the middle of the violin diagram represent the 75 % percentile , the mean , and the 25 % percentile in turn .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. FPKM of the 7 LncRNAs identified in the RNA-seq with high expression in PGCs. The solid lines at each end of the box plot represent the maximum and minimum values, respectively. The three dotted lines in the middle of the violin diagram represent the 75% percentile, the mean, and the 25% percentile in turn.


A . Up : The schematic diagram of PGCs induced from chicken ESCs in vitro . Down : Morphological observation of the number of embryoid bodies in the in vitro PGCs induction model after LncBMP4overexpression and interference ; oeLncBMP4 indicates the overexpression of LncBMP4 ; shLncBMP4 indicates the interference of LncBMP4 ; BMP4 induction was regarded as the control . Scale bar : 60 µm ( n = 3 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

A. Up: The schematic diagram of PGCs induced from chicken ESCs in vitro. Down: Morphological observation of the number of embryoid bodies in the in vitro PGCs induction model after LncBMP4overexpression and interference; oeLncBMP4 indicates the overexpression of LncBMP4; shLncBMP4 indicates the interference of LncBMP4; BMP4 induction was regarded as the control. Scale bar: 60 µm (n = 3 independent experiments).


B . The expression of reproductive marker genes ( Cvh and C - kit ) , LncBMP4 , and BMP4 were detected by qRT - PCR after LncBMP4 overexpression and interference , in vitro ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The expression of reproductive marker genes (Cvh and C-kit), LncBMP4, and BMP4 were detected by qRT-PCR after LncBMP4 overexpression and interference, in vitro (Data are shown as mean ± SEM, n = 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA) .


A . The results of IHC examining using anti - DDX4 antibody shows that the overexpression of LncBMP4 increases the number of PGCs in the genital ridge , and interference with LncBMP4 reduces the number of PGCs in the genital ridge . The number of PGCs was counted through the Segmentation module of Image J software . Scale bars : 200 μm ( top row ) , 40 μm ( bottom row ) ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 results of IHC examining using anti-DDX4 antibody shows that the overexpression of LncBMP4 increases the number of PGCs in the genital ridge, and interference with LncBMP4 reduces the number of PGCs in the genital ridge. The number of PGCs was counted through the Segmentation module of Image J software. Scale bars:200 μm (top row),40 μm (bottom row) (Data are shown as mean ± SEM, n = 3 independent experiments, * p < 0.05, ** p < 0.01, **** p < 0.0001, one-way ANOVA).


B . The number of PGCs in the genital ridge , following the overexpression or interference of LncBMP4 , as determined by flow cytometry ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The number of PGCs in the genital ridge, following the overexpression or interference of LncBMP4, as determined by flow cytometry (Data are shown as mean ± SEM, n = 3 independent experiments, **** p < 0.0001, one-way ANOVA).


C . The number of PGCs labeled with PkH26 ( red ) that migrated to the genital ridge in a frozen section , the number of PGCs labeled with PkH26 ( red ) was counted using Image J software . Scale bar : 70 µm ( Data are shown as mean ± SEM , n = 6 independent experiments , * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The number of PGCs labeled with PkH26 (red) that migrated to the genital ridge in a frozen section, the number of PGCs labeled with PkH26 (red) was counted using Image J software. Scale bar: 70 µm (Data are shown as mean ± SEM, n = 6 independent experiments, ** p < 0.01, **** p < 0.0001, one-way ANOVA.).


D . The expression of reproductive marker genes ( Cvh and C - kit ) , LncBMP4 , and BMP4 were detected by qRT - PCR in the genital ridge after gga - mir - 12211 overexpression and interference in vitro ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The expression of reproductive marker genes (Cvh and C-kit), LncBMP4, and BMP4 were detected by qRT-PCR in the genital ridge after gga-mir-12211 overexpression and interference in vitro (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA).


E . The efficiency of PGC differentiation was detected by IF , 4 d after induction , using C - KIT and CVH antibodies . Scale bar : 60 µm ( n = 3 independent experiments ) .

E. The efficiency of PGC differentiation was detected by IF, 4 d after induction, using C-KIT and CVH antibodies. Scale bar: 60 µm (n = 3 independent experiments).


A . A luciferase reporting system was used to detect the regulatory effects of gga - mir - 12211 on the activity of BMP4 - 3 ' UTR ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * * * p < 0 . 0001 , one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A luciferase reporting system was used to detect the regulatory effects of gga-mir-12211 on the activity of BMP4-3'UTR (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, **** p < 0.0001, one-way ANOVA).


B . qRT - PCR was used to detect the regulatory effects of gga - mir - 12211 on LncBMP4 expression ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. qRT-PCR was used to detect the regulatory effects of gga-mir-12211 on LncBMP4 expression (Data are shown as mean ± SEM, n =3 independent experiments, *** p < 0.001, **** p < 0.0001, one-way ANOVA).


Enrichment levels of m6A in LncBMP4 were detected by RIP - qPCR ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) .

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

Enrichment levels of m6A in LncBMP4 were detected by RIP-qPCR (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).


H and I . The effects of m6A modifications of LncBMP4 on the ceRNA system were detected by the luciferase reporting system ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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

H and I. The effects of m6A modifications of LncBMP4 on the ceRNA system were detected by the luciferase reporting system (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, **** p < 0.0001, one-way ANOVA.).


A . The influence of m6A on the stability of LncBMP4 was detected by qRT - PCR ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) .

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. The influence of m6A on the stability of LncBMP4 was detected by qRT-PCR (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).


D . The enrichment level of m6A , for LncBMP4 variants with the progressive accumulation of RRACH site mutations , was detected by RIP - qPCR ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , one - way ANOVA ) .

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

D. The enrichment level of m6A, for LncBMP4 variants with the progressive accumulation of RRACH site mutations, was detected by RIP-qPCR (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, one-way ANOVA).


E . The ability of LncBMP4 , modified with different levels of m6A , to bind gga - mir - 12211 was detected by the luciferase reporting system ( Data are shown as mean ± SEM , n = 3 independent experiments , * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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

E. The ability of LncBMP4, modified with different levels of m6A, to bind gga-mir-12211 was detected by the luciferase reporting system (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, **** p < 0.0001, one-way ANOVA.).


B . qRT - PCR was used to detect the expression of related genes in each group , during induction ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. qRT-PCR was used to detect the expression of related genes in each group, during induction (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA).


D . The number of CVH - positive cells was determined by flow cytometry ( Data are shown as mean ± SEM , n = 3 independent experiments , * * p < 0 . 01 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The number of CVH-positive cells was determined by flow cytometry (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA.).


C . Effects of m6A on small peptide expression , assessed by western blot ( Up ) , Image J software was used for gray level analysis ( Down ) ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) .

C. Effects of m6A on small peptide expression, assessed by western blot (Up), Image J software was used for gray level analysis(Down) (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).


D and E . Western blot ( Up ) was used to detect gga - mir - 12211 and m6A affect the ability of LncBMP4 to translate EPC5 . Image J software was used for gray level analysis ( Down ) ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and E. Western blot(Up) was used to detect gga-mir-12211 and m6A affect the ability of LncBMP4 to translate EPC5. Image J software was used for gray level analysis(Down) (Data are shown as mean ± SEM, n =3 independent experiments, *** p < 0.001,**** p < 0.0001, one-way ANOVA.).


( A ) Cardiac GATA4 protein abundance analyzed by immunoblotting . GAPDH was the loading control . + denotes positive control for GATA4 from cardiomyocytes infected with a GATA4 overexpressing adenovirus . ( B ) Densitometric quantification of the immunoblot shown under ( A ) ; * * * p = 0 . 0003 for P1 vs . P7 and P1 vs . P21 ; p < 0 . 0001 for P1 vs . P60 . The number within bars indicates the number of mice analyzed in that particular group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cardiac GATA4 protein abundance analyzed by immunoblotting. GAPDH was the loading control. + denotes positive control for GATA4 from cardiomyocytes infected with a GATA4 overexpressing adenovirus.(B) Densitometric quantification of the immunoblot shown under (A); ***p=0.0003 for P1 vs. P7 and P1 vs. P21; p<0.0001 for P1 vs. P60. The number within bars indicates the number of mice analyzed in that particular group.


( C ) Immunoblot of cardiac GATA4 abundance in control ( Con ) and cardiomyocyte specific Gata4 knock - out mice ( CM - G4 - KO ) at postnatal day ( P ) 1 . ( D ) Quantification of the immunoblot shown in ( C ) ; * p = 0 . 018 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot of cardiac GATA4 abundance in control (Con) and cardiomyocyte specific Gata4 knock-out mice (CM-G4-KO) at postnatal day (P) 1.(D) Quantification of the immunoblot shown in (C); *p=0.018.


( E ) Cardiac immunofluorescence staining and its quantification from Con and CM - G4 - KO mice at P1 . Scale bar : 25 µm ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O 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) Cardiac immunofluorescence staining and its quantification from Con and CM-G4-KO mice at P1. Scale bar: 25 µm; ****p<0.0001.


( F ) Transversal sections of mouse hearts 7 days after cryoinjury as indicated , stained with Sirius - Red . Scale bar : 500 µm . The quantification of the left ventricular ( LV ) scar area ( red , arrows ) 7 days after injury is shown on the right ; * * p = 0 . 0027 . ( G ) The quantification of the LV scar area of the indicated mice 21 days after cryoinjury ; * p = 0 . 0261 . ( H ) The quantification of the LV scar area of the indicated mice 60 days after cryoinjury ; * * * * p < 0 . 0001 .

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(F) Transversal sections of mouse hearts 7 days after cryoinjury as indicated, stained with Sirius-Red . Scale bar: 500 µm. The quantification of the left ventricular (LV) scar area (red, arrows) 7 days after injury is shown on the right; **p=0.0027.(G) The quantification of the LV scar area of the indicated mice 21 days after cryoinjury; *p=0.0261.(H) The quantification of the LV scar area of the indicated mice 60 days after cryoinjury; ****p<0.0001.


( I ) Echocardiography analysis of left ventricular systolic function ( measured as fractional area change ) in the indicated mice 7 days after cryoinjury ; * p = 0 . 033 between Con mice after sham or cryoinjury and p = 0 . 04 between Con and CM - G4 - KO mice after cryoinjury .

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(I) Echocardiography analysis of left ventricular systolic function (measured as fractional area change) in the indicated mice 7 days after cryoinjury; *p=0.033 between Con mice after sham or cryoinjury and p=0.04 between Con and CM-G4-KO mice after cryoinjury.


( A ) Representative pictures of TUNEL , WGA ( wheat germ agglutinin to mark cardiomyocyte membranes ) and DAPI immunofluorescence staining of mouse hearts as indicated . The white dashed line encircles the infarcted area ( arrows ) . Scale bar : 200µm . The quantification of the area of TUNEL positive cells 3 hours after cryoinjury is shown on the right ( A ´ ) . The number within bars indicates the number of mice analyzed in that particular group .

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(A) Representative pictures of TUNEL, WGA (wheat germ agglutinin to mark cardiomyocyte membranes) and DAPI immunofluorescence staining of mouse hearts as indicated. The white dashed line encircles the infarcted area (arrows). Scale bar: 200µm. The quantification of the area of TUNEL positive cells 3 hours after cryoinjury is shown on the right (A´). The number within bars indicates the number of mice analyzed in that particular group.


( B ) Representative merged and single channel pictures of cardiac CD31 , DAPI and WGA immunostaining in mouse hearts as indicated . The quantification of the myocardial capillary density as capillaries / cardiomyocyte ( CM ) ratio , 7 days ( d ) after cryoinjury or sham surgery is shown as bar graph ( B ´ ) . Scale bar : 20µm . * * p = 0 . 0039 .

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(B) Representative merged and single channel pictures of cardiac CD31, DAPI and WGA immunostaining in mouse hearts as indicated. The quantification of the myocardial capillary density as capillaries/cardiomyocyte (CM) ratio, 7 days (d) after cryoinjury or sham surgery is shown as bar graph (B´). Scale bar: 20µm. **p=0.0039.


( C ) Representative pictures of myocardial immunofluorescence staining for the mitosis marker phospho - histone H3 ( pH3 ) , troponin T and DAPI in mice as indicated . Enlarged pH3 positive cardiomyocyte nuclei are shown separately . The arrows indicate pH3 positive cardiomyocyte nuclei . Scale bar : 25µm . The quantification of pH3 positive ( + ) cardiomyocytes per microscopic field is shown as bar graph ( C ´ ) ; * p = 0 . 046 .

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(C) Representative pictures of myocardial immunofluorescence staining for the mitosis marker phospho-histone H3 (pH3), troponin T and DAPI in mice as indicated. Enlarged pH3 positive cardiomyocyte nuclei are shown separately. The arrows indicate pH3 positive cardiomyocyte nuclei. Scale bar: 25µm. The quantification of pH3 positive (+) cardiomyocytes per microscopic field is shown as bar graph (C´); *p=0.046.


( D ) Representative myocardial immunostaining for aurora B , DAPI and troponin T ( TropT ) . The arrow points towards a cardiomyocyte cell division , in which aurora B localizes at the midbody in cytokinesis . Scale bar : 25µm . The quantification of dividing cardiomyocytes per microscopic field is shown as bar graph ( D ´ ) ; * p = 0 . 0104 .

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(D) Representative myocardial immunostaining for aurora B, DAPI and troponin T (TropT). The arrow points towards a cardiomyocyte cell division, in which aurora B localizes at the midbody in cytokinesis. Scale bar: 25µm. The quantification of dividing cardiomyocytes per microscopic field is shown as bar graph (D´); *p=0.0104.


( E ) Determination of cardiac gene - expression by qPCR 7 days after sham or cryoinjury for the indicated genes ; * p = 0 . 039 for Il13 and p = 0 . 049 for Vegfa ; * * p = 0 . 0043 for Ccne1 , p = 0 . 0027 for Cdk4 , p = 0 . 0096 for Cenpa , p = 0 . 0015 for Zfp191 ; * * * p = 0 . 0009 for Ccna2 , p = 0 . 0002 for Cenpa .

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(E) Determination of cardiac gene-expression by qPCR 7 days after sham or cryoinjury for the indicated genes; *p=0.039 for Il13 and p=0.049 for Vegfa; **p=0.0043 for Ccne1, p=0.0027 for Cdk4, p=0.0096 for Cenpa, p=0.0015 for Zfp191; ***p=0.0009 for Ccna2, p=0.0002 for Cenpa.


( A ) Representative immunoblots for GATA4 and GAPDH ( as loading control ) of fetal cardiomyocytes treated with adenoviruses ( Ad ) expressing control shRNA ( shCon ) or shRNA against Gata4 ( shGATA4 ) for 48 hours .

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(A) Representative immunoblots for GATA4 and GAPDH (as loading control) of fetal cardiomyocytes treated with adenoviruses (Ad) expressing control shRNA (shCon) or shRNA against Gata4 (shGATA4) for 48 hours.


( B ) Number of cardiomyocytes ( CM ) in a defined low magnification ( 50x ) microscopic field before the adenoviral infection and 48h later . The number in the bars indicates the number of cell culture plates analyzed . * * * * p < 0 . 0001 .

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(B) Number of cardiomyocytes (CM) in a defined low magnification (50x) microscopic field before the adenoviral infection and 48h later. The number in the bars indicates the number of cell culture plates analyzed. ****p<0.0001.


( C ) Representative pictures and quantification of BrdU incorporation in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for BrdU , troponin I ( Trop I ) and DAPI . Scale bar : 50µm ; p = 0 . 0169 .

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(C) Representative pictures and quantification of BrdU incorporation in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for BrdU, troponin I (Trop I) and DAPI. Scale bar: 50µm; p=0.0169.


( D ) Representative pictures and quantification of cardiomyocytes positive for phospho - histone H3 ( pH3 ) in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for pH3 , troponin I and DAPI . Scale bar : 50µm ; * p = 0 . 0145 .

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(D) Representative pictures and quantification of cardiomyocytes positive for phospho-histone H3 (pH3) in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for pH3, troponin I and DAPI. Scale bar: 50µm; *p=0.0145.


( E ) Representative picture and quantification of cardiomyocyte cell division in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for aurora B ( Auro B ) , troponin I and DAPI . Scale bar : 50µm .

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(E) Representative picture and quantification of cardiomyocyte cell division in fetal cardiomyocytes 48 hours after application of the adenoviruses as indicated and immunostained for aurora B (Auro B), troponin I and DAPI. Scale bar: 50µm.


( F ) Representative pictures , single channel enlargements and quantification of cardiomyocyte apoptosis in fetal cardiomyocytes 48 hours after application of the adenoviruses and after immunostaining for cleaved caspase 3 , troponin I and DAPI . Scale bar : 100µm .

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(F) Representative pictures, single channel enlargements and quantification of cardiomyocyte apoptosis in fetal cardiomyocytes 48 hours after application of the adenoviruses and after immunostaining for cleaved caspase 3, troponin I and DAPI. Scale bar: 100µm.


( G ) Representative pictures of cardiac explants of the indicated mice embedded in Matrigel shortly after cryoinjury and immunostained for troponin I 7 days later . Quantification of outgrowing cardiomyocytes is shown on the right . The number within bars indicates the number of mice analyzed in that particular group . Scale bar : 50µm . * * p = 0 . 0037 .

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(G) Representative pictures of cardiac explants of the indicated mice embedded in Matrigel shortly after cryoinjury and immunostained for troponin I 7 days later. Quantification of outgrowing cardiomyocytes is shown on the right. The number within bars indicates the number of mice analyzed in that particular group. Scale bar: 50µm. **p=0.0037.


( H ) Representative pictures of cardiac explants like in ( G ) , stained for pH3 in addition . The quantification is shown on the right . Scale bar : 50µm . * p = 0 . 0215 .

(H) Representative pictures of cardiac explants like in (G), stained for pH3 in addition. The quantification is shown on the right. Scale bar: 50µm. *p=0.0215.


( A ) Representative immunoblot for GATA4 and actin from neonatal cardiomyocytes treated with control ( Ad . Con ) or GATA4 expressing adenovirus ( Ad . GATA4 ) .

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(A) Representative immunoblot for GATA4 and actin from neonatal cardiomyocytes treated with control (Ad.Con) or GATA4 expressing adenovirus (Ad.GATA4).


( B ) Quantification of BrdU incorporation , pH3 labelling and cytokinesis in neonatal cardiomyocytes treated as shown . * p = 0 . 0272 for BrdU incorporation , p = 0 . 0329 for pH3 labelling , p = 0 . 0217 for cytokinesis .

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(B) Quantification of BrdU incorporation, pH3 labelling and cytokinesis in neonatal cardiomyocytes treated as shown. *p=0.0272 for BrdU incorporation, p=0.0329 for pH3 labelling, p=0.0217 for cytokinesis.


( C ) Immunoblot for GATA4 and GAPDH of mouse hearts treated as shown in the experimental time scale above . ( D ) A quantification of the immunoblot from ( C ) is shown ; * * p = 0 . 0062 .

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(C) Immunoblot for GATA4 and GAPDH of mouse hearts treated as shown in the experimental time scale above.(D) A quantification of the immunoblot from (C) is shown; **p=0.0062.


( E ) Representative pictures of myocardial sections immunostained for the indicated proteins from mice 7 days after cryoinjury and treatment with Ad . Con or Ad . GATA4 . The quantification of cardiomyocytes ( CM ) staining positive for GATA4 vs . total cardiomyocytes is shown as bar graph ( E ´ ) . The number within bars indicates the number of mice analyzed in that particular group . Scale bar : 20µm . * * * * p < 0 . 0001 .

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(E) Representative pictures of myocardial sections immunostained for the indicated proteins from mice 7 days after cryoinjury and treatment with Ad.Con or Ad.GATA4. The quantification of cardiomyocytes (CM) staining positive for GATA4 vs. total cardiomyocytes is shown as bar graph (E´). The number within bars indicates the number of mice analyzed in that particular group. Scale bar: 20µm. ****p<0.0001.


( F ) Representative Sirius - Red stained heart sections of mice 7 days after cryoinjury ( performed at P7 ) and treated with adenoviruses as indicated . The scar is displayed in red ( arrows ) . Scale bar : 500 µm . The quantification of left ventricular ( LV ) scar area of mice 7 days after cryoinjury is shown as bar graph ( F ´ ) . * p = 0 . 0135 .

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(F) Representative Sirius-Red stained heart sections of mice 7 days after cryoinjury (performed at P7) and treated with adenoviruses as indicated. The scar is displayed in red (arrows). Scale bar: 500 µm. The quantification of left ventricular (LV) scar area of mice 7 days after cryoinjury is shown as bar graph (F´). *p=0.0135.


( G ) Representative picture of a myocardial section immunostained for the indicated proteins 7 days after cryoinjury . Scale bar : 10 µm . Quantification of Ki67 positive cardiomyocytes in these mice ( G ´ ) . * * p = 0 . 0023 .

(G) Representative picture of a myocardial section immunostained for the indicated proteins 7 days after cryoinjury. Scale bar: 10 µm. Quantification of Ki67 positive cardiomyocytes in these mice (G´). **p=0.0023.


( H ) Quantification of pH3 positive cardiomyocytes and cardiomyocyte ( CM ) cytokinesis in mice 7 days after cryoinjury ; * p = 0 . 0264 , * * * p = 0 . 0006 .

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(H) Quantification of pH3 positive cardiomyocytes and cardiomyocyte (CM) cytokinesis in mice 7 days after cryoinjury; *p=0.0264, ***p=0.0006.


( I ) Quantification of the capillary / cardiomyocyte ratio in mice 7 days after cryoinjury . * * * p = 0 .

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(I) Quantification of the capillary/cardiomyocyte ratio in mice 7 days after cryoinjury. ***p=0.0002


( B ) Quantification of left ventricular ( LV ) scar area of mice 7 days after cryoinjury and treated as indicated . The number within bars indicates the number of mice analyzed in that particular group . * * p = 0 . 0017 between Con and CM - G4 - KO after PBS treatment and p = 0 . 0078 between CM - G4 - KO mice with PBS or IL - 13 treatment .

(B) Quantification of left ventricular (LV) scar area of mice 7 days after cryoinjury and treated as indicated. The number within bars indicates the number of mice analyzed in that particular group. **p=0.0017 between Con and CM-G4-KO after PBS treatment and p=0.0078 between CM-G4-KO mice with PBS or IL-13 treatment.


( C ) Echocardiography analysis of left ventricular systolic function in the indicated mice 7 days after cryoinjury ; * p = 0 .

(C) Echocardiography analysis of left ventricular systolic function in the indicated mice 7 days after cryoinjury; *p=0.0218


( D ) Quantification of Ki67 or pH3 positive cardiomyocytes in mice treated as described in ( A ) ; * p = 0 . 0106 and * * p = 0 . 0052 between Con and CM - G4 - KO after PBS treatment and * p = 0 . 0439 or * * p = 0 . 0023 between CM - G4 - KO mice with PBS or IL - 13 treatment .

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(D) Quantification of Ki67 or pH3 positive cardiomyocytes in mice treated as described in (A); *p=0.0106 and **p=0.0052 between Con and CM-G4-KO after PBS treatment and *p=0.0439 or **p=0.0023 between CM-G4-KO mice with PBS or IL-13 treatment.


( E ) Quantification of capillary density as capillary / cardiomyocyte ( CM ) ratio in mice treated as described in ( A ) . * p = 0 . 0365 , * * p = 0 . 0031 .

(E) Quantification of capillary density as capillary/cardiomyocyte (CM) ratio in mice treated as described in (A). *p=0.0365, **p=0.0031.


( F ) Quantification of α - SMA positive small conductance vessel ( ~ 20 - 50µM in diameter ) in myocardial sections of the mice treated as indicated .

(F) Quantification of α-SMA positive small conductance vessel (~20-50µM in diameter) in myocardial sections of the mice treated as indicated.


( G ) Representative immunoblots for STAT6 and GAPDH from neonatal mouse hearts ( KO = CM - G4 - KO ) treated as indicated . The quantification of the blots is shown on the right . * p = 0 . 0114 .

(G) Representative immunoblots for STAT6 and GAPDH from neonatal mouse hearts (KO = CM-G4-KO) treated as indicated. The quantification of the blots is shown on the right. *p=0.0114.


HCT - 116 ( A ) and HT - 29 ( B ) cells were treated with the indicated concentrations of sodium butyrate ( NaB ) for 24 ( grey bars ) , 48 ( pink bars ) , or 72 ( yellow bars ) hours and cell proliferation was assessed . HCT -

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HCT-116 (A) and HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 (grey bars), 48 (pink bars), or 72 (yellow bars) hours and cell proliferation was assessed. HCT-116


( C , E ) and HT - 29 ( D , F ) cells were treated with the indicated concentrations of NaB for 24 h . C . D . The percentage of apoptotic cells in HCT - 116 ( C ) and HT - 29 ( D ) cells was quantified in three independent experiments using an annexinV / PI assay ( annexinV + / PI - ) by flow cytometry and expressed as mean ± SD . One - way ANOVA was used to compare between the control cells and NaB treatments . * p < 0 . 05 , * * p < 0 . 01 compared to control . E . F . Representative Western blots of full - length and cleaved PARP products in HCT - 116 ( E ) and HT - 29 ( F ) cells .

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(C, E) and HT-29 (D, F) cells were treated with the indicated concentrations of NaB for 24 h. C.D. The percentage of apoptotic cells in HCT-116 (C) and HT-29 (D) cells was quantified in three independent experiments using an annexinV/PI assay (annexinV+/PI-) by flow cytometry and expressed as mean ± SD. One-way ANOVA was used to compare between the control cells and NaB treatments. *p<0.05, ** p<0.01 compared to control. E.F. Representative Western blots of full-length and cleaved PARP products in HCT-116 (E) and HT-29 (F) cells.


HCT - 116 ( A , C ) and HT - 29 ( B , D ) cells were treated with 2mM sodium butyrate ( NaB ) for 24 h and then stained with acridine orange ( AO ; A - B ) or monodansylcadaverine ( MDC ; C - D ) .

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HCT-116 (A, C) and HT-29 (B, D) cells were treated with 2mM sodium butyrate (NaB) for 24 h and then stained with acridine orange (AO; A-B) or monodansylcadaverine (MDC; C-D).


Flow cytometry measured the percentage of AO + cells and the results of three independent AO fluorescence experiments for HTC - 116 ( E ) and HT - 29 ( F ) cells were expressed as mean ± SD . One - way ANOVA was used to compare control cells and NaB treatments . * p < 0 . 05 , * * p < 0 . 01 when compared to control .

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Flow cytometry measured the percentage of AO+ cells and the results of three independent AO fluorescence experiments for HTC-116 (E) and HT-29 (F) cells were expressed as mean ± SD. One-way ANOVA was used to compare control cells and NaB treatments. *p<0.05, ** p<0.01 when compared to control.


HCT - 116 ( A ) or HT - 29 ( B ) cells were treated with the indicated concentrations of sodium butyrate ( NaB ) for 24 h . Representative blots showed expression of Beclin1 , ATG3 , and LC3 as well as GAPDH , the loading control .

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HCT-116 (A) or HT-29 (B) cells were treated with the indicated concentrations of sodium butyrate (NaB) for 24 h. Representative blots showed expression of Beclin1, ATG3, and LC3 as well as GAPDH, the loading control.


C . Electron microscopy images of control and NaB ( 2mM ) treated HCT - 116 cells . ER , endoplasmic reticulum ; Mt , mitochondrion ; AL .

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C. Electron microscopy images of control and NaB (2mM) treated HCT-116 cells. ER, endoplasmic reticulum; Mt, mitochondrion; AL.