( b ) transcription of poxB over time ( E . coli MG1655 ∆ glnL ) , as calculated from - seq data ( Methods ) . colored bars indicate times at which glucose / acetate sensors will be on on metabolite measurements . It should be noted that these are right - shifted compared to Figure 2 due to slower growth of tested

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(b) The transcription of poxB over time (E. coli MG1655∆glnL), as calculated from RNA-seq data (Methods). The colored bars indicate the times at which the glucose/acetate sensors will be on based on metabolite measurements. It should be noted that these are right-shifted compared to Figure 2 due to slower growth of the tested strains


( c ) production of acetate is shown over time for E . coli MG1655 ∆ glnL ∆ pta poxB : : E170 ( black ) as compared to same containing complete circuit ( green ) . A version of circuit in which sgRNA is targeted to rfp ( not present in system ) is tested in MG1655 ∆ glnL ∆ pta containing an untagged poxB as a control ( red ) . Empty circles connected by dashed represent MG1655 ∆ glnL ∆ pta ∆

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(c) The production of acetate is shown over time for E. coli MG1655∆glnL∆pta poxB::E170 (black) as compared to the same strain containing the complete circuit (green). A version of the circuit in which the sgRNA is targeted to rfp (not present in the system) and is tested in MG1655∆glnL∆pta containing an untagged poxB as a control (red). Empty circles connected by the dashed line represent MG1655∆glnL∆pta∆poxB


( e ) transcription of pta over time ( E . coli MG1655 ∆ glnL ) , as calculated from - seq data ( Methods ) . colored bars indicate times at which glucose / oxygen sensors will be on on metabolite

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(e) The transcription of pta over time (E. coli MG1655∆glnL), as calculated from RNA-seq data (Methods). The colored bars indicate the times at which the glucose/oxygen sensors will be on based on metabolite measurements


( f ) production of acetate is shown over time for E . coli MG1655 ∆ glnL ∆ poxB pta : : pdt3 as compared to a containing complete circuit ( green ) tested in same . A version of circuit in which sgRNA is targeted to rfp ( not present in system ) is tested in MG1655 ∆ glnL ∆ poxB containing an untagged pta as a control ( red ) Empty circles connected by dashed represent MG1655 ∆ glnL ∆ pta ∆

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(f) The production of acetate is shown over time for E. coli MG1655∆glnL∆poxB pta::pdt3 as compared to a strain containing the complete circuit (green) tested in same strain. A version of the circuit in which the sgRNA is targeted to rfp (not present in the system) and is tested in MG1655∆glnL∆poxB containing an untagged pta as a control (red) Empty circles connected by the dashed line represent MG1655∆glnL∆pta∆poxB


BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with

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Primary BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with SM


BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with TAK1i for 6 or 4 h respectively .

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Primary BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with TAK1i for 6 or 4 h respectively.


Time - lapse confocal ( hour : min ) of BMDMs stimulated with recombinant murine TNF ( 100 ng / ml ) SM ( 250 nM ) stained with propidium iodide ( red ) for 6 h . Black arrowheads indicate ballooning while arrowheads indicate apoptotic bodies .

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Time-lapse confocal images (hour:min) of BMDMs stimulated with recombinant murine TNF (100 ng/ml) and SM (250 nM) stained with propidium iodide (red) for 6 h. Black arrowheads indicate membrane ballooning while white arrowheads indicate apoptotic bodies.


immortalized BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with

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immortalized BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with SM


immortalized BMDMs were stimulated with recombinant murine TNF ( 100 ng / ml ) in combination with TAK1i for 6 or 4 h respectively .

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immortalized BMDMs were stimulated with recombinant murine TNF (100 ng/ml) in combination with TAK1i for 6 or 4 h respectively.


BMDMs were stimulated with TNF ( 100 ng / ml ) in combination with TAK1i ( 125 nM ) for indicated time points . LDH

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BMDMs were stimulated with TNF (100 ng/ml) in combination with TAK1i (125 nM) for the indicated time points. LDH release


BMDMs were stimulated with TNF ( 100 ng / ml ) in combination with TAK1i ( 125 nM ) for indicated time points . mixed supernatant extracts were analysed .

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BMDMs were stimulated with TNF (100 ng/ml) in combination with TAK1i (125 nM) for the indicated time points. mixed supernatant and cell extracts were analysed.


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i ( 125 nM ) for 4 h in presence or absence of KCl ( 50 mM ) . Where indicated , were pre - incubated with MCC950 ( 10 μM ) 20 - 30 min prior to TNF / TAK1i stimulation .

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BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h in the presence or absence of KCl (50 mM). Where indicated, cells were pre-incubated with MCC950 (10 μM) 20-30 min prior to TNF/TAK1i stimulation.


BMDMs were costimulated with TNF ( 100 ng / ml ) SM ( 250 nM ; 6 h ) mixed supernatant extracts were analysed by

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BMDMs were costimulated with TNF (100 ng/ml) and SM (250 nM; 6 h) and mixed supernatant and cell extracts were analysed by immunoblot


BMDMs were costimulated with TNF ( 100 ng / ml ) SM LDH release in culture supernatant were quantified at indicated time points .

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BMDMs were costimulated with TNF (100 ng/ml) and SM LDH release in the cell culture supernatant were quantified at the indicated time points.


BMDMs were costimulated with TNF ( 100 ng / ml )

BMDMs were costimulated with TNF (100 ng/ml) and SM


HEK 293T were transfected with doxycycline - inducible DmrB - caspase - 8 indicated GSDMD constructs . were stimulated with doxycycline ( 10 μg / ml ) for 18 h to induce DmrB - caspase - 8 expression exposed to B / B homodimerizer ( 12 . 5 nM ) for another 2 h to activate caspase - 8 . Mixed supernatant extracts were analysed by immunoblot .

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HEK 293T cells were transfected with doxycycline-inducible DmrB-caspase-8 and the indicated GSDMD constructs. Cells were stimulated with doxycycline (10 μg/ml) for 18 h to induce DmrB-caspase-8 expression and exposed to B/B homodimerizer (12.5 nM) for another 2 h to activate caspase-8. Mixed supernatant and extracts were analysed by immunoblot.


Immortalized Gsdmd - / - BDMDs expressing GSDMDWT GSDMDD88A were costimulated with TNF ( 100 ng / ml ) SM for 6 h LDH release was quantified .

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Immortalized Gsdmd-/- BDMDs expressing GSDMDWT and GSDMDD88A were costimulated with TNF (100 ng/ml) and SM for 6 h and LDH release was quantified.


Immortalized Gsdmd - / - BDMDs expressing GSDMDWT GSDMDD88A were primed for 3 h with ultrapure E . coli K12 LPS ( 100 ng / ml ) stimulated with LCL161 ( 1 μM ) for 24 h LDH release was quantified .

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Immortalized Gsdmd-/- BDMDs expressing GSDMDWT and GSDMDD88A were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with LCL161 (1 μM) for 24 h and LDH release was quantified.


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i for 4 h ( D ) mixed supernatant extracts were analysed by

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BMDMs were costimulated with TNF (100 ng/ml) and TAK1i for 4 h and (D) mixed supernatant and extracts were analysed by immunoblot


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i for 4 h LDH release was quantified at indicated time points .

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

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i for 4 h and LDH release was quantified at the indicated time points.


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i ( 125 nM ) for 4 h LDH release was quantified Where indicated , were treated with inhibitors Nec - 1s ( 50 μM )

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BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and LDH release was quantified Where indicated, cells were treated with the inhibitors Nec-1s (50 μM)


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i ( 125 nM ) for 4 h LDH release was

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

BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and LDH release was quantified


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i ( 125 nM ) for 4 h mixed supernatant extracts were analysed by immunoblot . Where indicated , were treated with inhibitors MCC950 ( 10 μM ) , GSK ' 872 ( 1 μM ) , 20 - 30 min prior to stimulation . KCl ( 50 mM ) was added together with TNF TAK1i . To induce necroptosis , BMDMs were primed for 3 h with ultrapure E . coli K12 LPS ( 100 ng / ml ) Q - VD - OPh ( 10 μM ) was added at last 20 - 30 min of priming stimulated with SM ( 500 nM ) for 4 h .

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BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors MCC950 (10 μM), GSK'872 (1 μM), 20-30 min prior to cell stimulation. KCl (50 mM) was added together with TNF and TAK1i. To induce necroptosis, BMDMs were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and Q-VD-OPh (10 μM) was added at the last 20-30 min of priming and stimulated with SM (500 nM) for 4 h.


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i ( 125 nM ) for 4 h mixed supernatant extracts were analysed by immunoblot . Where indicated , were treated with inhibitors GSK ' 872 ( 1 μM ) , 20 - 30 min prior to stimulation .

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BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors GSK'872 (1 μM), 20-30 min prior to cell stimulation.


BMDMs were costimulated with TNF ( 100 ng / ml ) TAK1i ( 125 nM ) for 4 h mixed supernatant extracts were analysed by immunoblot . Where indicated , were treated with inhibitors probenecid ( 1 mM ) 20 - 30 min prior to stimulation . To activate NLRP3 inflammasome , BMDM were primed with ultrapure E . coli K12 LPS ( 100 ng / ml ) for 4 h stimulated with nigericin ( 10 μM ) for 1 h .

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BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors probenecid (1 mM) 20-30 min prior to cell stimulation. To activate the NLRP3 inflammasome, BMDM were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 4 h and stimulated with nigericin (10 μM) for 1 h.


BMDMs were primed for 3 h with ultrapure E . coli K12 LPS ( 100 ng / ml ) stimulated with SM ( 0 . 5 μM ) for a further 4 h . Probenecid ( 1 mM ) was added 20 - 30 min prior to stimulation mixed supernatant extracts were analysed by immunoblot .

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BMDMs were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with SM (0.5 μM) for a further 4 h. Probenecid (1 mM) was added 20-30 min prior to cell stimulation and mixed supernatant and extracts were analysed by immunoblot.


BMDMs were stimulated with TNF ( 100 ng / ml ) SM ( 0 . 5 μM ) for 6 h ( G ) mixed supernatant extracts were analysed by

O O O O B-CONTROLLED_VAR 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

BMDMs were stimulated with TNF (100 ng/ml) and SM (0.5 μM) for 6 h and (G) mixed supernatant and extracts were analysed by immunoblot


BMDMs were stimulated with TNF ( 100 ng / ml ) SM ( 0 . 5 μM ) for 6 h LDH release was quantified .

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BMDMs were stimulated with TNF (100 ng/ml) and SM (0.5 μM) for 6 h and LDH release was quantified.


BMDMs were stimulated with an increasing dose of S63845 in presence of ABT - 737 ( 0 . 5 μM ) LDH release was quantified at 6 h .

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BMDMs were stimulated with an increasing dose of S63845 in the presence of ABT-737 (0.5 μM) and LDH release was quantified at 6 h.


BMDMs were stimulated with ABT - 737 ( 0 . 5 μM ) S63845 ( 0 . 5 μM ) LDH release was

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

BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and LDH release was quantified


BMDMs were stimulated with ABT - 737 ( 0 . 5 μM ) S63845 ( 0 . 5 μM ) mixed supernatant extracts were analysed by immunoblot at 6

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

BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) mixed supernatant and extracts were analysed by immunoblot at 6 h


BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and mixed supernatant and extracts were analysed by immunoblot at 6 h


BMDMs were primed with ultrapure E . coli K12 LPS ( 100 ng / ml ) for 3 h further stimulated with ABT - 737 ( 1 μM ) S63845 ( 1 μM ) for 24 h mixed supernatant extracts were analysed by

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

BMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h and further stimulated with ABT-737 (1 μM) and S63845 (1 μM) for 24 h and mixed supernatant and extracts were analysed by immunoblot


BMDMs were primed with ultrapure E . coli K12 LPS ( 100 ng / ml ) for 3 h further stimulated with ABT - 737 ( 1 μM ) S63845 ( 1 μM ) for 24 h IL - 1β in - free supernatant were quantified by

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BMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h and further stimulated with ABT-737 (1 μM) and S63845 (1 μM) for 24 h and IL-1β in cell-free supernatant were quantified by ELISA


A Ccno mRNA is expressed in embryonic node at E8 as shown by in situ hybridization . Occasionally , additional expression is observed at posterior tip of ( asterisk ) . Double labelling by for LacZ expression from CcnoTA allele ( shown in B ) in situ hybridization for Dand5 , marking crown at circumference of node . Ccno expression is restricted to ciliated , ventral pit of node as detailed in transverse section at indicated plane .

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A Ccno mRNA is expressed in the embryonic node at E8 as shown by in situ hybridization. Occasionally, additional expression is observed at the posterior tip of the embryo (asterisk). Double labelling by staining for LacZ expression from the CcnoTA allele (shown in B) and in situ hybridization for Dand5, marking crown cells at the circumference of the node. Ccno expression is restricted to ciliated, ventral pit cells of the node as detailed in the transverse section at the indicated plane.


C Whole X ‐ Gal of E16 CcnoTA / + wild ‐ type control indicated section planes of ( D - F ' ) .

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C Whole embryo X‐Gal staining of E16 CcnoTA/+ and wild‐type control embryos and indicated section planes of (D-F').


D - F ' Histological sections of LacZ ‐ stained CcnoTA / + at E16 reveal Ccno expression in epithelium of ( D ) plexus choroideus ependyme , ( E ) snout epithelium , ( F , F ' ) trachea bronchi . pc , plexus choroideus ; se , snout epithelium ; tr , trachea ; br , bronchus .

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D-F' Histological sections of LacZ‐stained CcnoTA/+ embryo at E16 reveal Ccno expression in the epithelium of (D) plexus choroideus and ependyme, (E) snout epithelium, and (F, F') trachea and bronchi. pc, plexus choroideus; se, snout epithelium; tr, trachea; and br, bronchus.


G Co ‐ expression of Ccno Foxj1 in multiciliated of trachea shown by double for X ‐ Gal immunohistochemistry using a FOXJ1 ‐ specific antibody .

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G Co‐expression of Ccno and Foxj1 in multiciliated cells of the trachea shown by double staining for X‐Gal and immunohistochemistry using a FOXJ1‐specific antibody.


A Ccno ‐ deficient mice develop severe hydrocephalus resulting in characteristic head deformation at P21 ( arrow ) .

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A Ccno‐deficient mice develop severe hydrocephalus resulting in characteristic head deformation at P21 (arrow).


B , C Cranial MRI ( B ) shows drastically enlarged ventricular cavity ( marked by V ) diminished cerebral cortex tissue , ( C ) also seen when brains are cut in coronal orientation .

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B, C Cranial MRI analysis (B) shows the drastically enlarged ventricular cavity (marked by V) and diminished cerebral cortex tissue, (C) also seen when brains are cut in coronal orientation.


D , D ' Alcian ‐ blue of paranasal cavities reveals mucus congestion along nasal epithelium ( arrows ) .

D, D' Alcian‐blue staining of paranasal cavities reveals mucus congestion along the nasal epithelium (arrows).


F , F ' Scanning electron microscopy ( ) of P21 adult trachea shows reduced numbers of cilia of Ccno ‐ deficient MCCs . Remaining cilia are found in central regions of surface , margins frequently lack ciliary decoration ( arrows ) .

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F, F' Scanning electron microscopy (SEM) of P21 adult trachea shows reduced numbers of cilia of Ccno‐deficient MCCs. Remaining cilia are found in the central regions of the cell surface, and cell margins frequently lack the ciliary decoration (arrows).


G Transmission electron microscopy ( ) of wild ‐ type CcnoRA / RA MCCs from adult trachea . Ccno ‐ deficient MCCs show reduced numbers of basal bodies cilia that correctly docked to apical surface ( arrows ) . Ectopic electron ‐ dense material is found within cytoplasm of Ccno ‐ deficient MCCs ( arrowheads ) .

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G Transmission electron microscopy (TEM) of wild‐type and CcnoRA/RA MCCs from adult trachea. Ccno‐deficient MCCs show reduced numbers of basal bodies and cilia that correctly docked to the apical cell surface (arrows). Ectopic electron‐dense material is found within the cytoplasm of Ccno‐deficient MCCs (arrowheads).


A - H X ‐ Gal ‐ of ( A - C ) control ( D - H ) CcnoTA / + embryonic lungs at indicated stages showing onset of Ccno expression from E13 in proximal trachea in main bronchi . Expression is extending to more distal regions , from E16 is also found in bronchioli . I X ‐ Gal ‐ indicating LacZ expression from CcnoTA / + allele in mTEC cultures after 5 days of differentiation ‐ onset by switching to air - liquid interface ( ALI ) conditions ( ALI d5 ) .

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A-H X‐Gal‐staining of (A-C) control and (D-H) CcnoTA/+ embryonic lungs at indicated stages showing onset of Ccno expression from E13 in the proximal trachea and in the main bronchi. Expression is extending to more distal regions, and from E16 staining is also found in bronchioli.I X‐Gal‐staining indicating LacZ expression from the CcnoTA/+ allele in mTEC cultures after 5 days of differentiation‐onset by switching to air-liquid interface (ALI) conditions (ALI d5).


J Transcript of Ccno indicated genes with known functions for generation of multiple cilia during mTEC differentiation . mRNA from three independent experiments were measured by qRT - PCR of expression set as 1 on day 0 of ALI cultures . Scales for Ccno Deup1 are indicated on left , for Cep152 , Cep63 Ift57 on right side .

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J Transcript levels of Ccno and indicated genes with known functions for the generation of multiple cilia during mTEC differentiation. mRNA levels from three independent experiments were measured by qRT-PCR and levels of expression set as 1 on day 0 of ALI cultures. Scales for Ccno and Deup1 are indicated on the left, and for Cep152, Cep63 and Ift57 on the right side.


K Relative mRNA expression for indicated genes were measured by qRT - PCR in wild ‐ type Ccno ‐ deficient mTEC cultures at indicated days after differentiation ‐ onset . Relative values were calculated as in ( J ) relative to day 0 of ALI cultures .

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K Relative mRNA expression levels for indicated genes were measured by qRT-PCR in wild‐type and Ccno‐deficient mTEC cultures at indicated days after differentiation‐onset. Relative values were calculated as in (J) relative to day 0 of ALI cultures.


A - B ' of tracheae from ( A , A ' ) wild ‐ type ( B , B ' ) CcnoRA / RA at E17 . In comparison to wild ‐ type MCCs , Ccno ‐ deficient MCCs almost completely lack formation of multiple cilia only single , or doublet ( arrow ) cilia are observed at E17 .

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A-B' SEM of tracheae from (A, A') wild‐type and (B, B') CcnoRA/RA embryos at E17. In comparison to wild‐type MCCs, Ccno‐deficient MCCs almost completely lack formation of multiple cilia and only single, or doublet (arrow) cilia are observed at E17.


C - E lack of multiple cilia at E17 stages is reflected by almost complete absence of acetylated α ‐ tubulin as marker for axonemes in ( D ) Ccno ‐ deficient MCCs of E17 embryonic trachea as quantified in ( E ) . FOV , field of view .

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C-E The lack of multiple cilia at E17 stages is reflected by the almost complete absence of acetylated α‐tubulin staining as marker for axonemes in (D) Ccno‐deficient MCCs of E17 embryonic trachea as quantified in (E). FOV, field of view.


F - I of MCCs from E17 embryonic tracheae shows deuterosomes with forming procentrioles in ( F , F ' , H ) Ccno ‐ proficient ( G , G ' , I ) Ccno ‐ deficient MCCs . Asterisks in ( F , G ) indicate microvilli that extend from apical surface . ( F ' , G ' ) Deuterosomes in Ccno ‐ deficient MCCs are significantly enlarged ( identical bars in F ' G ' ) show irregular , which is different to annular shape of wild ‐ type deuterosomes as seen in ( F ' , H ) . Procentrioles ( arrowheads in F ' I ) that are found at deuterosomes of ( I ) Ccno ‐ deficient MCCs were recurrently found being shorter appeared less structured in comparison to ( F ' , H ) wild ‐ type .

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F-I TEM of MCCs from E17 embryonic tracheae shows deuterosomes with forming procentrioles in (F, F', H) Ccno‐proficient and (G, G', I) Ccno‐deficient MCCs. Asterisks in (F, G) indicate microvilli that extend from the apical cell surface. (F', G') Deuterosomes in Ccno‐deficient MCCs are significantly enlarged (identical size bars in F' and G') and show irregular morphology, which is different to the annular shape of wild‐type deuterosomes as seen in (F', H). Procentrioles (arrowheads in F' and I) that are found at deuterosomes of (I) Ccno‐deficient MCCs were recurrently found being shorter and appeared less structured in comparison to (F', H) wild‐type cells.


J average of procentrioles was quantified for wild ‐ type Ccno ‐ deficient MCCs of E17 tracheae ( 49 procentrioles in n = 3 independent samples for wild ‐ types 47 procentrioles in n = 3 independent samples for Ccno ‐ deficient MCCs ) . K diameter of deuterosomes ( 41 deuterosomes in n = 3 independent samples for wild ‐ types ; 38 deuterosomes in n = 5 independent samples for Ccno ‐ deficient MCCs ) was measured in wild ‐ type Ccno ‐ deficient MCCs of E17 tracheae .

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J The average length of procentrioles was quantified for wild‐type and Ccno‐deficient MCCs of E17 tracheae (49 procentrioles in n = 3 independent samples for wild‐types and 47 procentrioles in n = 3 independent samples for Ccno‐deficient MCCs).K The diameter of deuterosomes (41 deuterosomes in n = 3 independent samples for wild‐types; 38 deuterosomes in n = 5 independent samples for Ccno‐deficient MCCs) was measured in wild‐type and Ccno‐deficient MCCs of E17 tracheae.


Double IF of mTEC cultures at early stage of MCC differentiation centriole amplification ( ALI day 3 ) using antibodies for deuterosome DEUP1 early centriole marker SAS ‐ 6 . Deuterosome numbers are decreased deuterosome enlarged in Ccno ‐ deficient MCCs as quantified in ( C ) . Deuterosome ‐ dependent centriole biogenesis is severely reduced in Ccno ‐ deficient MCCs as shown by reduced for early centriole marker SAS ‐ 6 .

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Double IF analysis of mTEC cultures at early stage of MCC differentiation and centriole amplification (ALI day 3) using antibodies for the deuterosome protein DEUP1 and the early centriole marker SAS‐6. Deuterosome numbers are decreased and deuterosome size enlarged in Ccno‐deficient MCCs as quantified in (C). Deuterosome‐dependent centriole biogenesis is severely reduced in Ccno‐deficient MCCs as shown by reduced staining for the early centriole marker SAS‐6.


IFstaining for DEUP1 reveals a further increase of deuterosomes of Ccno ‐ deficient MCCs at later stages of centriole biogenesis ( ALI day 7 ) . Deuterosome number were quantified in ( C ) . IF using a Centrin antibody shows globally reduced presence of centrioles at ALI day 7 , which stay localized below level of deuterosomes fail to dock to apical cellmembrane in Ccno ‐ deficient MCCs as seen in z ‐ projection .

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IFstaining for DEUP1 reveals a further size increase of deuterosomes of Ccno‐deficient MCCs at later stages of centriole biogenesis (ALI day 7). Deuterosome number and size were quantified in (C). IF using a Centrin antibody shows globally reduced presence of centrioles at ALI day 7, which stay localized below the level of deuterosomes and fail to dock to the apical cellmembrane in Ccno‐deficient MCCs as seen in z‐projection.


IFstaining for DEUP1 in ( A , B ) was analysed by Imaris 7 . 7 . 2 software to calculate average deuterosome number of wild ‐ type ( n = 17 ) Ccno ‐ deficient ( n = 19 ) MCCs at ALI day 3 , wild ‐ type ( n = 12 ) Ccno ‐ deficient ( n = 14 ) MCCs at ALI day 7 . Deuterosome was measured using spot measurement tool in n = 10 MCCs for both genotypes at ALI day 3 , in wild ‐ type ( n = 12 ) Ccno ‐ deficient ( n = 14 ) MCCs at ALI day 7 .

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IFstaining for DEUP1 in (A, B) was analysed by Imaris 7.7.2 software to calculate average deuterosome number of wild‐type (n = 17) and Ccno‐deficient (n = 19) MCCs at ALI day 3, and wild‐type (n = 12) and Ccno‐deficient (n = 14) MCCs at ALI day 7. Deuterosome size was measured using the spot measurement tool in n = 10 MCCs for both genotypes at ALI day 3, and in wild‐type (n = 12) and Ccno‐deficient (n = 14) MCCs at ALI day 7.


A , B IF of mTEC cultures at ALI day 7 using antibodies against centriole marker Centrin CEP164 , which marks maturing centrioles . At ALI day 7 , centrioles of wild ‐ type MCCs are decorated with CEP164 are mostly localized to apical cellmembrane . fewer centrioles of Ccno ‐ deficient MCCs do not colocalize with CEP164 fail to reach apical surface as seen in z ‐ projection . Lack of CEP164 indicates immature centrioles in Ccno ‐ deficient MCCs . ( B ) Total centriole numbers per MCC were counted in n = 11 MCCs for both genotypes using Imaris 7 . 7 . 2 software .

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A, B IF analysis of mTEC cultures at ALI day 7 using antibodies against the centriole marker Centrin and CEP164, which marks maturing centrioles. At ALI day 7, centrioles of wild‐type MCCs are decorated with CEP164 and are mostly localized to the apical cellmembrane. The fewer centrioles of Ccno‐deficient MCCs do not colocalize with CEP164 and fail to reach the apical cell surface as seen in z‐projection. Lack of CEP164 indicates immature centrioles in Ccno‐deficient MCCs. (B) Total centriole numbers per MCC were counted in n = 11 MCCs for both genotypes using Imaris 7.7.2 software.


C , D Double IFstaining using Centrin ‐ CP110 ‐ specific antibodies shows an almost complete absence of CP110 from Centrin ‐ positive centrioles in majority of wild ‐ type MCCs at day 7 of ALI cultures . Most Ccno ‐ deficient MCCs exhibit pronounced CP110 that colocalizes with Centrin as seen in z ‐ projection . ( D ) CP110 ‐ positive MCCs were counted in wild ‐ type Ccno ‐ deficient MTEC cultures percentages of CP110 ‐ positive MCCs from total MCCs calculated .

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C, D Double IFstaining using Centrin‐ and CP110‐specific antibodies shows an almost complete absence of CP110 from Centrin‐positive centrioles in the majority of wild‐type MCCs at day 7 of ALI cultures. Most Ccno‐deficient MCCs exhibit pronounced CP110 staining that colocalizes with Centrin as seen in z‐projection. (D) CP110‐positive MCCs were counted in wild‐type and Ccno‐deficient MTEC cultures and the percentages of CP110‐positive MCCs from total MCCs calculated.


( A ) Representative whole mount of WT Cep63T / T brains at P60 . Scale bar = 0 . 2 cm . ( B ) Telencephalon of WT Cep63T / T brains . WT N = 4 , Cep63T / T N = 7 ; two - Mann - Whitney t - .

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(A) Representative whole mount images of WT and Cep63T/T brains at P60. Scale bar = 0.2 cm. (B) Telencephalon area of WT and Cep63T/T brains. WT littermates N = 4, Cep63T/T N = 7; two-tailed Mann-Whitney t-test.


( C ) Representative whole mount of WT Sas4cKO brains at P14 . Scale bar : 0 . 2 cm . ( D ) Telencephalon of WT Sas4cKO brains . WT N = 8 , Sas4cKO N = 7 ; two - Mann - Whitney t - . Black circles represent Nestin - Cre + .

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(C) Representative whole mount images of WT and Sas4cKO brains at P14. Scale bar: 0.2 cm. (D) Telencephalon area of WT and Sas4cKO brains. WT littermates N = 8, Sas4cKO N = 7; two-tailed Mann-Whitney t-test. Black circles represent Nestin-Cre+ animals.


( E ) Representative of E14 . 5 WT , Cep63T / T Sas4cKO cortices stained with antibodies against α - Tubulin ( green ) , γ - Tubulin ( red ) DAPI ( blue ) . Magenta arrows indicate dead ; yellow arrows indicate with bipolar monopolar spindles , respectively . Insets showing zoomed in view of 2 representative . Dashed lines showing orientation of cleavage plane relative to ventricular surface . Scale bar = 25 μm . ( F ) Graph showing of bipolar , monopolar multipolar at ventricular surface of E14 . 5 cortices . WT n = 201 , N = 3 ; Cep63T / T n = 312 , N = 3 ; Sas4cKO n = 296 , N = 3 ; # , chi - square with * , post - hoc , comparisons are made to WT .

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(E) Representative images of E14.5 WT, Cep63T/T and Sas4cKO cortices stained with antibodies against α-Tubulin (green), γ-Tubulin (red) and DAPI (blue). Magenta arrows indicate dead cells; white and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 25 μm. (F) Graph showing the percentage of bipolar, monopolar and multipolar cells at the ventricular surface of E14.5 cortices. WT n = 201 cells, N = 3 embryos; Cep63T/T n = 312 cells, N = 3 embryos; Sas4cKO n = 296 cells, N = 3 embryos; #, chi-square test with *, post-hoc analysis, comparisons are made to WT.


( E ) Representative of E14 . 5 WT , Cep63T / T Sas4cKO cortices stained with antibodies against α - Tubulin ( green ) , γ - Tubulin ( red ) DAPI ( blue ) . Magenta arrows indicate dead ; yellow arrows indicate with bipolar monopolar spindles , respectively . Insets showing zoomed in view of 2 representative . Dashed lines showing orientation of cleavage plane relative to ventricular surface . Scale bar = 25 μm .

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(E) Representative images of E14.5 WT, Cep63T/T and Sas4cKO cortices stained with antibodies against α-Tubulin (green), γ-Tubulin (red) and DAPI (blue). Magenta arrows indicate dead cells; white and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 25 μm.


( G ) E14 . 5 cortices stained with antibodies against Ki67 ( red ) phosphorylated - Histone H3 ( PH3 , yellow ) . Insets showing zoomed in view of in ventricular zone ( VZ ) sub - ventricular zone ( SVZ ) . Scale bar = 100 μm .

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(G) E14.5 cortices stained with antibodies against Ki67 (red) and phosphorylated-Histone H3 (PH3, yellow). Insets showing zoomed in view of cells in the ventricular zone (VZ) and sub-ventricular zone (SVZ). Scale bar = 100 μm.


( H ) of mitotic ( PH3 + ) to cycling ( Ki67 + ) in E14 . 5 cortices . WT N = 5 , Cep63T / T N = 3 , Sas4cKO N = 3 ; two - Mann - Whitney t - .

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(H) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Cep63T/T N = 3, Sas4cKO N = 3; two-tailed Mann-Whitney t-test.


( J ) Representative from live - movies of - mCherry + WT , Cep63T / T Sas4cKO NPCs . timing of mitosis began at envelope breakdown ( NEBD ) finished at envelope reformation . arrows track mitotic events of mother from NEBD to generation of daughter following cytokinesis . ( K ) Plot showing duration of mitosis in WT , Cep63T / T Sas4cKO NPCs . Triangles represent ; triangles of same color represent derived from same . Circles represent average mitotic duration of from each . WT n = 202 , N = 4 ; Cep63T / T n = 141 , N = 3 ; Sas4cKO n = 138 , N = 3 ; two - Welch ' s t - .

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(J) Representative images from live-cell movies of Nuclear-mCherry+ WT, Cep63T/T and Sas4cKO NPCs. The timing of mitosis began at nuclear envelope breakdown (NEBD) and finished at nuclear envelope reformation. White arrows track the mitotic events of the mother cell from NEBD to the generation of daughter cells following cytokinesis. (K) Plot showing the duration of mitosis in WT, Cep63T/T and Sas4cKO NPCs. Triangles represent individual cells; triangles of the same color represent cells derived from the same embryo. Circles represent the average mitotic duration of cells from each embryo. WT n = 202 cells, N = 4 embryos; Cep63T/T n = 141 cells, N = 3 embryos; Sas4cKO n = 138 cells, N = 3 embryos; two-tailed Welch's t-test.


( A - C ) Graph showing mitotic duration of NPCs fate of their progeny in cultures generated from E14 . 5 . Each bar represents one ; its height represents amount of time mother spent in mitosis ; bar color represents fate of progeny . Dashed is set at 30 mins . WT n = 330 , N = 4 ; Cep63T / T n = 260 , N = 3 ; Sas4cKO n = 223 , N = 3 . ( D - F ) of proliferating , arrested apoptotic progeny within each group of indicated mitotic duration , calculated from data shown in A - C ; groups with n < 20 were excluded from this ; # , chi - square with * , post - hoc , within each genotype , comparisons are made to 0 - 30min group .

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(A-C) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 mins. WT n = 330 cells, N = 4 embryos; Cep63T/T n = 260 cells, N = 3 embryos; Sas4cKO n = 223 cells, N = 3 embryos. (D-F) Percentage of proliferating, arrested and apoptotic progeny within each group of the indicated mitotic duration, calculated from data shown in A-C; groups with n < 20 cells were excluded from this quantification; #, chi-square test with *, post-hoc analysis, within each genotype, comparisons are made to the 0-30min group.


( G ) Genome - wide copy number plots of single sequenced from dissociated dorsolateral telencephalons of E14 . 5 . are represented in rows with copy number states indicated by colors ; arrows on zoomed in view ( right ) indicate aneuploid . WT n = 38 , N = 2 ; Cep63T / T n = 40 , N = 2 ; Sas4cKO n = 42 , N = 2 . ( H ) of aneuploid from single - sequencing data shown in G .

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(G) Genome-wide copy number plots of single cells sequenced from dissociated dorsolateral telencephalons of E14.5 embryos. Individual cells are represented in rows with copy number states indicated by colors; arrows on the zoomed in view (right) indicate aneuploid cells. WT n = 38 cells, N = 2 embryos; Cep63T/T n = 40 cells, N = 2 embryos; Sas4cKO n = 42 cells, N = 2 embryos. (H) Percentage of aneuploid cells from the single-cell sequencing data shown in G.


( A ) Representative whole mount of WT , Sas4cKO , Sas4cKO ; Trp53bp1 - / - , Sas4cKO ; Usp28cKO at P14 . Arrows indicate cerebellar hypoplasia resulting from lack of cilia . Scale bar = 0 . 2 cm . ( B ) Telencephalon of P14 brains of indicated genotypes . WT N = 8 , Sas4cKO N = 7 , Sas4cKO ; Trp53bp1 - / - N = 6 , Sas4cKO ; Usp28cKO N = 10 , Sas4cKO ; Trp53 - / - N = 5 ; one - way ANOVA with post - hoc . WT Sas4cKO data are from Figure 1D shown alongside for comparison . Black circles represent Nestin - Cre + . ( C ) Cortical thickness of P14 brains of indicated genotypes . WT N = 4 , Sas4cKO N = 4 , Sas4cKO ; Trp53bp1 - / - N = 4 , Sas4cKO ; Usp28cKO N = 4 , Sas4cKO ; Trp53 - / - N = 4 ; one - way ANOVA with post - hoc . WT Sas4cKO data are from Figure EV1D shown alongside for comparison . Black circles represent Nestin - Cre + .

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(A) Representative whole mount images of WT, Sas4cKO, Sas4cKO;Trp53bp1-/-, Sas4cKO;Usp28cKO animals at P14. Arrows indicate the cerebellar hypoplasia resulting from the lack of primary cilia. Scale bar = 0.2 cm. (B) Telencephalon area of P14 brains of the indicated genotypes. WT littermates N = 8, Sas4cKO N = 7, Sas4cKO;Trp53bp1-/- N = 6, Sas4cKO;Usp28cKO N = 10, Sas4cKO;Trp53-/- N = 5; one-way ANOVA with post-hoc analysis. WT and Sas4cKO data are from Figure 1D and shown alongside for comparison. Black circles represent Nestin-Cre+ animals. (C) Cortical thickness of P14 brains of the indicated genotypes. WT littermates N = 4, Sas4cKO N = 4, Sas4cKO;Trp53bp1-/- N = 4, Sas4cKO;Usp28cKO N = 4, Sas4cKO;Trp53-/- N = 4; one-way ANOVA with post-hoc analysis. WT and Sas4cKO data are from Figure EV1D and shown alongside for comparison. Black circles represent Nestin-Cre+ animals.


( D ) Representative whole mount of WT , Cep63T / T , Cep63T / T ; Trp53bp1 - / - , Cep63T / T ; Usp28 - / - at P60 . Scale bar = 0 . 2 cm . ( E ) Telencephalon of P60 brains of indicated genotypes . WT N = 4 , Cep63T / T N = 7 , Cep63T / T ; Trp53bp1 - / - N = 6 , Cep63T / T ; Usp28 - / - N = 5 ; one - way ANOVA with post - hoc . WT Cep63T / T data are from Figure 1B shown alongside for comparison . ( F ) Cortical thickness of P60 brains of indicated genotypes . WT N = 4 , Cep63T / T N = 5 , Cep63T / T ; Trp53bp1 - / - N = 3 , Cep63T / T ; Usp28 - / - N = 3 ; one - way ANOVA with post - hoc . WT Cep63T / T data are from Figure EV1B shown alongside for comparison .

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

(D) Representative whole mount images of WT, Cep63T/T, Cep63T/T;Trp53bp1-/-, Cep63T/T ;Usp28-/- animals at P60. Scale bar = 0.2 cm. (E) Telencephalon area of P60 brains of the indicated genotypes. WT littermates N = 4, Cep63T/T N = 7, Cep63T/T;Trp53bp1-/- N = 6, Cep63T/T;Usp28-/- N = 5; one-way ANOVA with post-hoc analysis. WT and Cep63T/T data are from Figure 1B and shown alongside for comparison. (F) Cortical thickness of P60 brains of the indicated genotypes. WT littermates N = 4, Cep63T/T N = 5, Cep63T/T;Trp53bp1-/- N = 3, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis. WT and Cep63T/T data are from Figure EV1B and shown alongside for comparison.


( G ) of P7 of indicated genotypes . WT N = 18 , Cep63T / T N = 16 , Cep63T / T ; Trp53bp1 - / - N = 2 , Cep63T / T ; Usp28 - / - N = 10 ; one - way ANOVA with post - hoc .

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(G) Body weight of P7 animals of the indicated genotypes. WT littermates N=18, Cep63T/T N=16, Cep63T/T;Trp53bp1-/- N=2, Cep63T/T;Usp28-/- N=10; one-way ANOVA with post-hoc analysis.


( H ) of total number of within a 500 μm - width column of cortices at P60 . WT N = 4 , Cep63T / T N = 4 , Cep63T / T ; Trp53bp1 - / - N = 4 , Cep63T / T ; Usp28 - / - N = 3 ; one - way ANOVA with post - hoc .

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(H) Quantification of total number of cells within a 500 μm-width column of cortices at P60. WT littermates N = 4, Cep63T/T N = 4, Cep63T/T;Trp53bp1-/- N = 4, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis .


( I - J ) of number of in ( I ) superficial layer ( CUX1 + ) ( J ) deep layer ( CTIP2 + ) within a 500 μm - width column of P60 cortices . WT N = 3 , Cep63T / T N = 4 , Cep63T / T ; Trp53bp1 - / - N = 4 , Cep63T / T ; Usp28 - / - N = 3 ; one - way ANOVA with post - hoc .

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

(I-J) Quantification of the number of cells in the (I) superficial layer (CUX1+) and (J) deep layer (CTIP2+) within a 500 μm-width column of P60 cortices. WT littermates N = 3, Cep63T/T N = 4, Cep63T/T;Trp53bp1-/- N = 4, Cep63T/T;Usp28-/- N = 3; one-way ANOVA with post-hoc analysis .


( K ) P60 cortices of indicated genotypes stained with antibodies against deep layer marker CTIP2 ( green ) , superficial layer marker CUX1 ( red ) DAPI ( blue ) . Scale bar = 200 μm .

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(K) P60 cortices of indicated genotypes stained with antibodies against the deep layer marker CTIP2 (green), superficial layer marker CUX1 (red) and DAPI (blue). Scale bar = 200 μm.


( A ) Cortices from E14 . 5 brains stained with antibodies against radial glial marker PAX6 ( yellow ) , intermediate progenitor marker TBR2 ( red ) DAPI ( blue ) . Scale bar = 50 μm . ( B C ) of number of ( B ) radial glial ( PAX6 + ) ( C ) intermediate progenitors ( TBR2 + ) within a 250 μm - width column of E14 . 5 cortices . WT N = 3 , Cep63T / T N = 3 , Cep63T / T ; Usp28 - / - N = 3 , Sas4cKO N = 3 , Sas4cKO ; Usp28cKO N = 3 ; one - way ANOVA with post - hoc .

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

(A) Cortices from E14.5 brains stained with antibodies against the radial glial cell marker PAX6 (yellow), intermediate progenitor marker TBR2 (red) and DAPI (blue). Scale bar = 50 μm. (B and C) Quantification of the number of (B) radial glial cells (PAX6+) and (C) intermediate progenitors (TBR2+) within a 250 μm-width column of E14.5 cortices. WT N=3, Cep63T/T N = 3, Cep63T/T;Usp28-/- N = 3, Sas4cKO N = 3, Sas4cKO;Usp28cKO N = 3; one-way ANOVA with post-hoc analysis.


( D ) Cortices from E14 . 5 stained with antibodies against - Caspase 3 ( CC3 , ) , TP53 ( red ) DAPI . Scale bar = 50 μm . ( E F ) of number of CC3 + apoptotic ( E ) with TP53 activation ( F ) within a 250 μm - width column of E14 . 5 cortices . WT N = 5 , Cep63T / T N = 3 , Cep63T / T ; Usp28 - / - N = 3 , Sas4cKO N = 4 , Sas4cKO ; Usp28cKO N = 4 ; one - way ANOVA with post - hoc .

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

(D) Cortices from E14.5 embryos stained with antibodies against Cleaved-Caspase 3 (CC3, white), TP53 (red) and DAPI. Scale bar = 50 μm. (E and F) Quantification of the number of CC3+ apoptotic cells (E) and cells with TP53 activation (F) within a 250 μm-width column of E14.5 cortices. WT N = 5, Cep63T/T N = 3, Cep63T/T;Usp28-/- N = 3, Sas4cKO N = 4, Sas4cKO;Usp28cKO N = 4; one-way ANOVA with post-hoc analysis.


( A ) Representative of E14 . 5 cortices stained with antibodies against Centrin ( green ) , γ - Tubulin ( red ) DAPI ( blue ) . yellow arrows indicate with bipolar monopolar spindles , respectively . Insets showing zoomed in view of 2 representative . Dashed lines showing orientation of cleavage plane relative to ventricular surface . Scale bar = 10 μm .

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(A) Representative images of E14.5 cortices stained with antibodies against Centrin (green), γ-Tubulin (red) and DAPI (blue). White and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 10 μm.


( B ) of bipolar , monopolar multipolar at ventricular surface of E14 . 5 cortices . WT n = 201 , N = 3 ; Cep63T / T ; Usp28 - / - n = 205 , N = 3 ; Sas4cKO ; Usp28cKO n = 308 , N = 3 ; # , chi - square with * , post - hoc , comparisons are made to WT . WT data are from Figure 1F shown alongside for comparison .

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(B) Percentage of bipolar, monopolar and multipolar cells at the ventricular surface of E14.5 cortices. WT n = 201 cells, N = 3 embryos; Cep63T/T;Usp28-/- n = 205 cells, N = 3 embryos; Sas4cKO;Usp28cKO n = 308 cells, N = 3 embryos; #, chi-square test with *, post-hoc analysis, comparisons are made to WT. WT data are from Figure 1F and shown alongside for comparison.


( C ) of mitotic ( PH3 + ) to cycling ( Ki67 + ) in E14 . 5 cortices . WT N = 5 , Cep63T / T ; Usp28 - / - N = 3 , Sas4cKO ; Usp28cKO N = 3 ; two - Mann - Whitney t - . 3 WT data points are from Figure 1H shown alongside for comparison .

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(C) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Cep63T/T;Usp28-/- N = 3, Sas4cKO;Usp28cKO N = 3; two-tailed Mann-Whitney t-test. 3 WT data points are from Figure 1H and shown alongside for comparison.


( D ) Representative from live - movies of - mCherry + WT , Sas4cKO Sas4cKO ; Usp28cKO NPCs . arrows showing nuclei of daughter following cytokinesis . ( E ) Plot showing of mitotic duration of WT , Sas4cKO Sas4cKO ; Usp28cKO NPCs . Triangles represent ; triangles of same color represent derived from same . Circles represent average mitotic duration of from each . WT n = 202 , N = 4 ; Sas4cKO n = 138 , N = 3 ; Sas4cKO ; Usp28cKO n = 191 , N = 3 ; two - Welch ' s t - . WT Sas4cKO data are from Figure 1K shown alongside for comparison . ( F ) Graph showing mitotic duration of NPCs fate of their progeny in cultures generated from Sas4cKO ; Usp28cKO E14 . 5 . Each bar represents one ; its height represents amount of time mother spent in mitosis ; bar color represents fate of progeny . Dashed is set at 30 min . Sas4cKO ; Usp28cKO n = 271 , N = 3 . ( G ) of proliferating , arrested apoptotic progeny within each group of indicated mitotic duration , calculated from data shown in F ; # , chi - square with * , post - hoc , comparisons are made to 0 - 30min group .

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

(D) Representative images from live-cell movies of Nuclear-mCherry+ WT, Sas4cKO and Sas4cKO;Usp28cKO NPCs. White arrows showing the nuclei of daughter cells following cytokinesis. (E) Plot showing quantification of mitotic duration of WT, Sas4cKO and Sas4cKO;Usp28cKO NPCs. Triangles represent individual cells; triangles of the same color represent cells derived from the same embryo. Circles represent the average mitotic duration of cells from each embryo. WT n = 202 cells, N = 4 embryos; Sas4cKO n = 138 cells, N = 3 embryos; Sas4cKO;Usp28cKO n = 191 cells, N = 3 embryos; two-tailed Welch's t-test. WT and Sas4cKO data are from Figure 1K and shown alongside for comparison. (F) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO;Usp28cKO E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 min. Sas4cKO;Usp28cKO n = 271 cells, N = 3 embryos. (G) Percentage of proliferating, arrested and apoptotic progeny within each group of the indicated mitotic duration, calculated from data shown in F; #, chi-square test with *, post-hoc analysis, comparisons are made to the 0-30min group.


Graph showing mitotic duration of NPCs fate of their progeny in cultures generated from Sas4cKO E14 . 5 . Each bar represents one ; its height represents amount of time mother spent in mitosis ; bar color represents fate of progeny . Dashed is set at 30 min . ( H I ) Data from Figures 2C 2F shown alongside for comparison with F G .

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

Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from Sas4cKO E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 min. (H and I) Data from Figures 2C and 2F shown alongside for comparison with F and G.


( a ) WT Smc5cKO cortices at E14 . 5 stained with antibodies against Centrin ( green ) , γ - Tubulin ( red ) DAPI ( blue ) . Insets showing zoomed in view of 2 representative . Scale bar : 25 μm . ( B C ) of number of ( B ) Centrin foci ( C ) γ - Tubulin foci in mitotic in VZ of E14 . 5 cortices . WT n = 175 , N = 4 ; Smc5cKO n = 134 , N = 3 . # , chi - square . WT data are from Figures EV1F EV1G shown alongside for comparison .

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(a) WT and Smc5cKO cortices at E14.5 stained with antibodies against Centrin (green), γ-Tubulin (red) and DAPI (blue). Insets showing zoomed in view of 2 representative cells. Scale bar: 25 μm. (B and C) Quantification of the number of (B) Centrin foci and (C) γ-Tubulin foci in mitotic cells in the VZ of E14.5 cortices. WT n = 175 cells, N = 4 embryos; Smc5cKO n = 134 cells, N = 3 embryos. #, chi-square test. WT data are from Figures EV1F and EV1G and shown alongside for comparison.


( D ) E14 . 5 cortices stained with antibodies against Ki67 ( red ) phosphorylated - Histone H3 ( PH3 , yellow ) . Insets showing zoomed in view of in VZ SVZ . Scale bar = 100 μm .

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(D) E14.5 cortices stained with antibodies against Ki67 (red) and phosphorylated-Histone H3 (PH3, yellow). Insets showing zoomed in view of cells in the VZ and SVZ. Scale bar = 100 μm.


( E ) of mitotic ( PH3 + ) to cycling ( Ki67 + ) in E14 . 5 cortices . WT N = 5 , Smc5cKO N = 5 ; two - Mann - Whitney t - . WT data are from Figure 1H shown alongside for comparison .

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(E) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Smc5cKO N = 5; two-tailed Mann-Whitney t-test. WT data are from Figure 1H and shown alongside for comparison.


( F of number of ( F ) apoptotic ( CC3 + ) within a 250 μm - width column of E14 . 5 cortices . WT N = 5 , Smc5cKO N = 4 ; two - Mann - Whitney t - . WT data are from Figures 4E 4G shown alongside for comparison .

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(F Quantification of the number of (F) apoptotic cells (CC3+) within a 250 μm-width column of E14.5 cortices. WT N = 5, Smc5cKO N = 4; two-tailed Mann-Whitney t-test. WT data are from Figures 4E and 4G and shown alongside for comparison.


G ) of number of ( G ) with TP53 activation within a 250 μm - width column of E14 . 5 cortices . WT N = 5 , Smc5cKO N = 4 ; two - Mann - Whitney t - . WT data are from Figures 4E 4G shown alongside for comparison .

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G) Quantification of the number of (G) cells with TP53 activation within a 250 μm-width column of E14.5 cortices. WT N = 5, Smc5cKO N = 4; two-tailed Mann-Whitney t-test. WT data are from Figures 4E and 4G and shown alongside for comparison.


( H ) Representative whole mount of WT , Smc5cKO , Smc5cKO ; Usp28cKO , Smc5cKO ; Trp53 - / - brains at P21 . Scale bar = 0 . 2 cm . ( I ) Telencephalon of P21 brains of indicated genotypes . WT N = 6 , Smc5cKO N = 5 , Smc5cKO ; Usp28cKO N = 4 , Smc5cKO ; Trp53 - / - N = 5 ; one - way ANOVA with post - hoc .

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(H) Representative whole mount images of WT, Smc5cKO, Smc5cKO;Usp28cKO, Smc5cKO;Trp53-/- brains at P21. Scale bar = 0.2 cm. (I) Telencephalon area of P21 brains of the indicated genotypes. WT littermates N = 6, Smc5cKO N = 5, Smc5cKO;Usp28cKO N = 4, Smc5cKO;Trp53-/- N = 5; one-way ANOVA with post-hoc analysis.


A . Total lysates from wild type ( WT ) HAT1 knockout ( KO ) Clone 1 HepG2 were analyzed for of Ksucc Kac by Western blot ( left ) . expression of HAT1 , Histone H3 ( H3 ) , beta - actin ( β - actin ) were measured by Western blot in ( right ) .

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

A. Total cell lysates from wild type (WT) and HAT1 knockout (KO) Clone 1 HepG2 cells were analyzed for the levels of Ksucc and Kac by Western blot analysis (left). The expression levels of HAT1, Histone H3 (H3), and beta-actin (β-actin) were measured by Western blot analysis in the cells (right).


D . HAT1 - catalyzed histone H3 succinylation was analyzed by mixing purified HAT1 , histone H3 , succinyl - CoA ( 2 μM ) with or without addition of indicated concentrations of CoA . Western blot was performed with indicated antibodies .

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

D. HAT1-catalyzed histone H3 succinylation was analyzed by mixing purified HAT1, histone H3, and succinyl-CoA (2 μM) with or without the addition of the indicated concentrations of CoA. Western blot analysis was performed with indicated antibodies.


E . HAT1 - mediated histone H3 succinylation was assessed by mixing purified HAT1 , histone H3 , succinyl - CoA ( 2 μM ) with or without acetyl - CoA ( 2 μM ) . Western blot was performed with indicated antibodies .

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

E. HAT1-mediated histone H3 succinylation was assessed by mixing purified HAT1, histone H3, and succinyl-CoA (2 μM) with or without acetyl-CoA (2 μM). Western blot analysis was performed with the indicated antibodies.


A . of histone H3K122 succinylation , histone H3K122 acetylation , histone H3K27 acetylation , histone H3 , HAT1 , β - actin were examined by Western blot in HepG2 , HAT1 knockout ( KO ) Clone 1 HepG2 , in HAT1 KO reconstituted with either HAT1 or mutant HAT1 ( T188A ) .

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A. The levels of histone H3K122 succinylation, histone H3K122 acetylation, histone H3K27 acetylation, histone H3, HAT1, and β-actin were examined by Western blot analysis in HepG2, HAT1 knockout (KO) Clone 1 HepG2 cells, and in HAT1 KO cells reconstituted with either HAT1 or mutant HAT1 (T188A).


D . effect of HAT1 on histone H3K122 succinylation histone H3K122 acetylation at indicated gene promoters was determined by ChIP assays with anti - H3K122 succinylation antibody anti - H3K122 acetylation antibody , followed by quantitative PCR with primers of promoter regions of CREBBP , BPTF RPTOR in WT HepG2 , HAT1 KO HepG2 HAT1 KO HepG2 reconstituted with either wildtype HAT1 or mutant HAT1 ( T188A ) . N = 3 biological replicates . Data were presented as mean ± SD . Student ' s t - , * * * P < 0 . 001 ; ns , no significance .

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

D. The effect of HAT1 on histone H3K122 succinylation and histone H3K122 acetylation at the indicated gene promoters was determined by ChIP assays with anti-H3K122 succinylation antibody and anti-H3K122 acetylation antibody, and followed by quantitative PCR with primers of the promoter regions of CREBBP, BPTF and RPTOR in WT HepG2 cells, HAT1 KO HepG2 cells and HAT1 KO HepG2 cells reconstituted with either wildtype HAT1 or mutant HAT1 (T188A). N = 3 biological replicates. Data were presented as mean ± SD. Student's t-test, ***P < 0.001; ns, no significance.


B . Bar graphs depicting relative of succinylation in HAT1 KO versus WT of 7 key enzymes in glycolysis pathway depicted in ( A ) . N = 3 biological replicates . Data were presented as mean ± SD .

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B. Bar graphs depicting the relative ratio of succinylation in HAT1 KO versus WT of the 7 key enzymes in the glycolysis pathway depicted in (A). N = 3 biological replicates. Data were presented as mean ± SD.


D . HAT1 - catalyzed PGAM1 succinylation was analyzed by mixing purified HAT1 , PGAM1 , succinyl - CoA / acetyl - CoA in in vitro succinylation assays . Western blot was performed with indicated antibodies .

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

D. HAT1-catalyzed PGAM1 succinylation was analyzed by mixing purified HAT1, PGAM1, and succinyl-CoA/acetyl-CoA in the in vitro succinylation assays. Western blot analysis was performed with indicated antibodies.


F . decrease of glucose increase of lactate in culture medium relative amounts of 2 - PG 3 - PG were analyzed by ELISA assays in HepG2 depleted endogenous PGAM1 reconstituted expression of PGAM1 or PGAM1 mutant ( K99R ) . N = 3 biological replicates . Data are presented as mean ± SD . Student ' s t - , * * P < 0 . 01 .

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

F. The decrease of glucose and increase of lactate in the culture medium and the relative amounts of 2-PG and 3-PG were analyzed by ELISA assays in HepG2 cells depleted endogenous PGAM1 and reconstituted expression of PGAM1 or PGAM1 mutant (K99R). N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.


A . HAT1 were assessed by immunohistochemical in clinical tissues of liver cancer , pancreatic cancer , cholangiocarcinoma . Scale bar = 100

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A. HAT1 levels were assessed by immunohistochemical staining in clinical tissues of liver cancer patients, pancreatic cancer patients, and cholangiocarcinoma patients. Scale bar = 100 μm


B . effect of HAT1 - mediated succinylation on cancer proliferation was measured by MTS assays in indicated . N = 3 biological replicates . Data are presented as mean ± SD . Student ' s t - , * * P < 0 . 01 . C . effect of PGAM1 succinylation on cancer proliferation was measured by MTS assays in indicated . N = 3 biological replicates . Data are presented as mean ± SD . Student ' s t - , * * P < 0 . 01 .

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

B. The effect of HAT1-mediated succinylation on cancer cell proliferation was measured by MTS assays in the indicated cells. N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01. C. The effect of PGAM1 succinylation on cancer cell proliferation was measured by MTS assays in the indicated cells. N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.


HepG2 , HAT1 KO HepG2 , HAT1 KO HepG2 reconstituted with either wild type HAT1 or mutant HAT1 ( T188A ) were subcutaneously injected into athymic nude mice volumes average were calculated . N = 6 biological replicates . Scale bar = 10 mm . In boxplots , boxes extend from 25th to 75th percentiles ( inter ‐ quartile range ( IQR ) ) , central band represents median , whiskers indicate lowest highest data within 1 . 5 × IQR from lower upper quartiles , respectively . Student ' s t - , * * * P < 0 .

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

HepG2 cells, HAT1 KO HepG2 cells, and HAT1 KO HepG2 cells reconstituted with either wild type HAT1 or mutant HAT1 (T188A) were subcutaneously injected into athymic nude mice Tumor volumes and average tumor weight were calculated. N = 6 biological replicates. Scale bar = 10 mm. In the boxplots, boxes extend from the 25th to 75th percentiles (inter‐quartile range (IQR)), central band represents the median, whiskers indicate the lowest and highest data within 1.5 × IQR from the lower and upper quartiles, respectively. Student's t-test, ***P < 0.001


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

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

(B, C) 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 WT mice from high regiment ( n = 5 mice , means ± , two - student ' s t - ) .

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

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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 ) analogous set of data is shown with WT MCK - KO GA . ( n = 3 , means ± , two - student ' s t - . 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

(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 ) were measured in WT MCK - KO soleus ( E ) at after a bout of exercise ( n = 6 per group , means ± , * p < 0 . 05 , two - student ' s t - 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

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

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

(A-D) 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 MCK - KO soleus tissue after a bout of low - exercise for 50 min under basal conditions in response to 4 mM oligomycin ( V inhibitor ) , 4 mM FCCP ( uncoupler ) , or 4 mM each of rotenone antimycin A ( I inhibitor ) ( n = 8 , means ± , * p < 0 . 05 , two - student ' s t - 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 in response to 4 mM oligomycin ( V inhibitor ) , 4 mM FCCP ( uncoupler ) , or 4 mM rotenone antimycin A ( I inhibitor ) ( n = 5 , means ± , * p < 0 . 05 , two - student ' s t - two - way ANOVA followed by Bonferroni post - hoc testing ) .

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

(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 expression in WT vs . MCK - KO soleus , GA EDL at sedentary normalizing to GAPDH using ImageJ .

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

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

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

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

(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 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 ± , * p < 0 . 05 , two - student ' s t - two - way ANOVA followed by Bonferroni post - hoc testing ) .

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

(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 MCK - KO with or without transfection of gCont vs . gAldh1l1 under low regimen ( n = 4 WT + gCont , n = 9 MCK - KO + gCont , MCK - KO + gAldh1l1 , * p < 0 . 05 , means ± , two - student ' s t - 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).


( a ) Western blot of CALM , actin , GAPDH LC3 - II in several lines ( HeLa , HEK , CAD murine embryonic fibroblast ( MEF ) ) where CALM was knocked down using shRNA or siRNA , as indicated , or knockout with rescue experiment . In experiments in this paper , we used a scramble siRNA or a luciferase shRNA as controls . were starved in Hanks balanced salt solution ( HBSS ) treated with Baf A1 as indicated . ( BC , basal conditions ; SE , short exposure ; LE , longer exposure ) . of LC3 - II / actin or GAPDH is shown in Supplementary Fig . 1A . * Not specific .

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

(a) Western blot analysis of CALM, actin, GAPDH and LC3-II in several cell lines (HeLa, HEK, CAD and murine embryonic fibroblast (MEF)) where CALM was knocked down using shRNA or siRNA, as indicated, or knockout with rescue experiment. In all experiments in this paper, we used a scramble siRNA or a luciferase shRNA as controls. The cells were starved in Hanks balanced salt solution (HBSS) and treated with Baf A1 as indicated. (BC, basal conditions; SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin or GAPDH ratio is shown in Supplementary Fig. 1A. *Not specific.


( b ) LC3 dot counting in CALM knockdown . CALM was knocked down in HeLa expressing GFP - LC3 with or without an siRNA - resistant form of wild - type CALM ( rescue ) , as indicated . were starved in HBSS for 4 h , fixed subjected to microscopy to score number of LC3 dots per . knockdown efficiency level of siRNA - resistant form of CALM are shown on left on western blotting . number of LC3 dots per ( shown as mean ± s . d . ) is shown on graph for each condition ( n ≥ 300 per condition ; BC , basal conditions ) . of number of LC3 dots per between starvation basal conditions is shown on right ( * P0 . 01 ; NS , not significant , two - t - ) . Pictures obtained from automated microscope are shown . Scale bars , 20 μm .

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

(b) LC3 dot counting in CALM knockdown cells. CALM was knocked down in HeLa cells expressing GFP-LC3 and with or without an siRNA-resistant form of wild-type CALM (rescue), as indicated. The cells were starved in HBSS for 4 h, fixed and subjected to fluorescence microscopy to score the number of LC3 dots per cell. The knockdown efficiency and the level of the siRNA-resistant form of CALM are shown on the left on the western blotting. The number of LC3 dots per cell (shown as mean ±s.d.) is shown on the graph for each condition (n≥300 cells per condition; BC, basal conditions). The ratio of the number of LC3 dots per cell between starvation and basal conditions is shown on the right (*P0.01; NS, not significant, two-tailed t-test). Pictures obtained from automated microscope are shown. Scale bars, 20 μm.


( a ) p62 vesicle formation in CALM knockdown HeLa . Confocal pictures are shown . # CALM - downregulated where p62 vesicles accumulate . Data are representative of three independent experiments shown as mean ± s . e . m . ( n ≥ 500 ; * P0 . 01 ; two - t - ) . Scale bars , 5 μm .

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(a) p62 vesicle formation in CALM knockdown HeLa cells. Confocal pictures are shown. #CALM-downregulated cells where p62 vesicles accumulate. Data are representative of three independent experiments and shown as mean ±s.e.m. (n≥500 cells; *P0.01; two-tailed t-test). Scale bars, 5 μm.


( b ) Western blot of p62 , actin GAPDH in HeLa HEK ( basal conditions , BC , or without serum for 1 h ) where CALM was knocked down using shRNA or siRNA , as indicated . Data are mean ± s . d ( n = 3 experiments for HeLa HEK ; * P0 . 05 ; two - t - ) .

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(b) Western blot analysis of p62, actin and GAPDH in HeLa cells and HEK cells (basal conditions, BC, or without serum for 1 h) where CALM was knocked down using shRNA or siRNA, as indicated. Data are mean ±s.d (n=3 experiments for HeLa cells and HEK cells; *P0.05; two-tailed t-test).


( c ) of huntingtinQ74 - expressing with in CALM knockdown HeLa . Data depict one representative experiments performed in triplicate , out of three independent experiments shown as mean ± s . d . ( * P0 . 05 ; two - t - ) .

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(c) Percentage of huntingtinQ74-expressing cells with aggregates in CALM knockdown HeLa cells. Data depict one representative experiments performed in triplicate, out of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).


( d ) Tau - positive tangle formation in CALM knockdown . HeLa transiently expressing DsRed - tau 4R were treated with Baf A1 for 4 h as indicated . were fixed analysed by confocal microscopy after immnunostaining for phosphorylated tau using PHF1 antibody ( green ) . Data represent number of with phosphorylated tau - positive tangles as mean ± s . e . m . ( n = 3 experiments ; * P0 . 01 ; two - t - ) . Scale bars , 5 μm .

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

(d) Tau-positive tangle formation in CALM knockdown cells. HeLa cells transiently expressing DsRed-tau 4R were treated with Baf A1 for 4 h as indicated. Cells were fixed and analysed by confocal microscopy after immnunostaining for phosphorylated tau using PHF1 antibody (green). Data represent the number of cells with phosphorylated tau-positive tangles as mean ±s.e.m. (n=3 experiments; *P0.01; two-tailed t-test). Scale bars, 5 μm.


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(b) Western blot analysis of p62, actin and GAPDH in HeLa cells and HEK cells (basal conditions, BC, or without serum for 1 h) where CALM was knocked down using shRNA or siRNA, as indicated. Data are mean ±s.d (n=3 experiments for HeLa cells and HEK cells; *P0.05; two-tailed t-test).


( a ) Formation of endogenous ATG12 vesicles in CALM knockdown HeLa in basal ( BC ) serum starvation conditions . Confocal pictures are shown with arrows indicating ATG12 vesicles in starvation conditions . Data are from one representative experiment , out of three independent experiments . Data shown as mean ± s . e . m . ( n ≥ 500 ; * P0 . 01 ; two - t - ) . Scale bars , 5 μm .

O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Formation of endogenous ATG12 vesicles in CALM knockdown HeLa cells in basal (BC) and serum starvation conditions. Confocal pictures are shown with arrows indicating ATG12 vesicles in starvation conditions. Data are from one representative experiment, out of three independent experiments. Data shown as mean ±s.e.m. (n≥500 cells; *P0.01; two-tailed t-test). Scale bars, 5 μm.


( b ) Co - localization between GFP - ATG16L1 internalized cholera toxin subunit B conjugated with Alexa555 ( CTX ; 20 min ) in CALM knockdown HeLa . Confocal pictures are presented with magnified areas showing co - localization between ATG16L1 CTX in greater detail . Scale bars , 5 μm . Pearson ' s coefficient between ATG16L1 cholera toxin is shown . Data are representative of three independent experiments shown as mean ± s . d . ( n ≥ 20 ; * P0 . 05 ; two - t - ) .

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

(b) Co-localization between GFP-ATG16L1 and internalized cholera toxin subunit B conjugated with Alexa555 (CTX; 20 min) in CALM knockdown HeLa cells. Confocal pictures are presented with magnified areas showing the co-localization between ATG16L1 and CTX in greater detail. Scale bars, 5 μm. The Pearson's coefficient between ATG16L1 and cholera toxin is shown. Data are representative of three independent experiments and shown as mean ±s.d. (n≥20 cells; *P0.05; two-tailed t-test).


( c ) of endogenous ATG12 vesicles in CALM knockdown HeLa in starvation conditions . Confocal pictures are shown with arrows indicating ATG12 vesicles # indicating CALM knockdown . Data are representative of three independent experiments shown as mean ± s . d . ( n ≥ 100 vesicles ; * P0 . 05 ; two - t - ) . ( AU , arbitrary unit ) . Scale bars , 5 μm .

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

(c) Size of endogenous ATG12 vesicles in CALM knockdown HeLa cells in starvation conditions. Confocal pictures are shown with arrows indicating ATG12 vesicles and # indicating CALM knockdown cells. Data are representative of three independent experiments and shown as mean ±s.d. (n≥100 vesicles; *P0.05; two-tailed t-test). (AU, arbitrary unit). Scale bars, 5 μm.


( d ) Live - imaging of ATG16L1 - GFP in CALM knockdown HeLa . ATG16L1 pictures from various time points of a 5 - min movie are shown in inverted grey style . Arrows indicate ATG16L1 vesicles . number of fusion events per 5 min is shown . Data are representative of five movies shown as mean ± s . d . ( * P0 . 05 ; two - t - ) . Scale bars , 5 μm .

O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O 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) Live-cell imaging of ATG16L1-GFP in CALM knockdown HeLa cells. ATG16L1 pictures from various time points of a 5-min movie are shown in inverted grey style. Arrows indicate ATG16L1 vesicles. The number of fusion events per 5 min is shown. Data are representative of five movies and shown as mean ±s.d. (*P0.05; two-tailed t-test). Scale bars, 5 μm.


( e ) In vitro fusion of post - supernatant from HeLa expressing either GFP - ATG16L1 or mStrawberry - ATG16L1 in control CALM knockdown conditions . Confocal pictures are shown where ATG16L1 - mStrawberry signal is shown in purple to enable better visualization . Fused vesicles appear in . ATP - negative condition , which prevents SNARE - dependent fusion , is also shown as a control for reaction . Magnified areas are shown to allow visualization of vesicles . of fused vesicles is represented . n = numbers of vesicles scored per field ( a minimum of five fields were analysed per condition ) . Data are representative of two independent experiments shown as mean ± s . d . ( n ≥ 100 vesicles ) . Scale bars , 5 μm . ( * P0 . 05 ; two - t - ) .

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

(e) In vitro fusion assay of post-nuclear supernatant from HeLa cells expressing either GFP-ATG16L1 or mStrawberry-ATG16L1 in control and CALM knockdown conditions. Confocal pictures are shown where ATG16L1-mStrawberry signal is shown in purple to enable better visualization. Fused vesicles appear in white. The ATP-negative condition, which prevents SNARE-dependent fusion, is also shown as a control for the reaction. Magnified areas are shown to allow visualization of the vesicles. The percentage of fused vesicles is represented. n=numbers of vesicles scored per field (a minimum of five fields were analysed per condition). Data are representative of two independent experiments and shown as mean ±s.d. (n≥100 vesicles). Scale bars, 5 μm. (*P0.05; two-tailed t-test).


( a ) LC3 in CALM knockdown . CALM was knocked down in expressing or not expressing an siRNA - resistant form of CALM wild type ( wt ) or CALM 219 mutant , as indicated . were lysed subjected to western blotting . knockdown efficiency , LC3 - II level of siRNA - resistant form of CALM are shown on western blotting .

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(a) LC3 levels in CALM knockdown cells. CALM was knocked down in cells expressing or not expressing an siRNA-resistant form of CALM wild type (wt) or CALM 219 mutant, as indicated. The cells were lysed and subjected to western blotting. The knockdown efficiency, LC3-II levels and the level of the siRNA-resistant form of CALM are shown on the western blotting.


( b ) LC3 dots counting in CALM knockdown . CALM was knocked down in expressing GFP - LC3 with or without an siRNA - resistant form of CALM wild type ( wt ) or CALM mutant ( 219 mutant ) as indicated . were kept in full medium or starved in Hanks balanced salt solution ( HBSS ) for 4 h , fixed subjected to microscopy to score number of LC3 dots per . of number of LC3 dots per ( shown as mean ± s . d . ) between starvation basal conditions is shown on graph ( * P0 . 01 ; NS , not significant , two - t - ; n ≥ 300 per condition ; BC , basal conditions ) .

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

(b) LC3 dots counting in CALM knockdown cells. CALM was knocked down in cells expressing GFP-LC3 with or without an siRNA-resistant form of CALM wild type (wt) or CALM mutant (219 mutant) as indicated. The cells were kept in full medium or starved in Hanks balanced salt solution (HBSS) for 4 h, fixed and subjected to microscopy to score the number of LC3 dots per cell. The ratio of the number of LC3 dots per cell (shown as mean ±s.d.) between starvation and basal conditions is shown on the graph (*P0.01; NS, not significant, two-tailed t-test; n≥300 cells per condition; BC, basal conditions).