IL - 1β ELISA of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 ng . ml - 1 IL - 10 , 10 µM beraprost or vehicle control ( Vh ) . Data shown are representative of at least 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 B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 ng.ml-1 IL-10, 10 µM beraprost or vehicle control (Vh). Data shown are representative of at least 3 independent experiments.


IL - 1β ELISA of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 µM beraprost , 5 µg . ml - 1 αIL - 10R or isotype antibody . Data shown are representative of at least 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM beraprost, 5 µg.ml-1 αIL-10R or isotype antibody. Data shown are representative of at least 3 independent experiments.


IL - 1β ( H ) and IL - 10 ( I ) ELISA of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 µM MCC950 , 5 µg . ml - 1 αIL - 10R or isotype antibody All stimulations were performed for 16 h . Data shown are representative of at least 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IL-1β (H) and IL-10 (I) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM MCC950, 5 µg.ml-1 αIL-10R or isotype antibody All stimulations were performed for 16 h. Data shown are representative of at least 3 independent experiments.


IL - 1β ELISA of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 ng . ml - 1 IL - 10 and 5 mM ATP . Data shown are a pool of 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 B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 ng.ml-1 IL-10 and 5 mM ATP. Data shown are a pool of 3 independent experiments.


Flow cytometry analysis of Mitotracker ( MT ) Green and Red integration in Gata6 - WT and Gata6 - KOmye naïve pMɸ ( gated on F4 / 80hi / + Tim4 + ) in vivo , 30 min after intraperitoneal injection ( i . p . ) of 1 µM of Mitotracker Green and Red . Data shown are representative of 3 to 5 independent mice of each genotype and are expressed as mean + / - SEM . Student ' s t test analysis was performed .

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

Flow cytometry analysis of Mitotracker (MT) Green and Red integration in Gata6-WT and Gata6-KOmye naïve pMɸ (gated on F4/80hi/+ Tim4+) in vivo, 30 min after intraperitoneal injection (i.p.) of 1 µM of Mitotracker Green and Red. Data shown are representative of 3 to 5 independent mice of each genotype and are expressed as mean +/- SEM. Student's t test analysis was performed.


Flow cytometry analysis of Mitotracker green and red staining in Gata6 - WT and Gata6 - KOmye pMɸ ( gated on F4 / 80hi / + Tim4 + ) unstimulated ( - ) or after LPS ( 100 ng . ml - 1 ) , IL - 10 ( 10 ng . ml - 1 ) , αIL - 10R ( 5 µg . ml - 1 ) or isotype ( 5 µg . ml - 1 ) stimulation for 16 h in vitro . Data shown are pooled from 3 independent experiments and normalized to WT unstimulated .

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

Flow cytometry analysis of Mitotracker green and red staining in Gata6-WT and Gata6-KOmye pMɸ (gated on F4/80hi/+ Tim4+) unstimulated (-) or after LPS (100 ng.ml-1), IL-10 (10 ng.ml-1), αIL-10R (5 µg.ml-1) or isotype (5 µg.ml-1) stimulation for 16 h in vitro. Data shown are pooled from 3 independent experiments and normalized to WT unstimulated.


Flow cytometry analysis of MitoSOX staining in Gata6 - WT and Gata6 - KOmye pMɸ ( gated on F4 / 80hi / + Tim4 + ) unstimulated ( - ) or after LPS ( 100 ng . ml - 1 ) , IL - 10 ( 10 ng . ml - 1 ) and αIL - 10R ( 5 µg . ml - 1 ) stimulation for 16 h in vitro . Data shown are pooled from 2 independent experiments and normalized to WT unstimulated .

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

Flow cytometry analysis of MitoSOX staining in Gata6-WT and Gata6-KOmye pMɸ (gated on F4/80hi/+ Tim4+) unstimulated (-) or after LPS (100 ng.ml-1), IL-10 (10 ng.ml-1) and αIL-10R (5 µg.ml-1) stimulation for 16 h in vitro. Data shown are pooled from 2 independent experiments and normalized to WT unstimulated.


IL - 1β ELISA of supernatants of Gata6 - WT and Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 500 µM MitoTempo , 10 mM NAC , 0 . 5 µM MitoQ for 16 h . Data are representative of at least 3 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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

IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 500 µM MitoTempo, 10 mM NAC, 0.5 µM MitoQ for 16 h. Data are representative of at least 3 experiments.


mRNA expression analysis of Il1b , Nlrp3 and Caspase1 ( Casp1 ) of Gata6 - WT and Gata6 - KOmye pMФ stimulated for 3 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 , 5 µg . ml - 1 αIL - 10R or 5 µg . ml - 1 isotype . Data are representative of at least 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mRNA expression analysis of Il1b, Nlrp3 and Caspase1 (Casp1) of Gata6-WT and Gata6-KOmye pMФ stimulated for 3 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10, 5 µg.ml-1 αIL-10R or 5 µg.ml-1 isotype. Data are representative of at least 3 independent experiments.


Western blot analysis ( left ) and quantification ( right ) of Gata6 - WT and Gata6 - KOmye pMФ stimulated for 6 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 or 5 µg . ml - 1 αIL - 10R . Data are representative of 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis (left) and quantification (right) of Gata6-WT and Gata6-KOmye pMФ stimulated for 6 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10 or 5 µg.ml-1 αIL-10R. Data are representative of 3 independent experiments.


Caspase1 activity analysis of Gata6 - WT and Gata6 - KOmye pMФ stimulated for 16 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 , 5 µg . ml - 1 αIL - 10R or 5 µg . ml - 1 isotype . n = 5 to 8 individual mice .

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

Caspase1 activity analysis of Gata6-WT and Gata6-KOmye pMФ stimulated for 16 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10, 5 µg.ml-1 αIL-10R or 5 µg.ml-1 isotype. n = 5 to 8 individual mice.


Mean fluorescence intensity ( MFI ) of pro - IL - 1β of Gata6 - WT and Gata6 - KOmye pMФ stimulated for 3 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 , 5 µg . ml - 1 αIL - 10R or 5 µg . ml - 1 isotype for 16 h or freshly isolated . n = 6 to 13 individual mice . Data were log transformed before performing statistical analysis .

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

Mean fluorescence intensity (MFI) of pro-IL-1β of Gata6-WT and Gata6-KOmye pMФ stimulated for 3 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10, 5 µg.ml-1 αIL-10R or 5 µg.ml-1 isotype for 16 h or freshly isolated. n = 6 to 13 individual mice. Data were log transformed before performing statistical analysis.


mRNA expression analysis of Il10 and Tnf of Gata6 - WT and Gata6 - KOmye pMФ stimulated for 3 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 or 5 µg . ml - 1 αIL - 10R . Data are representative of at least 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mRNA expression analysis of Il10 and Tnf of Gata6-WT and Gata6-KOmye pMФ stimulated for 3 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10 or 5 µg.ml-1 αIL-10R. Data are representative of at least 3 independent experiments.


B , C Colocalization of N1 - 54 : mVenus : OsCRT3 with OFP - HDEL . OFP - HDEL is an ER lumen marker harboring a C - terminal HDEL ER - retention sequence . ( C ) shows a magnification of the box represented in ( B ) . Bar : 20 μm . D Colocalization analyses along the white arrow in ( C ) .

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B, C Colocalization of N1-54:mVenus:OsCRT3 with OFP-HDEL. OFP-HDEL is an ER lumen marker harboring a C-terminal HDEL ER-retention sequence. (C) shows a magnification of the box represented in (B). Bar: 20 μm. D Colocalization analyses along the white arrow in (C).


E , F Colocalization of N1 - 54 : mVenus : OsCRT3 with the ER membrane marker AtCNX1 - mCherry . ( F ) shows a magnification of the box represented in ( E ) . Bar : 20 μm . G Colocalization analyses along the white arrow in ( F ) .

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E, F Colocalization of N1-54:mVenus:OsCRT3 with the ER membrane marker AtCNX1-mCherry. (F) shows a magnification of the box represented in (E). Bar: 20 μm. G Colocalization analyses along the white arrow in (F).


C Cold response of cytosolic free Ca2 + concentration in root cells expressing NES - YC3 . 6 . Superimposition of representative normalized ratio variations for wild - type , oscrt3 - 1 , and the complemented transgenic pOsCRT3 root cells . The rectangle represents the regions of interest ( ROIs ) for the ratio measurements . The blue background indicates duration of cold treatment . Bar : 50 μm .

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 O O O O O O O O O O O O O O O O O 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 Cold response of cytosolic free Ca2+ concentration in root cells expressing NES-YC3.6. Superimposition of representative normalized ratio variations for wild-type, oscrt3-1, and the complemented transgenic pOsCRT3 root cells. The rectangle represents the regions of interest (ROIs) for the ratio measurements. The blue background indicates duration of cold treatment. Bar: 50 μm.


B Yeast two - hybrid analysis to test the OsCRT3 - OsCIPK7 interaction . The interactions between OsCRT3 , OsCRT3N , OsCRT3P or OsCRT3C with OsCIPK7 , OsCIPK7N , OsCIPK7C , OsCIPK7NAF , or OsCIPK7PPI were verified .

O O O O O O O O O O O O O O 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 Yeast two-hybrid analysis to test the OsCRT3-OsCIPK7 interaction. The interactions between OsCRT3, OsCRT3N, OsCRT3P or OsCRT3C with OsCIPK7, OsCIPK7N, OsCIPK7C, OsCIPK7NAF, or OsCIPK7PPI were verified.


C Pull - down assay of the interaction between OsCRT3 and OsCIPK7 .

C Pull-down assay of the interaction between OsCRT3 and OsCIPK7.


E Localization of OsCIPK7 - GFP in N . benthamiana leaf epidermal cells . OFP - HDEL was used as an ER marker . Bar : 20 μm . F Magnification of the box represented in ( E ) . Colocalization analyses along the white arrow are shown . Bar : 20 μm .

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E Localization of OsCIPK7-GFP in N. benthamiana leaf epidermal cells. OFP-HDEL was used as an ER marker. Bar: 20 μm. F Magnification of the box represented in (E). Colocalization analyses along the white arrow are shown. Bar: 20 μm.


A OsCIPK7 interacts with OsCBLs in a yeast two - hybrid system . Interactions were verified by growing the yeast on selective medium ( SD / - Leu - Trp , SD / - Leu - Trp - His - Ade ) and conducting β - Galactosidase assays .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 OsCIPK7 interacts with OsCBLs in a yeast two-hybrid system. Interactions were verified by growing the yeast on selective medium (SD/-Leu-Trp, SD/-Leu-Trp-His-Ade) and conducting β-Galactosidase assays.


B Subcellular localization of OsCBL7 and OsCBL8 in N . benthamiana leaf epidermal cells . Pictures were taken after 2 and 3 days of expression . Bar : 50 μm .

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B Subcellular localization of OsCBL7 and OsCBL8 in N. benthamiana leaf epidermal cells. Pictures were taken after 2 and 3 days of expression. Bar: 50 μm.


C Bimolecular fluorescence complementation ( BiFC ) assay confirming the interaction between OsCBL7 / 8 and OsCIPK7 in N . benthamiana leaf epidermal cells . 19K was used as a negative control . Bar : 50 μm .

C Bimolecular fluorescence complementation (BiFC) assay confirming the interaction between OsCBL7/8 and OsCIPK7 in N. benthamiana leaf epidermal cells. 19K was used as a negative control. Bar: 50 μm.


D Interactions between OsCIPK7 and OsCBL7 or OsCBL8 were attenuated by OsCRT3 in a yeast three - hybrid assay .

D Interactions between OsCIPK7 and OsCBL7 or OsCBL8 were attenuated by OsCRT3 in a yeast three-hybrid assay.


E Constructs and OsCRT3 protein expression level in ( D ) .

E Constructs and OsCRT3 protein expression level in (D).


F Luciferase Complementation Imaging assay ( LCI ) to detect the interaction between OsCIPK7 - nluc and OsCBL7 - cluc or OsCBL8 - cluc , with co - expression of N1 - 54 : mVenus : OsCRT3 ( mVenus - OsCRT3 ) or GFP , respectively . Quantification of luciferase activity was measured and shown . Values are mean ± SD . At least 6 leaves were used for each sample . * P < 0 . 05 , Student ' s t - test . Experiments were repeated at least twice with similar results .

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F Luciferase Complementation Imaging assay (LCI) to detect the interaction between OsCIPK7-nluc and OsCBL7-cluc or OsCBL8-cluc, with co-expression of N1-54:mVenus:OsCRT3 (mVenus-OsCRT3) or GFP, respectively. Quantification of luciferase activity was measured and shown. Values are mean ± SD. At least 6 leaves were used for each sample. *P < 0.05, Student's t-test. Experiments were repeated at least twice with similar results.


G Immunoblot confirming the expression of OsCIPK7 , OsCBL7 , OsCBL8 , and OsCRT3 in ( F ) respectively .

G Immunoblot confirming the expression of OsCIPK7, OsCBL7, OsCBL8, and OsCRT3 in (F) respectively.


H Pull - down assay showing that the interaction between OsCIPK7 and OsCBL7 or OsCBL8 was weakened with increasing amounts of OsCRT3 . MBP was used as a negative control . Relative expression analysis was conducted using Image J software .

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H Pull-down assay showing that the interaction between OsCIPK7 and OsCBL7 or OsCBL8 was weakened with increasing amounts of OsCRT3. MBP was used as a negative control. Relative expression analysis was conducted using Image J software.


A OsCIPK7 showed autophosphorylation activity but could not phosphorylate OsCRT3 in vitro . The loaded protein is indicated by red arrows . Autoradiograph of γ - 32P - labeled OsCIPK7 indicates autophosphorylation of OsCIPK7 .

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A OsCIPK7 showed autophosphorylation activity but could not phosphorylate OsCRT3 in vitro. The loaded protein is indicated by red arrows. Autoradiograph of γ-32P-labeled OsCIPK7 indicates autophosphorylation of OsCIPK7.


B OsCIPK7 didn ' t phosphorylate OsCBL7 or OsCBL8 in vitro . Loaded proteins stained with Coomassie brilliant blue ( CBB ) are shown in the top panel and indicated by red arrows . Autoradiograph of γ - 32P - labeled myelin basic protein and OsCIPK7 are shown in the bottom panel .

B OsCIPK7 didn't phosphorylate OsCBL7 or OsCBL8 in vitro. Loaded proteins stained with Coomassie brilliant blue (CBB) are shown in the top panel and indicated by red arrows. Autoradiograph of γ-32P-labeled myelin basic protein and OsCIPK7 are shown in the bottom panel.


C The kinase activity OsCIPK7 was enhanced with increasing concentrations of OsCRT3 protein . SDS - PAGE gel showing CBB - stained OsCRT3 , myelin basic protein , and OsCIPK7 ( top panel ) . γ - 32P - labeled myelin basic protein and OsCIPK7 substrate phosphorylation ( bottom panel ) . The amount of OsCRT3 added was 0 μg , 0 . 2 μg , 0 . 4 μg , 0 . 6 μg , 0 . 8 μg in the SDS - PAGE gel lanes , respectively .

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C The kinase activity OsCIPK7 was enhanced with increasing concentrations of OsCRT3 protein. SDS-PAGE gel showing CBB-stained OsCRT3, myelin basic protein, and OsCIPK7 (top panel). γ-32P-labeled myelin basic protein and OsCIPK7 substrate phosphorylation (bottom panel). The amount of OsCRT3 added was 0 μg, 0.2 μg, 0.4 μg, 0.6 μg, 0.8 μg in the SDS-PAGE gel lanes, respectively.


A Sphere formation of GB2 cells transfected with an shRNA targeting SIRT2 was analyzed by an In Cell Analyzer 2000 . Primary spheres were re - plated to evaluate secondary sphere formation . Bars indicate mean ± s . d . of 10 wells .

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A Sphere formation of GB2 cells transfected with an shRNA targeting SIRT2 was analyzed by an In Cell Analyzer 2000. Primary spheres were re-plated to evaluate secondary sphere formation. Bars indicate mean ± s.d. of 10 wells.


B Knockdown of SIRT2 causes a decrease in the sphere formation capacity of GB16 . ( n = 3 ) .

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B Knockdown of SIRT2 causes a decrease in the sphere formation capacity of GB16. (n = 3).


C mRNA levels of the indicated genes in GB2 , GB4 and GB16 cells infected with a lentivirus expressing an shRNA targeting SIRT2 were measured by qRT - PCR . The results were normalized with the values for GAPDH . Bars indicate mean ± s . d . ( n = 3 - 4 ) .

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C mRNA levels of the indicated genes in GB2, GB4 and GB16 cells infected with a lentivirus expressing an shRNA targeting SIRT2 were measured by qRT-PCR. The results were normalized with the values for GAPDH. Bars indicate mean ± s.d. (n = 3-4).


The sphere formation capacity of CD133 - positive and CD133 - negative cells sorted by FACS directly from a tumor sample . GB17 was infected with a control ( Empty ) or shSIRT2 - expressing ( shS2 # 1 ) lentivirus . Bars indicate mean ± s . d . of 8 wells .

The sphere formation capacity of CD133-positive and CD133-negative cells sorted by FACS directly from a tumor sample. GB17 was infected with a control (Empty) or shSIRT2-expressing (shS2 #1) lentivirus. Bars indicate mean ± s.d. of 8 wells.


E The sphere formation capacity of CD133 - positive and CD133 - negative cells sorted by FACS directly from a tumor sample . ( Left panel ) GB18 was treated with AGK2 ( 10 μM ) or DMSO . ( Right panel ) Secondary sphere formation of GB18 was examined in the absence of AGK2 . Bars indicate mean ± s . d . of 8 wells .

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

E The sphere formation capacity of CD133-positive and CD133-negative cells sorted by FACS directly from a tumor sample. (Left panel) GB18 was treated with AGK2 (10 μM) or DMSO. (Right panel) Secondary sphere formation of GB18 was examined in the absence of AGK2. Bars indicate mean ± s.d. of 8 wells.


A Kaplan - Meier overall survival curves of mice transplanted with GB2 cells ( Left panel ) , GB13 cells ( Middle panel ) or GB16 cells ( Right panel ) infected with the indicated lentivirus ( 1 × 104 cells , 7 mice per group ) . The y - axis indicates the percent survival .

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A Kaplan-Meier overall survival curves of mice transplanted with GB2 cells (Left panel), GB13 cells (Middle panel) or GB16 cells (Right panel) infected with the indicated lentivirus (1 × 104 cells, 7 mice per group). The y-axis indicates the percent survival.


B Mice were transplanted with the indicated number of GB2 cells infected with a control ( Empty ) or shSIRT2 - expressing ( shS2 # 1 ) lentivirus . Six weeks after transplantation , mice ( 3 or 4 animals , see number of dots ) were sacrificed and the expression levels of human β - actin mRNA were quantified by qRT - PCR .

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B Mice were transplanted with the indicated number of GB2 cells infected with a control (Empty) or shSIRT2-expressing (shS2 #1) lentivirus. Six weeks after transplantation, mice (3 or 4 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA were quantified by qRT-PCR.


C H & E staining of tumors that were developed in mice implanted with GB16 cells that had been infected with a control ( Empty ) or shSIRT2 - expressing ( shS2 # 1 ) lentivirus . Scale bar , 1 mm . An image with higher magnification is shown on the right ( scale bar , 50 μm ) .

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C H&E staining of tumors that were developed in mice implanted with GB16 cells that had been infected with a control (Empty) or shSIRT2-expressing (shS2#1) lentivirus. Scale bar, 1 mm. An image with higher magnification is shown on the right (scale bar, 50 μm).


D The effect of AK7 on the deacetylation of acetyl - tubulin ( Left panel ) and the proliferation of GB2 cells ( Right panel ) . ( Left panel ) Immunoblotting analysis of the effect of AK7 ( 20 μM ) on deacetylation of acetyl - tubulin / tubulin were performed on day 3 in Right panel . Bars indicate mean ± s . d . ( n = 3 ) .

O 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 O O O O O B-CONTROLLED_VAR 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

D The effect of AK7 on the deacetylation of acetyl-tubulin (Left panel) and the proliferation of GB2 cells (Right panel). (Left panel) Immunoblotting analysis of the effect of AK7 (20 μM) on deacetylation of acetyl-tubulin/tubulin were performed on day 3 in Right panel. Bars indicate mean ± s.d. (n = 3).


E Ten days after intracranial transplantation of GB2 cells ( 1 . 0 × 104 cells ) , AK7 was intraperitoneally administrated for 4 weeks ( 15mg / kg , twice / week ) . After eight weeks , mice ( 4 or 5 animals , see number of dots ) were sacrificed and the expression levels of human β - actin mRNA was quantified by qRT - PCR .

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E Ten days after intracranial transplantation of GB2 cells (1.0 × 104 cells), AK7 was intraperitoneally administrated for 4 weeks (15mg/kg, twice/week). After eight weeks, mice (4 or 5 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA was quantified by qRT-PCR.


B Immunoblotting analysis of p73 in GB2 cells infected with a control ( Empty ) or shTP73 - expressing lentivirus for 96 h .

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B Immunoblotting analysis of p73 in GB2 cells infected with a control (Empty) or shTP73-expressing lentivirus for 96 h.


C Growth curve of GB16 cells infected with the indicated lentiviruses . Bars indicate mean ± s . d . ( n = 3 ) .

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C Growth curve of GB16 cells infected with the indicated lentiviruses. Bars indicate mean ± s.d. (n = 3).


Ectopic expression of the ∆ N isoform of p73β suppresses the expression levels of PUMA and GADD45 induced by SIRT2 knockdown in GB2 cells . Four days after lentiviral infection , the expression levels of PUMA and GADD45 mRNA were measured by qRT - PCR . Bars indicate mean ± s . d . ( n = 3 ) .

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

Ectopic expression of the ∆N isoform of p73β suppresses the expression levels of PUMA and GADD45 induced by SIRT2 knockdown in GB2 cells. Four days after lentiviral infection, the expression levels of PUMA and GADD45 mRNA were measured by qRT-PCR. Bars indicate mean ± s.d. (n = 3).


E GB2 and GB4 cells [ 5 × 104 and 2 × 104 cells , respectively ( indicated by the dashed lines ) ] were infected with the indicated lentiviruses . After 96 h , the number of viable cells was counted . Bars indicate mean ± s . d . ( n = 4 ) .

E GB2 and GB4 cells [5 × 104 and 2 × 104 cells, respectively (indicated by the dashed lines)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± s.d. (n = 4).


F HEK293 cells were transfected with TAp73α along with ΔNp73β . PUMA mRNA was measured by qRT - PCR analyses . Bars indicate mean ± s . d . ( n = 3 ) .

F HEK293 cells were transfected with TAp73α along with ΔNp73β. PUMA mRNA was measured by qRT-PCR analyses. Bars indicate mean ± s.d. (n = 3).


GB2 cells ( 5 × 104 cells , dashed line ) were infected with the indicated lentiviruses , respectively . After 96 h , the number of viable cells was counted . shTP73 corresponds to both α and β isoforms of p73 . Bars indicate mean ± s . d . ( n = 4 ) .

GB2 cells (5 × 104 cells, dashed line) were infected with the indicated lentiviruses, respectively. After 96 h, the number of viable cells was counted. shTP73 corresponds to both α and β isoforms of p73. Bars indicate mean ± s.d. (n = 4).


H GB2 cells were transfected with TAp73α along with NLS - SIRT2 ( nuclear - localizing mutant of SIRT2 ) or 3mut ( deacetylase - inactive , nuclear - localizing mutant of SIRT2 ) and a reporter construct consisting of the promoter region of PUMA fused to a luciferase gene ( Left panel ) . Reporter activities were determined by Dual luciferase assays ( Right panel ) . Bars indicate mean ± s . d . ( n = 3 ) .

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H GB2 cells were transfected with TAp73α along with NLS-SIRT2 (nuclear-localizing mutant of SIRT2) or 3mut (deacetylase-inactive, nuclear-localizing mutant of SIRT2) and a reporter construct consisting of the promoter region of PUMA fused to a luciferase gene (Left panel). Reporter activities were determined by Dual luciferase assays (Right panel). Bars indicate mean ± s.d. (n = 3).


I GB2 cells were electroporated with TAp73α along with NLS - SIRT2 ( nuclear - localizing mutant of SIRT2 ) or 3mut ( deacetylase - inactive , nuclear - localizing mutant of SIRT2 ) . The expression of PUMA and GADD45 was measured by qRT - PCR analysis . Bars indicate mean ± s . d . ( n = 3 )

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

I GB2 cells were electroporated with TAp73α along with NLS-SIRT2 (nuclear-localizing mutant of SIRT2) or 3mut (deacetylase-inactive, nuclear-localizing mutant of SIRT2). The expression of PUMA and GADD45 was measured by qRT-PCR analysis. Bars indicate mean ± s.d. (n = 3)


A Cells transfected with FLAG - tagged p73 isoforms were treated with 20 μM AGK2 or DMSO for 6 h and subjected to immunoprecipitation with anti - FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG .

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 B-MEASURED_VAR I-MEASURED_VAR O O O

A Cells transfected with FLAG-tagged p73 isoforms were treated with 20 μM AGK2 or DMSO for 6 h and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG.


Cells transfected with the indicated constructs were treated with 20 μM AGK2 or DMSO , respectively , for 6 h and subjected to immunoprecipitation with anti - FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG .

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 B-MEASURED_VAR I-MEASURED_VAR O O O

Cells transfected with the indicated constructs were treated with 20 μM AGK2 or DMSO, respectively, for 6 h and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG.


E HEK293T cells transfected with FLAG - tagged TAp73α were treated with 20 μM AGK2 ( pre - AGK2 + ) or DMSO ( pre - AGK2 - ) for 6 h . p73 was purified by immunoprecipitation and incubated with recombinant SIRT2 ( 10 U ) , NAD ( 1 mM ) and / or AGK2 ( 3 . 5 μM ) as indicated .

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E HEK293T cells transfected with FLAG-tagged TAp73α were treated with 20 μM AGK2 (pre-AGK2 +) or DMSO (pre-AGK2 -) for 6 h. p73 was purified by immunoprecipitation and incubated with recombinant SIRT2 (10 U), NAD (1 mM) and/or AGK2 (3.5 μM) as indicated.


F qRT - PCR analysis of PUMA and GADD45 mRNA in GB2 cells infected with the indicated lentiviruses . Bars indicate mean ± s . e . m . ( n = 4 - 5 ) .

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F qRT-PCR analysis of PUMA and GADD45 mRNA in GB2 cells infected with the indicated lentiviruses. Bars indicate mean ± s.e.m. (n = 4-5).


G Expression of p73 in ( F ) was determined by immunoblotting with anti - p73 antibody . RFP was used as a control .

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

G Expression of p73 in (F) was determined by immunoblotting with anti-p73 antibody. RFP was used as a control.


H GB glioblastima neurospheres [ 5 × 104 cells ( indicated by the dashed line ) ] were infected with the indicated lentiviruses . After 96 h , the number of viable cells was counted . Bars indicate mean ± s . d . ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 GB glioblastima neurospheres [5 × 104 cells (indicated by the dashed line)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± s.d. (n = 4).


GB2 cells infected with the indicated lentiviruses were intracranially transplanted into immunocompromised mice . After 8 weeks , mice ( 3 to 5 animals , see number of dots ) were sacrificed and the expression levels of human β - actin mRNA were quantified by qRT - PCR .

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

GB2 cells infected with the indicated lentiviruses were intracranially transplanted into immunocompromised mice. After 8 weeks, mice (3 to 5 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA were quantified by qRT-PCR.


( A ) Ribbon representation of the Atg12 - Atg5 conjugate bound to Atg16N . Atg12 , Atg5 and Atg6 are coloured salmon pink , green and orange , respectively . Atg12 Phe 185 and Gly 186 and Atg5 Lys 149 are shown with a stick model . The disordered C ‐ terminal region of Atg12 ( residues 182 - 184 ) is indicated with a broken line . The left figure was obtained by a 90 ° rotation of the right figure along the vertical axis . The amino ‐ and carboxy ‐ termini are denoted as N and C , respectively . All of the figures representing the molecular structures were generated with PyMOL [ 26 ] .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Ribbon representation of the Atg12-Atg5 conjugate bound to Atg16N. Atg12, Atg5 and Atg6 are coloured salmon pink, green and orange, respectively. Atg12 Phe 185 and Gly 186 and Atg5 Lys 149 are shown with a stick model. The disordered C‐terminal region of Atg12 (residues 182-184) is indicated with a broken line. The left figure was obtained by a 90° rotation of the right figure along the vertical axis. The amino‐ and carboxy‐termini are denoted as N and C, respectively. All of the figures representing the molecular structures were generated with PyMOL [ 26].


( B ) Stereo view of the detailed interactions between Atg12 and Atg5 . The side chains of the residues involved in the Atg12 - Atg5 interaction are shown with a stick model , in which nitrogen and oxygen atoms are coloured red and blue , respectively .

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

(B) Stereo view of the detailed interactions between Atg12 and Atg5. The side chains of the residues involved in the Atg12-Atg5 interaction are shown with a stick model, in which nitrogen and oxygen atoms are coloured red and blue, respectively.


( C ) Electron density map of the isopeptide linkage between Atg12 Gly 186 and Atg5 Lys 149 . The simulated annealing Fo - Fc difference Fourier map was calculated by omitting Atg12 Phe 185 and Gly 186 and Atg5 Lys 149 , and is shown with black meshes at 5 . 0σ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Electron density map of the isopeptide linkage between Atg12 Gly 186 and Atg5 Lys 149. The simulated annealing Fo-Fc difference Fourier map was calculated by omitting Atg12 Phe 185 and Gly 186 and Atg5 Lys 149, and is shown with black meshes at 5.0σ.


( D ) Superimposition of the Atg5 ‐ Atg16N complex structure ( PDB ID 2DYM ) on that of the Atg12 - Atg5 ‐ Atg16N complex . The Atg12 - Atg5 ‐ Atg16N complex is coloured red , while the Atg5 ‐ Atg16N complex is coloured grey . Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Superimposition of the Atg5‐Atg16N complex structure (PDB ID 2DYM) on that of the Atg12-Atg5‐Atg16N complex. The Atg12-Atg5‐Atg16N complex is coloured red, while the Atg5‐Atg16N complex is coloured grey. Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model.


( E ) In vivo analyses of the Atg12 and Atg5 mutants for studying the functional significance of the non ‐ covalent Atg12 ‐ Atg5 interactions . Yeast cells with or without starvation were lysed and subjected to urea SDS - polyacrylamide gel electrophoresis ( PAGE ) followed by western blotting . Ape1 , aminopeptidase I ; mApe1 , mature form of Ape1 ; PE , phosphatidylethanolamine ; prApe1 , preform of Ape1 ; WT , wild ‐ type .

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(E) In vivo analyses of the Atg12 and Atg5 mutants for studying the functional significance of the non‐covalent Atg12‐Atg5 interactions. Yeast cells with or without starvation were lysed and subjected to urea SDS-polyacrylamide gel electrophoresis (PAGE) followed by western blotting. Ape1, aminopeptidase I; mApe1, mature form of Ape1; PE, phosphatidylethanolamine; prApe1, preform of Ape1; WT, wild‐type.


( A ) In vitro pull ‐ down assay using recombinant plant Atg homologues . CBB , Coomassie Brilliant Blue staining ; GST , S ; HA , haemagglutinin ; PE , phosphatidylethanolamine ; WT , wild ‐ type .

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(A) In vitro pull‐down assay using recombinant plant Atg homologues.CBB, Coomassie Brilliant Blue staining; GST, S; HA, haemagglutinin; PE, phosphatidylethanolamine; WT, wild‐type.


( B ) Mapping of the mutated residues on the Atg12 - Atg5 ‐ Atg16N complex structure . Atg5 and Atg16N are shown with a ribbon model , whereas Atg12 is shown with both surface and ribbon models . The side chains of Tyr 147 , Tyr 149 and Phe 154 of Atg12 are shown with a stick model and coloured grey ( Tyr 147 ) or blue ( Tyr 149 and Phe 154 ) . Corresponding AtAtg12b residues are indicated in parentheses . Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mapping of the mutated residues on the Atg12-Atg5‐Atg16N complex structure. Atg5 and Atg16N are shown with a ribbon model, whereas Atg12 is shown with both surface and ribbon models. The side chains of Tyr 147, Tyr 149 and Phe 154 of Atg12 are shown with a stick model and coloured grey (Tyr 147) or blue (Tyr 149 and Phe 154). Corresponding AtAtg12b residues are indicated in parentheses. Atg12 Phe 185 and Gly 186 and the side chain of Atg5 Lys 149 are shown with a stick model.


( B , C ) Exercise capacity of MCK - KO and WT mice from the low intensity regiment was conducted . ( n = 4 WT , n = 5 KO mice , mean ± SEM , * p < 0 . 05 , two - tailed student ' s t - test ) .

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(B, C) Exercise capacity of MCK-KO and WT mice from the low intensity regiment was conducted. (n = 4 WT, n = 5 KO mice, mean ±SEM, * p < 0.05, two-tailed student's t-test).


( E , F ) Exercise capacity of MCK - KO and WT mice from the high intensity regiment ( n = 5 mice , means ± SEM , two - tailed student ' s t - test ) .

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(E, F) Exercise capacity of MCK-KO and WT mice from the high intensity regiment (n = 5 mice, means ± SEM, two-tailed student's t-test).


( A ) The H & E staining of MCK - KO and WT soleus muscle after a bout of low intensity treadmill running ( top 40X , bottom 100X magnifications ) . Black arrow indicates centralized nuclei . ( B ) The percentage of myofibers with centralized nuclei was determined by manual counting 300 myofibers in 20X magnification . ( n = 3 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test ) .

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(A) The H&E staining of MCK-KO and WT soleus muscle after a bout of low intensity treadmill running (top 40X, bottom 100X magnifications). Black arrow indicates centralized nuclei. (B) The percentage of myofibers with centralized nuclei was determined by manual counting 300 myofibers in 20X magnification. (n = 3, means ± SEM, * p < 0.05, two-tailed student's t-test).


( C - D ) The analogous set of data is shown with WT and MCK - KO GA muscles . ( n = 3 , means ± SEM , two - tailed student ' s t - test . GA : Gastrocnemius )

O O O O O O O O O O O O O O O O O O O O O O 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) The analogous set of data is shown with WT and MCK-KO GA muscles. (n = 3, means ± SEM, two-tailed student's t-test. GA: Gastrocnemius)


Hydrogen peroxide ( H2O2 ) levels were measured in WT and MCK - KO soleus ( E ) at rest and after a bout of exercise ( n = 6 per group , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) .

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

Hydrogen peroxide (H2O2) levels were measured in WT and MCK-KO soleus (E) at rest and after a bout of exercise (n = 6 per group, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).


( A - D ) Succinate dehydrogenase staining was performed in WT and MCK - KO soleus at sedentary ( A , B ) and after a bout of low - intensity exercise for 50 min ( C , D ) ( 10X , 20X magnifications ) , and the staining intensity was quantified using ImageJ ( n = 6 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test ) . ( E - H ) The analogous set of data is shown with WT and MCK - KO GA muscles . ( n = 6 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test ) . ( I - L ) The analogous set of data is shown with WT and MCK - KO EDL muscles . ( n = 6 , means ± SEM , two - tailed student ' s t - test . EDL : Extensor digitorum longus )

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(A-D) Succinate dehydrogenase staining was performed in WT and MCK-KO soleus at sedentary (A, B) and after a bout of low-intensity exercise for 50 min (C, D) (10X, 20X magnifications), and the staining intensity was quantified using ImageJ (n = 6, means ± SEM, * p < 0.05, two-tailed student's t-test). (E-H) The analogous set of data is shown with WT and MCK-KO GA muscles. (n = 6, means ± SEM, * p < 0.05, two-tailed student's t-test). (I-L) The analogous set of data is shown with WT and MCK-KO EDL muscles. (n = 6, means ± SEM, two-tailed student's t-test. EDL: Extensor digitorum longus)


( M , N ) Mitochondrial respiration was measured in WT and MCK - KO soleus tissue after a bout of low - intensity exercise for 50 min under basal conditions and in response to 4 mM oligomycin ( complex V inhibitor ) , 4 mM FCCP ( uncoupler ) , or 4 mM each of rotenone and antimycin A ( complex I inhibitor ) ( n = 8 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) . ( O , P ) Mitochondrial respiration was measured in Dnmt3a knocked down L6 myotubes which were transduced with lentiviral under basal conditions and in response to 4 mM oligomycin ( complex V inhibitor ) , 4 mM FCCP ( uncoupler ) , or 4 mM rotenone and antimycin A ( complex I inhibitor ) ( n = 5 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) .

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(M, N) Mitochondrial respiration was measured in WT and MCK-KO soleus tissue after a bout of low-intensity exercise for 50 min under basal conditions and in response to 4 mM oligomycin (complex V inhibitor), 4 mM FCCP (uncoupler), or 4 mM each of rotenone and antimycin A (complex I inhibitor) (n = 8, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing). (O, P) Mitochondrial respiration was measured in Dnmt3a knocked down L6 myotubes which were transduced with lentiviral under basal conditions and in response to 4 mM oligomycin (complex V inhibitor), 4 mM FCCP (uncoupler), or 4 mM rotenone and antimycin A (complex I inhibitor) (n = 5, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).


( E , F ) ALDH1L1 protein expression and the quantification in WT vs . MCK - KO soleus , GA and EDL muscles at sedentary and normalizing to GAPDH using ImageJ .

O O O O O O O O O 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, F) ALDH1L1 protein expression and the quantification in WT vs. MCK-KO soleus, GA and EDL muscles at sedentary and normalizing to GAPDH using ImageJ.


Single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes were achieved by lentiviral transduction . NADPH levels ( H ) and NOX activity ( I ) were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes . ( n = 3 , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) .

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Single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes were achieved by lentiviral transduction. NADPH levels (H) and NOX activity (I) were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes. (n = 3, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).


( J , K ) H2O2 levels were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes . ( J : n = 3 and K : n = 6 for Control , Aldh1l1 KD , Aldh1l1 KD + Dnmt3a KD , n = 5 for Dnmt3a KD , means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) .

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(J, K) H2O2 levels were measured in single and double knockdowns of Dnmt3a and Aldh1l1 in L6 myotubes. (J: n = 3 and K: n = 6 for Control, Aldh1l1 KD, Aldh1l1 KD + Dnmt3a KD, n = 5 for Dnmt3a KD, means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).


( D , E ) mitochondrial respiration in Dnmt3a knockdown and control L6 myotubes treated with NAC ( 3mM ) or vehicle treatment for 24hrs ( n = 5 Control groups n = 3 Dnmt3a KD , n = 4 Dnmt3a KD with NAC treatment groups . means ± SEM , * p < 0 . 05 , two - tailed student ' s t - test and two - way ANOVA followed by Bonferroni post - hoc testing ) .

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(D, E) mitochondrial respiration in Dnmt3a knockdown and control L6 myotubes treated with NAC (3mM) or vehicle treatment for 24hrs (n = 5 Control groups n = 3 Dnmt3a KD, n = 4 Dnmt3a KD with NAC treatment groups. means ± SEM, * p < 0.05, two-tailed student's t-test and two-way ANOVA followed by Bonferroni post-hoc testing).


( D , E ) Exercise capacity of WT and MCK - KO with or without transfection of gCont vs . gAldh1l1 under the low intensity regimen ( n = 4 WT + gCont , n = 9 MCK - KO + gCont , and MCK - KO + gAldh1l1 , * p < 0 . 05 , means ± SEM , two - tailed student ' s t - test and one - way Anova ) .

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(D, E) Exercise capacity of WT and MCK- KO with or without transfection of gCont vs. gAldh1l1 under the low intensity regimen (n = 4 WT + gCont, n = 9 MCK- KO + gCont, and MCK- KO + gAldh1l1, *p<0.05, means ± SEM, two-tailed student's t-test and one-way Anova).


Number of genes detected on protein and mRNA level in each tissue . The colouring the bars indicates the fractions of transcripts and proteins that are expressed everywhere or enriched in certain tissues . The full classification is provided in the text .

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Number of genes detected on protein and mRNA level in each tissue. The colouring the bars indicates the fractions of transcripts and proteins that are expressed everywhere or enriched in certain tissues. The full classification is provided in the text.


Abundance distribution of all transcripts detected in all tissues ( grey ) ; the fraction of detected proteins is shown in blue and the fraction of transcripts for which no protein was detected is shown in orange .

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Abundance distribution of all transcripts detected in all tissues (grey); the fraction of detected proteins is shown in blue and the fraction of transcripts for which no protein was detected is shown in orange.


Distribution of global transcript and protein abundance in all tissues . It is apparent that the dynamic range of protein expression ( iBAQ scale ) exceeds that of mRNA expression ( FPKM scale ; see Appendix Fig S10 for the corresponding plot for RNA and protein copy numbers ) .

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Distribution of global transcript and protein abundance in all tissues. It is apparent that the dynamic range of protein expression (iBAQ scale) exceeds that of mRNA expression (FPKM scale; see Appendix Fig S10 for the corresponding plot for RNA and protein copy numbers).


Protein to mRNA abundance plot for brain tissue . The slope of the regression line indicates that high abundance mRNAs give rise to more protein copies per mRNA than low abundance mRNAs .

Protein to mRNA abundance plot for brain tissue. The slope of the regression line indicates that high abundance mRNAs give rise to more protein copies per mRNA than low abundance mRNAs.


Ranked abundance plot of proteins and transcripts in human heart . While the 10 most abundant transcripts cover almost 70 % of all transcripts in this tissue , the corresponding proteins only represent about 20 % of the total protein .

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Ranked abundance plot of proteins and transcripts in human heart. While the 10 most abundant transcripts cover almost 70% of all transcripts in this tissue, the corresponding proteins only represent about 20% of the total protein.


Analysis of the number of genes that are shared among the 100 most abundant transcripts and proteins . Regardless of the tissue , the fraction of shared genes rarely exceeds 20 % .

Analysis of the number of genes that are shared among the 100 most abundant transcripts and proteins. Regardless of the tissue, the fraction of shared genes rarely exceeds 20%.


Correlation analysis of protein to RNA abundance ( in log10 scale ) across tissues , resulting in almost 90 % positive correlations . The protein highlighted in the next panel are marked .

Correlation analysis of protein to RNA abundance (in log10 scale) across tissues, resulting in almost 90% positive correlations. The protein highlighted in the next panel are marked.


Examples for proteins that show high ( SYK , left panel ) or no ( EIF4A3 , right panel ) correlation of protein / RNA ratios across tissues . While the former indicates that different tissues express different quantities of SYK , EIF4A3 expression appears to be similar in all tissues .

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Examples for proteins that show high (SYK, left panel) or no (EIF4A3, right panel) correlation of protein/RNA ratios across tissues. While the former indicates that different tissues express different quantities of SYK, EIF4A3 expression appears to be similar in all tissues.


Global correlation analysis of proteomes vs proteomes and transcriptomes vs transcriptomes across human tissues . It is apparent that proteomes correlate stronger across tissues than transcriptomes .

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Global correlation analysis of proteomes vs proteomes and transcriptomes vs transcriptomes across human tissues. It is apparent that proteomes correlate stronger across tissues than transcriptomes.


Average cellular compositions of tissues highlighted in panel ( B ) showing that the molecular similarities in their transcriptomes and proteomes are driven by similarities in cell types .

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Average cellular compositions of tissues highlighted in panel (B) showing that the molecular similarities in their transcriptomes and proteomes are driven by similarities in cell types.


( A ) Growth assay of the indicated cells with and without doxycycline - inducible overexpression of PLK4 . Experiments were performed in wild - type or SAS6 monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(A) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in wild-type or SAS6 monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( D ) Top hits that emerged from the screens ranked by MaGeCK FDR value . Blue hits are genes required for centriole duplication or stability . Green hits are genes predicted to be required to arrest the growth of cells with extra centrosomes .

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(D) Top hits that emerged from the screens ranked by MaGeCK FDR value. Blue hits are genes required for centriole duplication or stability. Green hits are genes predicted to be required to arrest the growth of cells with extra centrosomes.


( G ) Top : Graph showing the efficiency of frameshifting INDELs measured using TIDE . N . D . = Not determined . Data shown is from n = 1 biological replicate . Bottom : Graph showing the relative growth of doxycycline treated PLK4Dox cells expressing an sgRNA targeting the indicated genes . Each dot displays measurements from a single experiment . Experiments were performed in polyclonal knockout cells . Data acquired across n ≥ 3 biological replicates . Mean ± s . e . m .

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(G) Top: Graph showing the efficiency of frameshifting INDELs measured using TIDE. N.D. = Not determined. Data shown is from n = 1 biological replicate. Bottom: Graph showing the relative growth of doxycycline treated PLK4Dox cells expressing an sgRNA targeting the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.


( H ) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes . Experiments were performed in polyclonal knockout cells . Data acquired across n ≥ 3 biological replicates . Mean ± s . e . m .

(H) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.


( A ) Top , widefield image showing the localization of CEP164 and PIDD1 at the mature - mother centriole . Bottom : Representative 3D STORM image of the same centriole showing PIDD1 localization . STORM image colors correspond to the Z - depth with red being the closest to the coverslip and blue being the most distant .

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(A) Top, widefield image showing the localization of CEP164 and PIDD1 at the mature-mother centriole. Bottom: Representative 3D STORM image of the same centriole showing PIDD1 localization. STORM image colors correspond to the Z-depth with red being the closest to the coverslip and blue being the most distant.


( B ) 3D STORM image of PIDD1 at the mature mother centriole . The overlaid mask represents the inner and outer diameters of the PIDD1 signal .

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(B) 3D STORM image of PIDD1 at the mature mother centriole. The overlaid mask represents the inner and outer diameters of the PIDD1 signal.


( C ) Inner and outer diameter measurements for distal appendage proteins and PIDD1 . Measurements in the shaded region were previously reported in ( Bowler et al . , 2019 ) . Data displayed as box and whisker plots , where the box represents the upper and lower quartile and the whiskers represent the s . d . Scale bars : 1 μm for all wide - field images of centrioles and 500 nm for STORM images . Data acquired across n ≥ 6 cells .

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(C) Inner and outer diameter measurements for distal appendage proteins and PIDD1. Measurements in the shaded region were previously reported in (Bowler et al., 2019). Data displayed as box and whisker plots, where the box represents the upper and lower quartile and the whiskers represent the s.d. Scale bars: 1 μm for all wide-field images of centrioles and 500 nm for STORM images. Data acquired across n ≥6 cells.


( D ) Quantification of the fraction of cells with PIDD1 localized to the mature mother centriole in PLK4Dox cells expressing an sgRNA targeting the indicated genes . A dot displays measurements from each experiment . Experiments were performed in PLK4Dox polyclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(D) Quantification of the fraction of cells with PIDD1 localized to the mature mother centriole in PLK4Dox cells expressing an sgRNA targeting the indicated genes. A dot displays measurements from each experiment. Experiments were performed in PLK4Dox polyclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( F ) Graph showing the relative growth of doxycycline treated PLK4Dox cells that were knocked out for the indicated genes . Each dot displays measurements from a single experiment . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n ≥ 3 biological replicates . Mean ± s . e . m .

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(F) Graph showing the relative growth of doxycycline treated PLK4Dox cells that were knocked out for the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.


( G ) Quantification of the fraction of cells with PIDD1 localized at the mature mother centriole . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(G) Quantification of the fraction of cells with PIDD1 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( A ) Quantification of the fraction of cells with ANKRD26 localized at the mature mother centriole . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

(A) Quantification of the fraction of cells with ANKRD26 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( B - D ) Representative images of WT or ANKRD26 - / - PLK4Dox cells immunostained with the indicated antibodies . Scale bar = 5 µm .

(B-D) Representative images of WT or ANKRD26-/- PLK4Dox cells immunostained with the indicated antibodies. Scale bar = 5 µm.


( E ) Quantification of the fraction of WT or ANKRD26 - / - hTERT RPE1 cells with cilia . Each dot displays measurements from a single experiment . Data acquired across n = 5 biological replicates . Mean ± s . e . m .

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(E) Quantification of the fraction of WT or ANKRD26-/- hTERT RPE1 cells with cilia. Each dot displays measurements from a single experiment. Data acquired across n = 5 biological replicates. Mean ± s.e.m.


( F ) Growth assay of the indicated cells with and without doxycycline - inducible overexpression of PLK4 . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

(F) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( G ) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

(G) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( H ) Western blot showing expression of pro - CASP2 and P21 following treatment with dox for the specified number of days . Experiments were performed in PLK4Dox monoclonal knockout cells . ( I ) Quantification of pro - CASP2 levels following treatment with dox for the specified number of days . Experiments were performed in PLK4Dox monoclonal knockout cells . Each dot displays measurements from a single experiment . Data acquired across n = 6 biological replicates . Mean ± s . e . m .

(H) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. (I) Quantification of pro-CASP2 levels following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. Each dot displays measurements from a single experiment. Data acquired across n = 6 biological replicates. Mean ± s.e.m.


( A ) Top : schematic representation of PIDD1 and its various cleavage products . Bottom : HEK293FT cells were transfected with the indicated constructs , subjected to co - immunoprecipitation and immunoblotted with the indicated antibodies .

(A) Top: schematic representation of PIDD1 and its various cleavage products. Bottom: HEK293FT cells were transfected with the indicated constructs, subjected to co- immunoprecipitation and immunoblotted with the indicated antibodies.


( B ) HEK293FT cells were transfected with the indicated constructs , subjected to co - immunoprecipitation and immunoblotted with the indicated antibodies .

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(B) HEK293FT cells were transfected with the indicated constructs, subjected to co- immunoprecipitation and immunoblotted with the indicated antibodies.


( C ) Western blot showing the expression of ANKRD26 . Experiments were performed in monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated ANKRD26 transgenes .

(C) Western blot showing the expression of ANKRD26. Experiments were performed in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated ANKRD26 transgenes.


( D ) Quantification of the fraction of cells with ANKRD26 ( left ) and PIDD1 ( right ) localized to the mature mother centriole in monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated transgenes . Each dot displays measurements from a single experiment . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(D) Quantification of the fraction of cells with ANKRD26 (left) and PIDD1 (right) localized to the mature mother centriole in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated transgenes. Each dot displays measurements from a single experiment. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( E ) Graph showing the relative growth of doxycycline - treated , monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated ANKRD26 transgenes . Each dot displays measurements from a single experiment . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

(E) Graph showing the relative growth of doxycycline-treated, monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated ANKRD26 transgenes. Each dot displays measurements from a single experiment. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( F ) Western blot showing expression of pro - CASP2 and P21 following treatment with dox for 4 days . Experiments were performed in monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated transgenes .

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(F) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for 4 days. Experiments were performed in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated transgenes.


( A and B ) NIH3T3 cells stably expressing GFP - ULK1 ( A ) and - ULK2 ( B ) were cultured in complete or starvation medium for 30 min . They were then cultured in fresh complete medium for an additional 30 min ( starvation → complete ) .

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(A and B) NIH3T3 cells stably expressing GFP-ULK1 (A) and -ULK2 (B) were cultured in complete or starvation medium for 30 min. They were then cultured in fresh complete medium for an additional 30 min (starvation→complete).


( C and D ) NIH3T3 cells stably expressing GFP - ULK1 ( C ) and - ULK2 ( D ) were cultured in starvation medium for 120 min . The cells were fixed , permeabilized , and subjected to immunofluorescence microscopy using anti - Atg16L1 antibody and Alexa Fluor 660 - conjugated secondary antibody . More than 90 % of GFP - ULKs dots were positive for Atg16L1 .

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(C and D) NIH3T3 cells stably expressing GFP-ULK1 (C) and -ULK2 (D) were cultured in starvation medium for 120 min. The cells were fixed, permeabilized, and subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 660-conjugated secondary antibody. More than 90% of GFP-ULKs dots were positive for Atg16L1.


( E - H ) Wild - type ( E and G ) and Atg5 − / − ( F and H ) MEFs were transfected with retroviral vectors encoding GFP - ULK1 and - ULK2 . MEFs stably expressing GFP - ULK1 ( E and F ) and - ULK2 ( G and H ) were cultured in complete or starvation medium for 120 min . The cells were fixed and examined by fluorescence microscopy . Bars , 20 μm .

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(E-H) Wild-type (E and G) and Atg5−/− (F and H) MEFs were transfected with retroviral vectors encoding GFP-ULK1 and -ULK2. MEFs stably expressing GFP-ULK1 (E and F) and -ULK2 (G and H) were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bars, 20 μm.


( A ) In vitro kinase assay of endogenous ULK1 . MEFs were cultured in complete or starvation medium for 60 min . ULK1 kinase activity was determined as described in Materials and methods . Relative kinase activity is shown . Data are the mean ± SE of five independent experiments .

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(A) In vitro kinase assay of endogenous ULK1. MEFs were cultured in complete or starvation medium for 60 min. ULK1 kinase activity was determined as described in Materials and methods. Relative kinase activity is shown. Data are the mean ± SE of five independent experiments.


( B and C ) NIH3T3 cells were transiently transfected with HA - ULK1 , HA - ULK2 , or their kinase - dead mutants and subjected to immunofluoresence microscopy using monoclonal anti - HA and polyclonal anti - Atg16L1 antibodies for primary staining and Alexa Fluor 488 - conjugated goat anti - mouse IgG and Alexa Fluor 568 - conjugated goat anti - rabbit IgG antibodies for secondary antibodies . Transfected cells are indicated by arrows . Bars , 20 μm . ( D ) NIH3T3 cells stably expressing GFP - ULK1K46N and GFP - ULK2K39T were cultured in starvation medium for 120 min . The cells were subjected to immunofluorescence microscopy using anti - Atg16L1 antibody . Bar , 20 μm .

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(B and C) NIH3T3 cells were transiently transfected with HA-ULK1, HA-ULK2, or their kinase-dead mutants and subjected to immunofluoresence microscopy using monoclonal anti-HA and polyclonal anti-Atg16L1 antibodies for primary staining and Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG antibodies for secondary antibodies. Transfected cells are indicated by arrows. Bars, 20 μm. (D) NIH3T3 cells stably expressing GFP-ULK1K46N and GFP-ULK2K39T were cultured in starvation medium for 120 min. The cells were subjected to immunofluorescence microscopy using anti-Atg16L1 antibody. Bar, 20 μm.


( E ) NIH3T3 cells were transfected with the retroviral vectors encoding HA - ULK1K46N or with the corresponding empty retrovirus ( mock ) . They were cultured in complete or starvation medium for 120 min . Cell lysates were then analyzed by immunoblot analysis with the indicated antibodies .

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(E) NIH3T3 cells were transfected with the retroviral vectors encoding HA-ULK1K46N or with the corresponding empty retrovirus (mock). They were cultured in complete or starvation medium for 120 min. Cell lysates were then analyzed by immunoblot analysis with the indicated antibodies.


( A ) HEK293T cells were cotransfected with FLAG - FIP200 and HA - ULKs . Cell lysates were subjected to immunoprecipitation ( IP ) using antibodies against FLAG . The resulting precipitates were examined by immunoblot ( IB ) analysis with the indicated antibodies . The asterisk indicates nonspecific band .

(A) HEK293T cells were cotransfected with FLAG-FIP200 and HA-ULKs. Cell lysates were subjected to immunoprecipitation (IP) using antibodies against FLAG. The resulting precipitates were examined by immunoblot (IB) analysis with the indicated antibodies. The asterisk indicates nonspecific band.


( B ) Lysates of MEFs were immunoprecipitated with anti - ULK1 or anti - FIP200 antibody or preimmune rabbit serum , and the resulting precipitates were subjected to immunoblot analysis with antibodies against ULK1 and FIP200 .

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(B) Lysates of MEFs were immunoprecipitated with anti-ULK1 or anti-FIP200 antibody or preimmune rabbit serum, and the resulting precipitates were subjected to immunoblot analysis with antibodies against ULK1 and FIP200.


( D ) HEK293T cells were cotransfected with FLAG - FIP200 and various ULK1 mutants and analyzed as in A using anti - HA and anti - FLAG antibodies .

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(D) HEK293T cells were cotransfected with FLAG-FIP200 and various ULK1 mutants and analyzed as in A using anti-HA and anti-FLAG antibodies.


( E ) NIH3T3 cells stably expressing GFP - ULK1 ( left ) and GFP - ULK1ΔC ( right ) were cultured in starvation medium for 120 min . Bar , 20 μm .

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(E) NIH3T3 cells stably expressing GFP-ULK1 (left) and GFP-ULK1ΔC (right) were cultured in starvation medium for 120 min. Bar, 20 μm.


( A ) NIH3T3 cells stably expressing GFP - FIP200 were cultured in complete or starvation medium for 120 min and the GFP signal was observed .

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(A) NIH3T3 cells stably expressing GFP-FIP200 were cultured in complete or starvation medium for 120 min and the GFP signal was observed.


( B ) NIH3T3 cells stably expressing GFP - FIP200 were cultured in starvation medium for 120 min and then subjected to immunofluorescence microscopy using anti - Atg16L1 antibody and Alexa Fluor 568 - conjugated secondary antibody . Bars , 20 μm . More than 90 % of GFP - FIP200 dots were positive for Atg16L1 .

(B) NIH3T3 cells stably expressing GFP-FIP200 were cultured in starvation medium for 120 min and then subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 568-conjugated secondary antibody. Bars, 20 μm. More than 90% of GFP-FIP200 dots were positive for Atg16L1.


( C ) NIH3T3 cells stably expressing GFP - ULK1 were starved for 120 min and then subjected to immunofluorescence microscopy using anti - FIP200 antibody and Alexa Fluor 568 - conjugated secondary antibody . Black squares indicate the enlarged areas shown in insets . Bar , 20 μm .

(C) NIH3T3 cells stably expressing GFP-ULK1 were starved for 120 min and then subjected to immunofluorescence microscopy using anti-FIP200 antibody and Alexa Fluor 568-conjugated secondary antibody. Black squares indicate the enlarged areas shown in insets. Bar, 20 μm.


( A ) FIP200 + / + , FIP200 − / − , Atg5 + / + , and Atg5 − / − MEFs were cultured in complete or starvation medium for 60 min . In the recovery experiments , starved MEFs were cultured in fresh complete medium for an additional 60 min ( replenishment ) . The cell lysates were subjected to immunoblot analysis with the indicated antibodies .

(A) FIP200+/+, FIP200−/−, Atg5+/+, and Atg5−/− MEFs were cultured in complete or starvation medium for 60 min. In the recovery experiments, starved MEFs were cultured in fresh complete medium for an additional 60 min (replenishment). The cell lysates were subjected to immunoblot analysis with the indicated antibodies.


( B ) Wild - type and FIP200 − / − MEFs were cultured in the complete or starvation medium for indicated time with or without 100 nM bafilomycin A1 . The cell lysates were subjected to immunoblot analysis with anti - LC3 antibody .

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(B) Wild-type and FIP200−/− MEFs were cultured in the complete or starvation medium for indicated time with or without 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody.


( C ) Wild - type and FIP200 − / − MEFs were transfected with retroviral vectors encoding GFP - Atg5 or GFP - LC3 . Resulting cells were cultured in the starvation medium for 120 min . The cells were fixed and examined by fluorescence microscopy . Bar , 20 μm .

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(C) Wild-type and FIP200−/− MEFs were transfected with retroviral vectors encoding GFP-Atg5 or GFP-LC3. Resulting cells were cultured in the starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm.


( D - G ) Wild - type ( D and E ) and FIP200 − / − ( F and G ) MEFs were cultured in complete ( D and F ) or starvation ( E and G ) medium for 120 min and then fixed and subjected to EM analysis . Autophagosome - like structures ( open arrowheads ) , and autolysosomes ( closed arrowheads ) are indicated . Bar , 1 μm . ( H ) The ratio of total area of autophagosomes ( AP ) and autolysosomes ( AL ) to total cytoplasmic area in D - G was determined by morphometric analysis .

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(D-G) Wild-type (D and E) and FIP200−/− (F and G) MEFs were cultured in complete (D and F) or starvation (E and G) medium for 120 min and then fixed and subjected to EM analysis. Autophagosome-like structures (open arrowheads), and autolysosomes (closed arrowheads) are indicated. Bar, 1 μm. (H) The ratio of total area of autophagosomes (AP) and autolysosomes (AL) to total cytoplasmic area in D-G was determined by morphometric analysis.


( A ) FAK + / − and FAK − / − MEFs were cultured in the complete or starvation medium for the indicated times with or without 100 nM bafilomycin A1 . The cell lysates were subjected to immunoblot analysis with the indicated antibodies .

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(A) FAK+/− and FAK−/− MEFs were cultured in the complete or starvation medium for the indicated times with or without 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with the indicated antibodies.


( B ) FAK + / − and FAK − / − MEFs stably expressing GFP - LC3 were cultured in complete or starvation medium for 120 min . The cells were fixed and examined by fluorescence microscopy .

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(B) FAK+/− and FAK−/− MEFs stably expressing GFP-LC3 were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy.


( C ) Wild - type MEFs stably expressing GFP - FAK were cultured in complete or starvation medium for 120 min . The cells were fixed and subjected to immunofluorescence microscopy using anti - Atg16 antibody . Black squares indicate the enlarged areas shown in insets . Bars , 20 μm .

(C) Wild-type MEFs stably expressing GFP-FAK were cultured in complete or starvation medium for 120 min. The cells were fixed and subjected to immunofluorescence microscopy using anti-Atg16 antibody. Black squares indicate the enlarged areas shown in insets. Bars, 20 μm.


( A ) Wild - type and FIP200 − / − MEFs were treated with 100 ng / ml rapamycin ( rapa ) or vehicle ( DMSO ) for 120 min in the presence or absence of 100 nM bafilomycin A1 . The cell lysates were subjected to immunoblot analysis with anti - LC3 antibody .

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(A) Wild-type and FIP200−/− MEFs were treated with 100 ng/ml rapamycin (rapa) or vehicle (DMSO) for 120 min in the presence or absence of 100 nM bafilomycin A1. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody.


( B ) Wild - type and FIP200 − / − MEFs stably expressing GFP - Atg5 or GFP - LC3 were cultured in the presence of 100 ng / ml rapamycin for 120 min . The formation of GFP - Atg5 ( top ) and GFP - LC3 ( bottom ) puncta was examined by fluorescence microscopy . Bar , 20 μm .

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(B) Wild-type and FIP200−/− MEFs stably expressing GFP-Atg5 or GFP-LC3 were cultured in the presence of 100 ng/ml rapamycin for 120 min. The formation of GFP-Atg5 (top) and GFP-LC3 (bottom) puncta was examined by fluorescence microscopy. Bar, 20 μm.


( C ) Wild - type and FIP200 − / − MEFs were treated with 10 mM lithium chloride for 24 h or 100 μM C2 - ceramide for 2 h .

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(C) Wild-type and FIP200−/− MEFs were treated with 10 mM lithium chloride for 24 h or 100 μM C2-ceramide for 2 h.


( A ) FIP200 + / − and FIP200 − / − MEFs stably expressing GFP - ULK1 or - ULK2 were cultured in starvation medium for 120 min . The cells were fixed and examined by fluorescence microscopy . Bar , 20 μm .

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(A) FIP200+/− and FIP200−/− MEFs stably expressing GFP-ULK1 or -ULK2 were cultured in starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm.


( B ) FIP200 + / + and FIP200 − / − MEFs were cultured in complete medium . The cell lysates were subjected to immunoblot analysis with antibodies against ULK1 or HSP90 ( loading control ) .

(B) FIP200+/+ and FIP200−/− MEFs were cultured in complete medium. The cell lysates were subjected to immunoblot analysis with antibodies against ULK1 or HSP90 (loading control).


( C ) Phosphatase sensitivity of ULK1 . FIP200 + / + and FIP200 − / − MEFs were cultured in complete medium . ULK1 was immunoprecipitated from cell lysates and treated with λ phosphatase for 30 min at 30 ° C in the absence or presence of phosphatase inhibitors ( 1 mM Na2VO4 , 50 mM KF , 15 mM Na4P2O7 , and 1 mM EGTA ) .

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(C) Phosphatase sensitivity of ULK1. FIP200+/+ and FIP200−/− MEFs were cultured in complete medium. ULK1 was immunoprecipitated from cell lysates and treated with λ phosphatase for 30 min at 30°C in the absence or presence of phosphatase inhibitors (1 mM Na2VO4, 50 mM KF, 15 mM Na4P2O7, and 1 mM EGTA).


Example images showing the localization of SOX2 - SNAP and OCT4 - HALO by fluorescence microscopy of SBROS cells . Scale bar : 50µm .

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Example images showing the localization of SOX2-SNAP and OCT4-HALO by fluorescence microscopy of SBROS cells. Scale bar: 50µm.


Correlation between OCT4 - HALO and total OCT4 levels determined by immunofluorescence and HALO labelling . R is Pearson ' s correlation coefficient .

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Correlation between OCT4-HALO and total OCT4 levels determined by immunofluorescence and HALO labelling. R is Pearson's correlation coefficient.


Distributions of OCT4 ( n = 2682 ) , SOX2 ( n = 1236 ) and NANOG ( n = 2416 ) levels in WT E14 and SBROS cell lines as determined by quantitative immunofluorescence . Dashed lines : mean protein levels ; Dotted lines : median protein levels .

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Distributions of OCT4 (n=2682), SOX2 (n=1236) and NANOG (n=2416) levels in WT E14 and SBROS cell lines as determined by quantitative immunofluorescence. Dashed lines: mean protein levels; Dotted lines: median protein levels.


Protein half - lives of OCT4 - HALO and SOX2 - SNAP in SBROS cells . Whiskers : minimum and maximum values ; Box : lower and upper quartiles ; Solid line : median . Black dots : outliers .

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Protein half-lives of OCT4-HALO and SOX2-SNAP in SBROS cells. Whiskers: minimum and maximum values; Box: lower and upper quartiles; Solid line: median. Black dots: outliers.


Cell cycle duration of E14 WT ( n = 6 ) and SBROS ( n = 8 ) cells . Error bars : SE . p - value : two - sided t - test with unequal variance .

Cell cycle duration of E14 WT (n=6) and SBROS (n=8) cells. Error bars: SE. p-value: two-sided t-test with unequal variance.


Endogenous SOX2 levels in single cells ( turquoise ) normalized to the value at t = 0 in SNSF cells upon induction of SOX2 - SNAP ( n = 37 , left ) or a truncated SOX2 version missing the DNA binding domain ( YPET - SOX2 - delDBD , n = 47 , right ) . Black line : average SOX2 - SNAP levels after induction normalized to the maximum level ( n = 22 ) ; Yellow line : average YPET - SOX2 - delDBD levels after induction , normalized to the maximum level ( n = 20 ) .

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Endogenous SOX2 levels in single cells (turquoise) normalized to the value at t=0 in SNSF cells upon induction of SOX2-SNAP (n=37, left) or a truncated SOX2 version missing the DNA binding domain (YPET-SOX2-delDBD, n=47, right). Black line: average SOX2-SNAP levels after induction normalized to the maximum level (n=22); Yellow line: average YPET-SOX2-delDBD levels after induction, normalized to the maximum level (n=20).


Sox2 and Oct4 mRNA levels upon overexpression of YPet - SOX2 or YPET - SOX2 - delDBD ( n = 3 ) . p - values : two - sided t - test with unequal variance .

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Sox2 and Oct4 mRNA levels upon overexpression of YPet-SOX2 or YPET-SOX2-delDBD (n=3). p-values: two-sided t-test with unequal variance.


Changes of OCT4 half - life upon SOX2 - SNAP or YPET - SOX2 - delDBD overexpression ( n = 20 cells each ) . p - values : Mann - Whitney U test . SBROS cells harbouring inducible SOX2 - SNAP or YPET - SOX2 - delDBD were treated with 0 or 500 ng / ml dox for 6 - 8 hours followed by pulse - labelling of OCT4 - HALO and live - cell imaging .

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Changes of OCT4 half-life upon SOX2-SNAP or YPET-SOX2-delDBD overexpression (n=20 cells each). p-values: Mann-Whitney U test. SBROS cells harbouring inducible SOX2-SNAP or YPET-SOX2-delDBD were treated with 0 or 500 ng/ml dox for 6-8 hours followed by pulse-labelling of OCT4-HALO and live-cell imaging.


Sorting strategy to evaluate the impact of endogenous fluctuations of SOX2 and OCT4 on OCT4 and SOX2 protein levels , respectively .

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Sorting strategy to evaluate the impact of endogenous fluctuations of SOX2 and OCT4 on OCT4 and SOX2 protein levels, respectively.


Changes of SOX2 and OCT4 levels after sorting for high and low SOX2 levels in cells expressing intermediate OCT4 levels ( n = 4 ) . Error bars : SE ; p - values : one - sided t - test with unequal variance .

Changes of SOX2 and OCT4 levels after sorting for high and low SOX2 levels in cells expressing intermediate OCT4 levels (n=4). Error bars: SE; p-values: one-sided t-test with unequal variance.


Changes of SOX2 and OCT4 levels after sorting for high and low OCT4 levels in cells expressing intermediate SOX2 levels ( n = 4 ) . p - values : one - sided t - test with unequal variance .

Changes of SOX2 and OCT4 levels after sorting for high and low OCT4 levels in cells expressing intermediate SOX2 levels (n=4). p-values: one-sided t-test with unequal variance.


Representative traces of the absolute number of SOX2 - NLUC molecules ( top ) and inferred concentration in nM ( bottom ) in in - silico synchronized cells ( n = 10 ) . Single cells were ranked according to their SOX2 expression at t = 0 and assigned a color code for their initial rank from low ( blue ) to high ( red ) levels , and changes in ranks over time are shown ( n = 59 ) . Rank - based autocorrelation function of the SOX2 ranks ( n = 59 ) . Error bars : SE estimated by bootstrapping .

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Representative traces of the absolute number of SOX2-NLUC molecules (top) and inferred concentration in nM (bottom) in in-silico synchronized cells (n=10). Single cells were ranked according to their SOX2 expression at t=0 and assigned a color code for their initial rank from low (blue) to high (red) levels, and changes in ranks over time are shown (n=59). Rank-based autocorrelation function of the SOX2 ranks (n=59). Error bars: SE estimated by bootstrapping.


Representative single cell traces of the integrated intensity of OCT4 - HALO in single cells ( top ) and the corresponding inferred concentration ( bottom ) in in - silico synchronized cells ( n = 10 ) . Representation of the ranks as in G . Rank - based autocorrelation function as in I ( n = 47 ) . Error bars : SE estimated by bootstrapping .

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Representative single cell traces of the integrated intensity of OCT4-HALO in single cells (top) and the corresponding inferred concentration (bottom) in in-silico synchronized cells (n=10). Representation of the ranks as in G. Rank-based autocorrelation function as in I (n=47). Error bars: SE estimated by bootstrapping.


Snapshot from luminescence movie of SNSF cells in differentiation . White arrowheads : cells starting to express SOX1 - P2A - FLUC ; Orange arrowheads : cells staying SOX1 - negative throughout the experiment . Red : SOX2 - NLUC , turquoise : Sox1 - P2A - FLUC . Scale bar : 50µm .

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Snapshot from luminescence movie of SNSF cells in differentiation. White arrowheads: cells starting to express SOX1-P2A-FLUC; Orange arrowheads: cells staying SOX1-negative throughout the experiment. Red: SOX2-NLUC, turquoise: Sox1-P2A-FLUC. Scale bar: 50µm.


Single cell traces of SOX2 levels in differentiating cells . Turquoise traces : cells becoming SOX1 - positive in the middle cell cycle , population average is shown in bold black ; Red traces : cells remaining SOX1 - negative throughout the experiment , population average is shown in bold ; Green line : population average FLUC signal in cells that become SOX1 - positive . * p < 0 . 05 determined by two - sided t - test with unequal variance .

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Single cell traces of SOX2 levels in differentiating cells. Turquoise traces: cells becoming SOX1-positive in the middle cell cycle, population average is shown in bold black; Red traces: cells remaining SOX1-negative throughout the experiment, population average is shown in bold; Green line: population average FLUC signal in cells that become SOX1-positive. * p<0.05 determined by two-sided t-test with unequal variance.


Strategies to sort SBROS cell populations with different SOX2 and OCT4 levels . Differentiation outcome for cells sorted for different SOX2 levels ( n = 5 for low and high , n = 4 for medium ) . p - values : two - sided t - test with unequal variance . Differentiation outcome for cells sorted for different OCT4 levels ( n = 4 ) . p - values : two - sided t - test with unequal variance .

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Strategies to sort SBROS cell populations with different SOX2 and OCT4 levels. Differentiation outcome for cells sorted for different SOX2 levels (n=5 for low and high, n=4 for medium). p-values: two-sided t-test with unequal variance. Differentiation outcome for cells sorted for different OCT4 levels (n=4). p-values: two-sided t-test with unequal variance.


Differentiation outcome of uninduced cells or cells induced 12 hours for OCT4 - SNAP overexpression before sorting in G1 phase ( n = 6 ) . p - values : two - sided t - test with unequal variance .

Differentiation outcome of uninduced cells or cells induced 12 hours for OCT4-SNAP overexpression before sorting in G1 phase (n=6). p-values: two-sided t-test with unequal variance.


Sorting strategy for SBROS cells in G1 or S phase and into the four following subpopulations : SOX2 high & OCT4 high ( SHOH ) , SOX2 high & OCT4 low ( SHOL ) , SOX2 low & OCT4 high ( SLOH ) or SOX2 low & OCT4 low ( SLOL ) .

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Sorting strategy for SBROS cells in G1 or S phase and into the four following subpopulations: SOX2 high & OCT4 high (SHOH), SOX2 high & OCT4 low (SHOL), SOX2 low & OCT4 high (SLOH) or SOX2 low & OCT4 low (SLOL).


Differentiation outcome for cells sorted in G1 phase ( n = 5 ) . p - values : two - sided t - test with unequal variance ( SOX1 ) and Mann - Whitney U test ( BRA ) . Differentiation outcome for cells sorted in S phase ( n = 5 ) . p - values : Mann - Whitney U test ( SOX1 ) and two - sided t - test with unequal variance ( BRA ) .

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Differentiation outcome for cells sorted in G1 phase (n=5). p-values: two-sided t-test with unequal variance (SOX1) and Mann-Whitney U test (BRA). Differentiation outcome for cells sorted in S phase (n=5). p-values: Mann-Whitney U test (SOX1) and two-sided t-test with unequal variance (BRA).


Differentiation outcome for cells sorted in G1 phase differentiated in the presence of 3μM CHIR ( n = 7 ) . p - values : two - sided t - test with unequal variance .

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Differentiation outcome for cells sorted in G1 phase differentiated in the presence of 3μM CHIR (n=7). p-values: two-sided t-test with unequal variance.


Differentiation outcome for cells sorted in G1 phase differentiated in the presence of 1 μM SB431542 and 25 ng / ml bFGF ( n = 7 ) . p - values : two - sided t - test with unequal variance .

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Differentiation outcome for cells sorted in G1 phase differentiated in the presence of 1 μM SB431542 and 25 ng/ml bFGF (n=7). p-values: two-sided t-test with unequal variance.


Example tracks of the ATAC - seq signal in SHOH , SHOL , SLOH and SLOL populations for loci where chromatin accessibility is unaffected , affected by OCT4 levels , SOX2 levels , or affected by both .

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Example tracks of the ATAC-seq signal in SHOH, SHOL, SLOH and SLOL populations for loci where chromatin accessibility is unaffected, affected by OCT4 levels, SOX2 levels, or affected by both.


Heatmap of relative chromatin accessibility in SHOH / SHOL / SLOH / SLOL samples for all loci with significantly altered accessibility , with examples of genes close to affected regulatory regions . Groups are defined by their significance ( OHvsOL / SHvsSL / SHOHvsSLOL ) and fold - change direction .

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Heatmap of relative chromatin accessibility in SHOH/SHOL/SLOH/SLOL samples for all loci with significantly altered accessibility, with examples of genes close to affected regulatory regions. Groups are defined by their significance (OHvsOL/SHvsSL/SHOHvsSLOL) and fold-change direction.


Percentage of regions in each group showing an overlap with OCT4 , SOX2 and H3K4me3 ChIP - seq peaks .

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Percentage of regions in each group showing an overlap with OCT4, SOX2 and H3K4me3 ChIP-seq peaks.


B . Comparison of ΔPrP expression levels . Representative immunoblot is shown for detection of ( 3F4 ) MoPrP and ΔPrPs in transiently transfected N2a cells using mAb 3F4 . ΔPrPs are similar in expression level glycosylation appearance , except for Δ159 - 167 which has an extra fragment larger than the diglycoform ( lane 7 , arrow ) .

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B. Comparison of ΔPrP expression levels. Representative immunoblot is shown for detection of (3F4)MoPrP and ΔPrPs in transiently transfected N2a cells using mAb 3F4. ΔPrPs are similar in expression level glycosylation appearance, except for Δ159-167 which has an extra fragment larger than the diglycoform (lane 7, arrow).


C . ΔPrPs are not converted into PK - resistant PrP in prion - infected cells . 22L - ScN2a cells were transiently transfected with ( 3F4 ) MoPrP , Δ159 or Δ159 - 175 , and cell lysates digested with PK or not . Immunoblot was done using mAb 3F4 .

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C. ΔPrPs are not converted into PK-resistant PrP in prion-infected cells. 22L-ScN2a cells were transiently transfected with (3F4)MoPrP, Δ159 or Δ159-175, and cell lysates digested with PK or not. Immunoblot was done using mAb 3F4.