( H ) Epg5 − / − females gradually develop the limb - clasping phenotype .

(H) Epg5−/− females gradually develop the limb-clasping phenotype.


( A ) Nissl staining of the fifth layer of the cortex in 10 - mo - old Epg5 + / − and Epg5 − / − mice . ( B ) The number of pyramidal cells in the fifth layer per millimeters squared .

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(A) Nissl staining of the fifth layer of the cortex in 10-mo-old Epg5+/− and Epg5−/− mice. (B) The number of pyramidal cells in the fifth layer per millimeters squared.


( C and D ) H & amp ; amp ; E staining of the hippocampus shows reduced pyramidal cell numbers in Epg5 − / − mice . Means ± SEM of three mice are shown in B and D .

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(C and D) H&amp;E staining of the hippocampus shows reduced pyramidal cell numbers in Epg5−/− mice. Means ± SEM of three mice are shown in B and D.


( E ) The normally developed cerebellum in Epg5 − / − mice . ( F ) The thickness of the molecular layer in the cerebellum in mutant and control mice .

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(E) The normally developed cerebellum in Epg5−/− mice. (F) The thickness of the molecular layer in the cerebellum in mutant and control mice.


( G and H ) Purkinje cells , stained by calbindin , are not reduced in number in Epg5 − / − mice . Means ± SEM of five mice are shown in F and H .

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(G and H) Purkinje cells, stained by calbindin, are not reduced in number in Epg5−/− mice. Means ± SEM of five mice are shown in F and H.


( I ) Arrows indicate Pyknotic neurons in the anterior horn of Epg5 − / − mice .

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(I) Arrows indicate Pyknotic neurons in the anterior horn of Epg5−/−mice.


( J ) The number of motor neurons in the spinal cord ( SC ) in Epg5 + / − and Epg5 − / − mice .

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(J) The number of motor neurons in the spinal cord (SC) in Epg5+/− and Epg5−/−mice.


( K ) The numbers of Nissl - stained interneurons in the spinal cord are similar in control and Epg5 − / − mice . Means ± SEM of three mice are shown in J and K .

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(K) The numbers of Nissl-stained interneurons in the spinal cord are similar in control and Epg5−/− mice. Means ± SEM of three mice are shown in J and K.


( L ) Eosinophilic spheroids ( arrows ) accumulate in the thoracic spinal cord of Epg5 − / − mice .

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(L) Eosinophilic spheroids (arrows) accumulate in the thoracic spinal cord of Epg5−/− mice.


( M ) Eosinophilic spheroids are swollen degenerating axons ( arrow ) .

(M) Eosinophilic spheroids are swollen degenerating axons (arrow).


( N ) EM pictures showing degenerated axons ( arrows ) in the spinal cord of Epg5 − / − mice .

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(N) EM pictures showing degenerated axons (arrows) in the spinal cord of Epg5−/− mice.


( O and P ) GFAP staining in Epg5 + / − and Epg5 − / − mice . DCST , dorsal corticospinal tract .

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(O and P) GFAP staining in Epg5+/− and Epg5−/−mice. DCST, dorsal corticospinal tract.


( A ) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5 + / − and Epg5 − / − mice . SC , spinal cord .

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(A) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5+/− and Epg5−/− mice. SC, spinal cord.


( B ) Polyubiquitinated proteins in detergent - soluble ( S ) and - insoluble ( I ) fractions from brain and spinal cord homogenates of Epg5 + / − and Epg5 − / − mice .

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(B) Polyubiquitinated proteins in detergent-soluble (S) and -insoluble (I) fractions from brain and spinal cord homogenates of Epg5+/− and Epg5−/− mice.


( C ) p62 aggregates and ubiquitin - positive aggregates are absent from Epg5 + / − mice but dramatically accumulate and are colocalized ( arrows ) in Epg5 − / − mice .

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(C) p62 aggregates and ubiquitin-positive aggregates are absent from Epg5+/− mice but dramatically accumulate and are colocalized (arrows) in Epg5−/− mice.


( D - F ) p62 aggregates are detected in neurons ( arrows ; D ) and oligodendrocytes ( arrows ; E ) but absent from astrocytes ( F ) in Epg5 − / − mice .

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(D-F) p62 aggregates are detected in neurons (arrows; D) and oligodendrocytes (arrows; E) but absent from astrocytes (F) in Epg5−/− mice.


( G ) Cytoplasmic TDP - 43 aggregates accumulate in motor neurons in the anterior horn of spinal cord in Epg5 − / − mice . Arrows show cytoplasmic TDP - 43 aggregates . DSCT , dorsal corticospinal tract . ( H ) Percentages of cytoplasmic TDP - 43 aggregate - positive motor neurons in the fifth layer of cortex and anterior horn of the spinal cord in Epg5 + / − and Epg5 − / − mice . Means ± SEM of three mice are shown . LC , lateral column . Bars , 10 µm .

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(G) Cytoplasmic TDP-43 aggregates accumulate in motor neurons in the anterior horn of spinal cord in Epg5−/− mice. Arrows show cytoplasmic TDP-43 aggregates. DSCT, dorsal corticospinal tract. (H) Percentages of cytoplasmic TDP-43 aggregate-positive motor neurons in the fifth layer of cortex and anterior horn of the spinal cord in Epg5+/− and Epg5−/− mice. Means ± SEM of three mice are shown. LC, lateral column. Bars, 10 µm.


( A and B ) Electromyography of the gastrocnemius muscle from a 3 - mo - old Epg5 − / − mouse showing fibrillations ( A ) and positive sharp waves ( B ) . These defects were absent in control mice .

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(A and B) Electromyography of the gastrocnemius muscle from a 3-mo-old Epg5−/− mouse showing fibrillations (A) and positive sharp waves (B). These defects were absent in control mice.


( C ) At 10 mo , Epg5 − / − mice showed high - amplitude and long - duration action potentials when conducting MUAP tests . The blue lines show the start and end of an action potential . Dur , duration ; Amp , amplitude .

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(C) At 10 mo, Epg5−/−mice showed high-amplitude and long-duration action potentials when conducting MUAP tests. The blue lines show the start and end of an action potential. Dur, duration; Amp, amplitude.


( D - F ) H & amp ; amp ; E staining of gastrocnemius muscles showed features of muscle degeneration in Epg5 − / − mice . The arrow in E indicates centrally nucleated fibers .

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(D-F) H&amp;E staining of gastrocnemius muscles showed features of muscle degeneration in Epg5−/−mice. The arrow in E indicates centrally nucleated fibers.


( G ) The transcription levels of the atrophy - related genes were up - regulated in gastrocnemius muscles of Epg5 − / − mice .

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(G) The transcription levels of the atrophy-related genes were up-regulated in gastrocnemius muscles of Epg5−/−mice.


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(A) Levels of LC3 and p62 in brain and spinal cord extracts from Epg5+/− and Epg5−/−mice. SC, spinal cord.


( J ) Levels of LC3 - II and p62 in muscle extracts from Epg5 + / − and Epg5 − / − mice .

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(J) Levels of LC3-II and p62 in muscle extracts from Epg5+/− and Epg5−/− mice.


( K ) Percentage of p62 aggregate - positive myofibers in Epg5 + / − and Epg5 − / − mice . Means ± SEM of three mice are shown .

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(K) Percentage of p62 aggregate-positive myofibers in Epg5+/− and Epg5−/− mice. Means ± SEM of three mice are shown.


( L ) Accumulation and colocalization of p62 and ubiquitin aggregates in the gastrocnemius muscles of Epg5 − / − mice but not controls . Bars : ( D - F and L ) 10 µm ; ( H and I ) 1 µm .

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(L) Accumulation and colocalization of p62 and ubiquitin aggregates in the gastrocnemius muscles of Epg5−/− mice but not controls. Bars: (D-F and L) 10 µm; (H and I) 1 µm.


( A ) Number of LC3 puncta in Epg5 + / − ( control ) and Epg5 − / − MEFs .

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(A) Number of LC3 puncta in Epg5+/− (control) and Epg5−/− MEFs.


( B ) Western blot showing levels of LC3 - I and LC3 - II in control and Epg5 − / − MEFs upon indicated treatment ( Starv . , starvation ; Rapa . , rapamycin ; Bafilo . , bafilomycin A1 ) .

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(B) Western blot showing levels of LC3-I and LC3-II in control and Epg5−/− MEFs upon indicated treatment (Starv., starvation; Rapa., rapamycin; Bafilo., bafilomycin A1).


( C ) Proteinase K ( ProK ) protection assay in different MEFs . PNS , postnuclear supernatant ; P , pellet fractions .

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(C) Proteinase K (ProK) protection assay in different MEFs. PNS, postnuclear supernatant; P, pellet fractions.


( D ) Colocalization ratio of LC3 and LAMP - 1 in control and Epg5 − / − MEFs . 50 cells were examined per time point in A and D .

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(D) Colocalization ratio of LC3 and LAMP-1 in control and Epg5−/− MEFs. 50 cells were examined per time point in A and D.


( E ) Colocalization of LC3 puncta and lysosomes in control and Epg5 − / − MEFs 4 h after starvation .

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(E) Colocalization of LC3 puncta and lysosomes in control and Epg5−/− MEFs 4 h after starvation.


( F ) Under nutrient repletion conditions , almost no autophagic elements are detected in Epg5 + / − MEFs . RER , rough ER ; M , mitochondrion ; N , nucleus . The arrowhead indicates a vacuole with a late residual body - like appearance ( aAV - III ) . ( G ) Under nutrient repletion conditions , Epg5 − / − MEFs accumulate a large number of autophagic vacuoles . Red arrow , a likely autophagosome ; red arrowheads , complex vacuoles of the aAV - I type ; white arrowheads , aAV - II

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(F) Under nutrient repletion conditions, almost no autophagic elements are detected in Epg5+/− MEFs. RER, rough ER; M, mitochondrion; N, nucleus. The arrowhead indicates a vacuole with a late residual body-like appearance (aAV-III). (G) Under nutrient repletion conditions, Epg5−/− MEFs accumulate a large number of autophagic vacuoles. Red arrow, a likely autophagosome; red arrowheads, complex vacuoles of the aAV-I type; white arrowheads, aAV-II vacuoles


( H and I ) High magnification of autophagic elements in unstarved Epg5 − / − MEFs . ( H ) The red arrow indicates a complex autophagic vacuole containing multilayered membrane structures ( white arrowheads ) and a lipid - like region ( L ) . ( I ) The red arrow indicates an autophagosome containing cytoplasm and ribosomes . The red arrowhead points to an autophagosome containing a mitochondrion and cytoplasmic material with membranous structures . The white arrow shows an aAV - III vacuole .

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(H and I) High magnification of autophagic elements in unstarved Epg5−/− MEFs. (H) The red arrow indicates a complex autophagic vacuole containing multilayered membrane structures (white arrowheads) and a lipid-like region (L). (I) The red arrow indicates an autophagosome containing cytoplasm and ribosomes. The red arrowhead points to an autophagosome containing a mitochondrion and cytoplasmic material with membranous structures. The white arrow shows an aAV-III vacuole.


( J ) Compared with control cells , Rab5 - labeled early endosomes are significantly enlarged in Epg5 siRNA - treated cells .

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(J) Compared with control cells, Rab5-labeled early endosomes are significantly enlarged in Epg5 siRNA-treated cells.


( K and L ) EGFR degradation in control and Epg5 knockdown cells .

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(K and L) EGFR degradation in control and Epg5 knockdown cells.


( M and N ) Distribution of Alexa Fluor 488 - conjugated transferrin at different time points in control siRNA - and Epg5 siRNA - treated cells . Higher magnification views are shown in the insets . Error bars indicate SEMs .

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(M and N) Distribution of Alexa Fluor 488-conjugated transferrin at different time points in control siRNA- and Epg5 siRNA-treated cells. Higher magnification views are shown in the insets. Error bars indicate SEMs.


( A ) Co - immunoprecipitation ( Co - IP ) of Beclin1 with WT or mutant vBcl - 2 . 293T cells were transiently transfected with the indicated constructs , followed by immunoprecipitation of HA - tagged vBcl - 2 and immunoblotting of V5 - tagged Beclin1 .

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(A) Co-immunoprecipitation (Co-IP) of Beclin1 with WT or mutant vBcl-2. 293T cells were transiently transfected with the indicated constructs, followed by immunoprecipitation of HA-tagged vBcl-2 and immunoblotting of V5-tagged Beclin1.


( B ) Co - IP of WT or mutant vBcl - 2 with endogenous Beclin1 . 293T cells were transfected with the indicated vBcl - 2 constructs , followed by immunoprecipitation of HA - tagged vBcl - 2 and immunoblotting of endogenous Beclin1 . 1 % whole - cell lysates ( WCLs ) was used as the input .

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(B) Co-IP of WT or mutant vBcl-2 with endogenous Beclin1. 293T cells were transfected with the indicated vBcl-2 constructs, followed by immunoprecipitation of HA-tagged vBcl-2 and immunoblotting of endogenous Beclin1. 1% whole-cell lysates (WCLs) was used as the input.


( C ) vBcl - 2 interaction with Bak protein . 293T cells were transfected with WT and mutant forms of vBcl - 2 as indicated . At 48 h posttransfection , WCLs were mixed either with GST - BakΔTM fusion protein ( left panel ) or with GST alone ( right panel ) for an in vitro GST pull - down ( GST PD ) assays . GST fusion proteins used for the pulldown assay are indicated ( bottom panel ) . 1 % WCL was used as the input . Data are representative of at least three experiments yielding similar results .

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(C) vBcl-2 interaction with Bak protein. 293T cells were transfected with WT and mutant forms of vBcl-2 as indicated. At 48 h posttransfection, WCLs were mixed either with GST-BakΔTM fusion protein (left panel) or with GST alone (right panel) for an in vitro GST pull-down (GST PD) assays. GST fusion proteins used for the pulldown assay are indicated (bottom panel). 1% WCL was used as the input. Data are representative of at least three experiments yielding similar results.


( A ) NIH3T3 cells stably expressing the WT and mutant forms of vBcl - 2 were transfected with GFP - LC3 . At 18 h posttransfection , cells were incubated under normal or starvation conditions for 4 h . Autophagy was quantified as mean ± SEM of the combined results from three independent experiments . * * , P < 0 . 0001 .

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(A) NIH3T3 cells stably expressing the WT and mutant forms of vBcl-2 were transfected with GFP-LC3. At 18 h posttransfection, cells were incubated under normal or starvation conditions for 4 h. Autophagy was quantified as mean±SEM of the combined results from three independent experiments. **, P<0.0001.


( B ) NIH3T3 cells expressing the WT or mutant forms of vBcl - 2 as indicated were transfected with GFP - LC3 and treated with 2 µM rapamycin for 6 h . GFP - LC3 was detected using an inverted fluorescence microscope ( top ) . Autophagy was quantified as mean ± SEM of the combined results from three independent experiments . Scale bar , 5 µm ; * * , P < 0 . 01 .

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(B) NIH3T3 cells expressing the WT or mutant forms of vBcl-2 as indicated were transfected with GFP-LC3 and treated with 2 µM rapamycin for 6 h. GFP-LC3 was detected using an inverted fluorescence microscope (top). Autophagy was quantified as mean±SEM of the combined results from three independent experiments. Scale bar, 5 µm; **, P<0.01.


( C ) NIH3T3 cells stably expressing the WT or mutant forms of vBcl - 2 , as indicated , were treated with 2 µM rapamycin . LC3 - I and LC3 - II levels were then determined by immunoblotting with an antibody against LC3 ( top ) . Densitometric quantification of the LC3 - II / LC3 - I ratios under normal and rapamycin treatment conditions is shown at the bottom . Similar results were obtained from three independent experiments .

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(C) NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2, as indicated, were treated with 2 µM rapamycin. LC3-I and LC3-II levels were then determined by immunoblotting with an antibody against LC3 (top). Densitometric quantification of the LC3-II/LC3-I ratios under normal and rapamycin treatment conditions is shown at the bottom. Similar results were obtained from three independent experiments.


( top ) Representative confocal images of GFP - LC3 and HA - tagged vBcl - 2 ( WT and mutants ) in NIH3T3 cells infected with the indicated viruses ( MOI = 5 ) . Nuclei were counterstained with 4 ′ , 6 ′ - diamidino - 2 - phenylindole ( DAPI ) . Scale bar , 5 µm . ( bottom ) Quantification of the percentage of virally infected cells with GFP - LC3 punctate staining . Results shown represent mean ± SEM of combined results from three independent experiments ( 200 cells per experimental condition ) . * , P < 0 . 01 versus HA - WT ; * * , P < 0 . 001 versus HA - WT .

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(top) Representative confocal images of GFP-LC3 and HA-tagged vBcl-2 (WT and mutants) in NIH3T3 cells infected with the indicated viruses (MOI = 5). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). Scale bar, 5 µm. (bottom) Quantification of the percentage of virally infected cells with GFP-LC3 punctate staining. Results shown represent mean±SEM of combined results from three independent experiments (200 cells per experimental condition). *, P<0.01 versus HA-WT; **, P<0.001 versus HA-WT.


NIH3T3 cells stably expressing the WT or mutant forms of vBcl - 2 were treated with TNFα and cycloheximide ( CHX ) for 12 h , then assayed for cell viability by trypan blue exclusion assay ( A ) ,

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NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A),


NIH3T3 cells stably expressing the WT or mutant forms of vBcl - 2 were treated with TNFα and cycloheximide ( CHX ) for 12 h , then assayed for cell viability by trypan blue exclusion assay ( A ) , or for apoptosis by TUNEL staining ( B )

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NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A), or for apoptosis by TUNEL staining (B)


NIH3T3 cells stably expressing the WT or mutant forms of vBcl - 2 were treated with TNFα and cycloheximide ( CHX ) for 12 h , then assayed for cell viability by trypan blue exclusion assay ( A ) , or for apoptosis by TUNEL staining ( B ) or for the caspase 3 activation using flow cytometry ( C ) . Apoptotic cells in ( B ) were counted under high power magnification ( 60 × magnification ) . Mock , untreated condition . Data represents mean ± SEM of combined results from three independent experiments . Scale bars , 100 µm ( A ) , 5 µm ( B ) . * * , P < 0 . 0001 versus vector cells .

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NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A), or for apoptosis by TUNEL staining (B) or for the caspase 3 activation using flow cytometry (C). Apoptotic cells in (B) were counted under high power magnification (60×magnification). Mock, untreated condition. Data represents mean±SEM of combined results from three independent experiments. Scale bars, 100 µm (A), 5 µm (B). **, P<0.0001 versus vector cells.


( A ) Single - step ( right ) and multiple - step ( left ) growth curves of the WT and recombinant γHV68 viruses in BHK21 cells .

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(A) Single-step (right) and multiple-step (left) growth curves of the WT and recombinant γHV68 viruses in BHK21 cells.


( B ) Acute replication of the WT and mutant vBcl - 2 γHV68 viruses in the lungs of BALB / c mice at 5 dpi ( up ) and 7 dpi ( down ) after intranasal infection determined by viral titers in the lungs of the infected mice ( left ) or by real - time PCR of the viral genomic DNA ( right ) . Mean ± SEM of five mice per group / experiment . Data of 7 dpi is pooled from two separate experiments . The vBcl - 2 Δα1 and ΔBH2 mutants did not yield significantly different results when compared to the WT in infectious virus titers [ for Δα1 , P = 0 . 82 ( day 5 ) ; P = 0 . 08 ( day 7 ) ; for ΔBH2 , P = 0 . 49 ( day 5 ) ; P = 0 . 54 ( day 7 ) ; unpaired t - tests ] and in viral genome loads in the lungs [ for Δα1 , P = 0 . 25 ( day 5 ) ; P = 0 . 28 ( day 7 ) ; for ΔBH2 , P = 0 . 21 ( day 5 ) ; P = 0 . 37 ( day 7 ) ; unpaired t - tests ] . n . s . , not significant .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Acute replication of the WT and mutant vBcl-2 γHV68 viruses in the lungs of BALB/c mice at 5 dpi (up) and 7 dpi (down) after intranasal infection determined by viral titers in the lungs of the infected mice (left) or by real-time PCR of the viral genomic DNA (right). Mean±SEM of five mice per group/experiment. Data of 7 dpi is pooled from two separate experiments. The vBcl-2 Δα1 and ΔBH2 mutants did not yield significantly different results when compared to the WT in infectious virus titers [for Δα1, P = 0.82 (day 5); P = 0.08 (day 7); for ΔBH2, P = 0.49 (day 5); P = 0.54 (day 7); unpaired t-tests] and in viral genome loads in the lungs [for Δα1, P = 0.25 (day 5); P = 0.28 (day 7); for ΔBH2, P = 0.21 (day 5); P = 0.37 (day 7); unpaired t-tests]. n.s., not significant.


Splenic infectious centers measured at 12 dpi ( A , left ) , 14 dpi ( B , left ) , 21 dpi ( C , left ) , 28 dpi ( D , up ) , 35 dpi ( E , up ) , or 42 dpi ( F , up ) and viral genome load measured by real - time PCR at 12 dpi ( A , right ) , 14 dpi ( B , right ) , 21 dpi ( C , right ) , 28 dpi ( D , down ) , 35 dpi ( E , down ) , or 42 dpi ( F , down ) , in the BALB / cmice intranasally infected with the WT or recombinant γHV68 mutants , as indicated ( Mean ± SEM of five mice per group / time point / experiment ) . Data of 14 dpi and 28 dpi is pooled from two and three separate experiments , respectively . Preformed infectious virus was negligible in all spleen samples . No significant difference was detected at 12 dpi ( A ) , 14 dpi ( B ) , and 21 dpi ( C ) with the Δα1 and ΔBH2 mutant viruses when compared to the WT in infectious center titers [ for Δα1 , P = 0 . 58 ( day 12 ) ; P = 0 . 75 ( day 14 ) ; P = 0 . 18 ( day 21 ) ; for ΔBH2 , P = 0 . 64 ( day 12 ) ; P = 0 . 73 ( day 14 ) ; P = 0 . 81 ( day 21 ) ; unpaired t - tests ] and in viral genome loads [ for Δα1 , P = 0 . 85 ( day 12 ) ; P = 0 . 76 ( day 14 ) ; P = 0 . 96 ( day 21 ) ; for ΔBH2 , P = 0 . 56 ( day 12 ) ; P = 0 . 73 ( day 14 ) ; P = 0 . 25 ( day 21 ) ; unpaired t - tests ] . At 28 dpi ( D ) , 35 dpi ( E ) , and 42 dpi ( F ) , the decreased infectious center titers ( top ) of vBcl - 2 mutant viruses , as compared to WT γHV68 , were statistically significant [ vBcl - 2Δα1 versus vBcl - 2AAA ( 42 dpi ) , P = 0 . 46 ; vBcl - 2Δα1 versus WT γHV68 ( 42 dpi ) , P = 0 . 08 ; unpaired t - tests ] . At 28 dpi ( D ) , 35 dpi ( E ) , and 42 dpi ( F ) , the decreased viral DNA loads ( bottom ) of the vBcl - 2Δα1 , vBcl - 2AAA , and vBcl - 2 null mutants , as compared to WT γHV68 and the vBcl - 2ΔBH2 mutant , were statistically significant as follows ( unpaired t - tests ) : at day 28 , vBcl - 2Δα1 versus WT γHV68 , P < 0 . 01 ; vBcl - 2AAA versus WT γHV68 , P < 0 . 01 ; vBcl - 2 - null versus WT γHV68 , P < 0 . 01 ; vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2 - null versus vBcl - 2ΔBH2 , P < 0 . 001 ; at day 35 , vBcl - 2Δα1 versus WT γHV68 , P < 0 . 05 ; vBcl - 2AAA versus WT γHV68 , P < 0 . 05 ; vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 01 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 05 ; at day 42 , vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2ΔBH2 versus WT γHV68 , P = 0 . 33 . I . C . , infectious center . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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

Splenic infectious centers measured at 12 dpi (A, left), 14 dpi (B, left), 21 dpi (C, left), 28 dpi (D, up), 35 dpi (E, up), or 42 dpi (F, up) and viral genome load measured by real-time PCR at 12 dpi (A, right), 14 dpi (B, right), 21 dpi (C, right), 28 dpi (D, down), 35 dpi (E, down), or 42 dpi (F, down), in the BALB/cmice intranasally infected with the WT or recombinant γHV68 mutants, as indicated (Mean±SEM of five mice per group/time point/experiment). Data of 14 dpi and 28 dpi is pooled from two and three separate experiments, respectively. Preformed infectious virus was negligible in all spleen samples. No significant difference was detected at 12 dpi (A), 14 dpi (B), and 21 dpi (C) with the Δα1 and ΔBH2 mutant viruses when compared to the WT in infectious center titers [for Δα1, P = 0.58 (day 12); P = 0.75 (day 14); P = 0.18 (day 21); for ΔBH2, P = 0.64 (day 12); P = 0.73 (day 14); P = 0.81 (day 21); unpaired t-tests] and in viral genome loads [for Δα1, P = 0.85 (day 12); P = 0.76 (day 14); P = 0.96 (day 21); for ΔBH2, P = 0.56 (day 12); P = 0.73 (day 14); P = 0.25 (day 21); unpaired t-tests]. At 28 dpi (D), 35 dpi (E), and 42 dpi (F), the decreased infectious center titers (top) of vBcl-2 mutant viruses, as compared to WT γHV68, were statistically significant [vBcl-2Δα1 versus vBcl-2AAA (42 dpi), P = 0.46; vBcl-2Δα1 versus WT γHV68 (42 dpi), P = 0.08; unpaired t-tests]. At 28 dpi (D), 35 dpi (E), and 42 dpi (F), the decreased viral DNA loads (bottom) of the vBcl-2Δα1, vBcl-2AAA, and vBcl-2 null mutants, as compared to WT γHV68 and the vBcl-2ΔBH2 mutant, were statistically significant as follows (unpaired t-tests): at day 28, vBcl-2Δα1 versus WT γHV68, P<0.01; vBcl-2AAA versus WT γHV68, P<0.01; vBcl-2-null versus WT γHV68, P<0.01; vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.001; vBcl-2AAA versus vBcl-2ΔBH2, P<0.001; vBcl-2-null versus vBcl-2ΔBH2, P<0.001; at day 35, vBcl-2Δα1 versus WT γHV68, P<0.05; vBcl-2AAA versus WT γHV68, P<0.05; vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.01; vBcl-2AAA versus vBcl-2ΔBH2, P<0.05; at day 42, vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.001; vBcl-2AAA versus vBcl-2ΔBH2, P<0.001; vBcl-2ΔBH2 versus WT γHV68, P = 0.33. I.C., infectious center. *, P<0.05; **, P<0.01; ***, P<0.001.


( A ) Volcano plot displaying the associations between the mutational status of genes and the activity of kinases . The x - axis contains the mutation coefficients ( effect sizes ) and the y - axis the adjusted P - values . The vertical line in the x - axis is at x = 0 and separates the positive ( right ) from the negative ( left ) associations . The horizontal lines in the y - axis represent the points of statistical significance in the plot ( adjusted P - values < 0 . 05 and < 0 . 01 ) . The associations are represented in the form of a mutated gene - kinase . The colour gradient represents the string network edge weight interval of the pair ( grey if the pair is not in the string network ) . ( B ) Same as ( A ) for the TFs . The inner plot shows the effects of TF mutations on their own activities . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plot displaying the associations between the mutational status of genes and the activity of kinases. The x-axis contains the mutation coefficients (effect sizes) and the y-axis the adjusted P-values. The vertical line in the x-axis is at x=0 and separates the positive (right) from the negative (left) associations. The horizontal lines in the y-axis represent the points of statistical significance in the plot (adjusted P-values < 0.05 and < 0.01).The associations are represented in the form of a mutated gene - kinase. The colour gradient represents the string network edge weight interval of the pair (grey if the pair is not in the string network). (B) Same as (A) for the TFs. The inner plot shows the effects of TF mutations on their own activities. (


( C ) ( D ) Examples of the genetic associations highlighted in the volcano plots . The x - axis represents the associations and the y - axis the protein activities . The colours stratify the samples by their mutational status in the respective genes . The outliers ( defined as the data points beyond Q1 - 1 . 5 * IQR and Q3 + 1 . 5 * IQR , where Q1 and Q3 are the first and third quartiles and IQR is the interquartile range ) were removed from the distributions for representation purposes . The number of protein activity quantifications ( including outliers ) are shown beneath each boxplot . The P - values from Wilcoxon rank sum tests comparing both distributions are shown . All data points ( including outliers ) were used to calculate the P - values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Examples of the genetic associations highlighted in the volcano plots. The x-axis represents the associations and the y-axis the protein activities. The colours stratify the samples by their mutational status in the respective genes. The outliers (defined as the data points beyond Q1-1.5*IQR and Q3+1.5*IQR, where Q1 and Q3 are the first and third quartiles and IQR is the interquartile range) were removed from the distributions for representation purposes. The number of protein activity quantifications (including outliers) are shown beneath each boxplot. The P-values from Wilcoxon rank sum tests comparing both distributions are shown. All data points (including outliers) were used to calculate the P-values.


( C ) Kinase activity gradient along the samples for a selection of the kinases shown in ( B ) .

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

(C) Kinase activity gradient along the samples for a selection of the kinases shown in (B).


( F ) Linear regression between the percentage of samples where the kinase is regulated in the perturbed conditions and in the tumour samples . The trend line and the Pearson ' s r , with the respective P - value , are shown . In red and green are the kinases preferably regulated in the tumours and in the conditions , respectively . In blue are the kinases regulated in both . The 95 % confidence interval around the regression line is represented by the shaded area .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Linear regression between the percentage of samples where the kinase is regulated in the perturbed conditions and in the tumour samples. The trend line and the Pearson's r, with the respective P-value, are shown. In red and green are the kinases preferably regulated in the tumours and in the conditions, respectively. In blue are the kinases regulated in both. The 95% confidence interval around the regression line is represented by the shaded area.


( C - F ) Kaplan - Meier survival plots comparing the survival probabilities ( y - axes ) as a function of time in days ( x - axes ) for proteins and cancer types shown in ( A ) and ( B ) . ( C ) MYC / brain cancer ( inactive = 71 , neutral = 39 , active = 67 ) . ( D ) MYC / liver cancer ( inactive = 58 , neutral = 59 , active = 40 ) . ( E ) MAP3K8 / renal cancer ( neutral = 95 , active = 7 ) . ( F ) PRKCA / renal cancer ( inactive = 3 , neutral = 92 , active = 7 ) . The log - rank P - values are shown in the plots .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Kaplan-Meier survival plots comparing the survival probabilities (y-axes) as a function of time in days (x-axes) for proteins and cancer types shown in (A) and (B). (C) MYC/brain cancer (inactive = 71, neutral = 39, active = 67). (D) MYC/liver cancer (inactive = 58, neutral = 59, active = 40). (E) MAP3K8/renal cancer (neutral = 95, active = 7). (F) PRKCA/renal cancer (inactive = 3, neutral = 92, active = 7). The log-rank P-values are shown in the plots.


( c ) Shown are the responses of the glucose sensor promoter ( PgluA7 ) , a negative control lacking the CRP operator ( PgluA7 * ) , and a constitutive promoter ( BBa _ J23101 ) to the presence ( + ) and absence ( - ) of

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

(c) Shown are the responses of the glucose sensor promoter (PgluA7), a negative control lacking the CRP operator (PgluA7*), and a constitutive promoter (BBa_J23101) to the presence (+) and absence (-) of glucose


( d ) Shown are the responses of the oxygen sensor promoter ( PfnrF8 ) , a negative control lacking the FNR operator ( PfnrF8 * ) , and a constitutive promoter ( BBa _ J23101 ) to the presence ( + ) and absence ( - ) of

(d) Shown are the responses of the oxygen sensor promoter (PfnrF8), a negative control lacking the FNR operator (PfnrF8*), and a constitutive promoter (BBa_J23101) to the presence (+) and absence (-) of oxygen


Shown are the schematics and responses for the glucose sensor The response functions ( center ) are shown for each sensor ( black circles ) compared to promoter variants where the operators are removed ( PgluA7 * , PfnrF8 * ) ( open circles ) text ) . Representative cytometry florescence distributions for Figures

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

Shown are the schematics and responses for the glucose sensor The response functions (center) are shown for each sensor (black circles) compared to promoter variants where the operators are removed (PgluA7*, PfnrF8*) (open circles) text). Representative cytometry florescence distributions for Figures 1f


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

Shown are the schematics and responses for th oxygen sensors Horizontal error bars in the PfnrF8 response reflect one standard deviation of three DO measurements Representative cytometry florescence distributions for Figure 1g


Shown are the schematics and responses for th acetate sensors For the acetate sensor , the response of the sensor is shown in unmodified E . coli MG1655 with glnL intact ( open diamonds ) . The dynamics of induction are shown ( right graph ) where cells are induced at the time indicated by the dashed line ( see text ) . Representative cytometry florescence distributions for Figure 1h are shown

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

Shown are the schematics and responses for th acetate sensors For the acetate sensor, the response of the sensor is shown in unmodified E. coli MG1655 with glnL intact (open diamonds). The dynamics of induction are shown (right graph) where cells are induced at the time indicated by the dashed line (see text). Representative cytometry florescence distributions for Figure 1h are shown i


The responses of the glucose sensors during growth in shake flasks are shown


The responses of th e , oxygen sensors during growth in shake flasks are

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

The responses of th e, oxygen sensors during growth in shake flasks are shown


The responses of th acetate sensors during growth in shake flasks are shown


( e ) The responses of a 3 - input , 2 - output circuit are shown . ( f ) Shown are the circuit ( left ) and genetic diagram ( right ) of the circuit corresponding to part e . In the genetic diagram , the dashed line and * indicates a second copy of the PgluA7 promoter that drives rfp expression and is repressed by PhlF via an immediately downstream PhlF operator . ( g ) The response of the circuit in parts e and f to different combinations of stimuli under the same conditions as Figure 1 ( Methods ) . The ( + ) and ( - ) indicate whether the output promoter of each sensor is active under those conditions . Bars where the circuit is predicted to be on are shown in grey and white when predicted to be

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

(e) The responses of a 3-input, 2-output circuit are shown. (f) Shown are the circuit (left) and genetic diagram (right) of the circuit corresponding to part e. In the genetic diagram, the dashed line and * indicates a second copy of the PgluA7 promoter that drives rfp expression and is repressed by PhlF via an immediately downstream PhlF operator. (g) The response of the circuit in parts e and f to different combinations of stimuli under the same conditions as Figure 1 (Methods). The (+) and (-) indicate whether the output promoter of each sensor is active under those conditions. Bars where the circuit is predicted to be on are shown in grey and white when predicted to be off


( h ) The response of a 3 - input 1 - output circuit is shown . ( i ) Shown are the circuit ( left ) and genetic diagram ( right ) of the circuit corresponding to part h . ( j ) The response of the circuit in parts h and i to different combinations of stimuli under the same conditions as Figure 1 ( Methods ) . Representative cytometry florescence distributions for Figure 2a / b / c and Figure 2e / g / h / j are shown in Appendix Figures S7 and S10 , respectively . Error bars represent one standard deviation of three independent experiments done on different

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The response of a 3-input 1-output circuit is shown. (i) Shown are the circuit (left) and genetic diagram (right) of the circuit corresponding to part h. (j) The response of the circuit in parts h and i to different combinations of stimuli under the same conditions as Figure 1 (Methods). Representative cytometry florescence distributions for Figure 2a/b/c and Figure 2e/g/h/j are shown in Appendix Figures S7 and S10, respectively. Error bars represent one standard deviation of three independent experiments done on different days


( b ) Reduction of fluorescence of RFP by the different mechanisms of repression after 18 hours of growth ( Methods ) . The inducers are either 1 mM IPTG ( sRNA ) or 25 μM DAPG ( sgRNA , mf - LON ) . sgRNA and mf - LON are co - transcribed on a single transcript , processed by ribozymes ( Appendix Figure S13 ) . ( c ) The dynamics of repression by each of the mechanisms is shown . Empty circles are uninduced and black circles are induced ( 1 mM IPTG or 25 µM DAPG ) at the 2 - hour time point ( dashed line ) ( Methods )

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 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 O O O O O 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) Reduction of fluorescence of RFP by the different mechanisms of repression after 18 hours of growth (Methods). The inducers are either 1 mM IPTG (sRNA) or 25 μM DAPG (sgRNA, mf-LON). sgRNA and mf-LON are co-transcribed on a single transcript, processed by ribozymes (Appendix Figure S13). (c) The dynamics of repression by each of the mechanisms is shown. Empty circles are uninduced and black circles are induced (1 mM IPTG or 25 µM DAPG) at the 2-hour time point (dashed line) (Methods)


( f ) The impact on acetate production for a double mutant where poxB is complemented on a BAC ( Methods ) . Note that complementation causes a ~ 3 - fold reduction in acetate compared to WT in part d . N - ter and E170 refer to the location of the SuMMV tag . ( g ) Reduction of acetate production by the expression of SuMMV from an DAPG - inducible promoter ( - no inducer , + 25 µM ) . The PoxB variant with the degron inserted at position E170 is either carried on a BAC or at its native location in the

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

(f) The impact on acetate production for a double mutant where poxB is complemented on a BAC (Methods). Note that complementation causes a ~3-fold reduction in acetate compared to WT in part d. N-ter and E170 refer to the location of the SuMMV tag. (g) Reduction of acetate production by the expression of SuMMV from an DAPG-inducible promoter (- no inducer, + 25 µM). The PoxB variant with the degron inserted at position E170 is either carried on a BAC or at its native location in the genome


( 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

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 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

(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 ) 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 ∆

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

(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 ) 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

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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 ) 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 ∆

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

(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


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

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

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


Primary 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 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 .

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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

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 .

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 the indicated time points . LDH

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

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 the indicated time points . mixed supernatant and cell extracts were analysed .

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

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 ) 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 .

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

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 ) and SM ( 250 nM ; 6 h ) and mixed supernatant and cell extracts were analysed by

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

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 ) and SM LDH release in the cell culture supernatant were quantified at the indicated time points .

O O O O B-CONTROLLED_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

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 ) and

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


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 .

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

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 and GSDMDD88A were costimulated with TNF ( 100 ng / ml ) and SM for 6 h and LDH release was quantified .

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

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 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 .

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

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 ) and TAK1i for 4 h and ( D ) mixed supernatant and extracts were analysed by

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 O O O O O

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 ) and TAK1i for 4 h and LDH release was quantified at the indicated time points .

O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O 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 ) and TAK1i ( 125 nM ) for 4 h and LDH release was quantified Where indicated , cells were treated with the inhibitors Nec - 1s ( 50 μM )

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

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 ) and TAK1i ( 125 nM ) for 4 h and LDH release was

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 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 ) 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 .

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

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 ) 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 .

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

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 ) 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 .

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

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 ) 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 .

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

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 ) and SM ( 0 . 5 μM ) for 6 h and ( G ) mixed supernatant and extracts were analysed by

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

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 ) and SM ( 0 . 5 μM ) for 6 h and LDH release was quantified .

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

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 the presence of ABT - 737 ( 0 . 5 μM ) and 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 ) and S63845 ( 0 . 5 μM ) and LDH release was

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

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 ) and S63845 ( 0 . 5 μM ) mixed supernatant and extracts were analysed by immunoblot at 6

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

O O O O O O O O O O O O O O O O O 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) 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 and further stimulated with ABT - 737 ( 1 μM ) and S63845 ( 1 μM ) for 24 h and mixed supernatant and extracts were analysed by

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

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

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 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 .

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-MEASURED_VAR 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 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

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 embryo X ‐ Gal staining of E16 CcnoTA / + and wild ‐ type control embryos and 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 / + 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 and Foxj1 in multiciliated cells of the trachea shown by double staining for X ‐ Gal and 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 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 .

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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 staining of paranasal cavities reveals mucus congestion along the nasal epithelium ( arrows ) .

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


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 ) .

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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 ( 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 ) .

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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 ‐ 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 ) .

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

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 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 .

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

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 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 .

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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 ' 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 ‐ 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.