( C ) Northern ( top panel ) and western ( middle , bottom panels ) blot analyses of total RNA and protein from SOD1G85R strains , with ( M1 / M1 ) or without ( + / + ) suppressor mutations , demonstrating that the levels of SOD1G85R mRNA and total protein are not changed by the suppressor mutation ( M1 / M1 ) . The western blot lanes are from the same gel and exposure .

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(C) Northern (top panel) and western (middle, bottom panels) blot analyses of total RNA and protein from SOD1G85R strains, with (M1/M1) or without (+/+) suppressor mutations, demonstrating that the levels of SOD1G85R mRNA and total protein are not changed by the suppressor mutation (M1/M1). The western blot lanes are from the same gel and exposure.


( E ) C . elegans SPR - 5 and UFD - 2 and their mammalian homologs lysine - specific demethylase 1 ( LSD1 ) and ubiquitination factor E4 B ( UBE4B ) share all major protein domains . These include the Swi3 - Rsc8 - Moira ( SWIRM ) , amine oxidase - like ( AOL ) , and TOWER domains in LSD1 / SPR - 5 and the Ufd2 Core and U - box domains in UBE4B / UFD - 2 . Positions of the missense , nonsense , and deletion mutations in mutant C . elegans are indicated .

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(E) C. elegans SPR-5 and UFD-2 and their mammalian homologs lysine-specific demethylase 1 (LSD1) and ubiquitination factor E4 B (UBE4B) share all major protein domains. These include the Swi3-Rsc8-Moira (SWIRM), amine oxidase-like (AOL), and TOWER domains in LSD1/SPR-5 and the Ufd2 Core and U-box domains in UBE4B/UFD-2. Positions of the missense, nonsense, and deletion mutations in mutant C. elegans are indicated.


( F ) Locomotor behavior , measured by thrashing rates , of the C . elegans carrying the SOD1G85R transgene on the normal background ( WT ) , with the null mutation of either ufd - 2 ( tm1380 ) or spr - 5 ( by134 ) , the double mutation ufd - 2 ( tm1380 ) ; spr - 5 ( by134 ) , or the M1 suppressor ufd - 2 ( W824X ) ; spr - 5 ( R646Q ) ( n = 16 ) .

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(F) Locomotor behavior, measured by thrashing rates, of the C. elegans carrying the SOD1G85R transgene on the normal background (WT), with the null mutation of either ufd-2(tm1380) or spr-5(by134), the double mutation ufd-2(tm1380);spr-5(by134), or the M1 suppressor ufd-2(W824X);spr-5(R646Q) (n = 16).


( G ) Western blotting of the supernatant ( S ) and pellet ( P ) protein fractions from the C . elegans carrying the SOD1G85R transgene with the double mutation ufd - 2 ( tm1380 ) ; spr - 5 ( by134 ) compared with controls . While SOD1G85R protein levels are unchanged in the S fraction , those in the P fraction are decreased in the double suppressor mutant . The P fraction represents only about 1 . 7 % of total SOD1G85R in the WT sample ; therefore , a higher ratio of the P fraction relative to the S fraction ( approximately 2 : 1 ) is used for the western analysis .

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(G) Western blotting of the supernatant (S) and pellet (P) protein fractions from the C. elegans carrying the SOD1G85R transgene with the double mutation ufd-2(tm1380);spr-5(by134) compared with controls. While SOD1G85R protein levels are unchanged in the S fraction, those in the P fraction are decreased in the double suppressor mutant. The P fraction represents only about 1.7% of total SOD1G85R in the WT sample; therefore, a higher ratio of the P fraction relative to the S fraction (approximately 2:1) is used for the western analysis.


( H ) The overexpression ( OE ) of WT UFD - 2 or SPR - 5 in the C . elegans nervous system blocks the protection conferred by the double mutation ufd - 2 ( tm1380 ) ; spr - 5 ( by134 ) ( n = 16 ) . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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(H) The overexpression (OE) of WT UFD-2 or SPR-5 in the C. elegans nervous system blocks the protection conferred by the double mutation ufd-2(tm1380);spr-5(by134) (n = 16). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) Top : schematic drawing depicts pan - neuronal expression of YFP in head and ventral neurons in the context of the C . elegans body plan . Left : micrographs show the SOD1G85R - YFP proteins expressed in WT or the spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) mutant background . The double - mutant worms show a marked decrease in protein aggregation in neurons . Enlarged sections of headneurons ( red framed ) and ventral cord neurons ( white framed ) are shown . Middle : quantification of locomotion in the spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) and the WT C . elegans with neuronal expression of SOD1G85R - YFP ( n = 30 ) . Right : a decrease in the protein levels of SOD1G85R - YFP in the presence of spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) is shown by western blots of the supernatant ( S ) and the pellet ( P ) fractions . ( B and C ) Analyses of the spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) and the WT C . elegans with neuronal expression of TDP - 43 - c25 - YFP ( n = 30 ) or PolyQ - YFP ( n = 12 ) as in ( A ) .

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(A) Top: schematic drawing depicts pan-neuronal expression of YFP in head and ventral neurons in the context of the C. elegans body plan. Left: micrographs show the SOD1G85R-YFP proteins expressed in WT or the spr-5(by134);ufd-2(tm1380) mutant background. The double-mutant worms show a marked decrease in protein aggregation in neurons. Enlarged sections of headneurons (red framed) and ventral cord neurons (white framed) are shown. Middle: quantification of locomotion in the spr-5(by134);ufd-2(tm1380) and the WT C. elegans with neuronal expression of SOD1G85R-YFP (n = 30). Right: a decrease in the protein levels of SOD1G85R-YFP in the presence of spr-5(by134);ufd-2(tm1380) is shown by western blots of the supernatant (S) and the pellet (P) fractions. (B and C) Analyses of the spr-5(by134);ufd-2(tm1380) and the WT C. elegans with neuronal expression of TDP-43-c25-YFP (n = 30) or PolyQ-YFP (n = 12) as in (A).


( D ) Neurodegenerative rough - eye phenotype in adults is alleviated by the knockdown of the Drosophila homologs of ufd - 2 / UBE4B and spr - 5 / LSD1 - CG9934 and Su ( Var ) 3 - 3 , respectively , compared to the control ( CTRL ) . Eye - specific expression of TDP - 43M337V , FUSR521C , and RNA interference ( RNAi ) was driven by GMR - Gal4 .

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(D) Neurodegenerative rough-eye phenotype in adults is alleviated by the knockdown of the Drosophila homologs of ufd-2/UBE4B and spr-5/LSD1-CG9934 and Su(Var)3-3, respectively, compared to the control (CTRL). Eye-specific expression of TDP-43M337V, FUSR521C, and RNA interference (RNAi) was driven by GMR-Gal4.


( E ) Drosophila eye pigment quantitation . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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(E) Drosophila eye pigment quantitation. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) Western blots of cell lysates derived from mock ( CTRL ) , single UBE4B or LSD1 , or double UBE4B and LSD1 knockdowns . Supernatant ( S ) and pellet ( P ) fractions were probed with indicated antibodies . While the LSD1 or UBE4B single - knockdown reduces the SOD1G85R aggregates in both supernatant and pellet fractions , the combined knockdown produces the strongest reduction in the aggregates . ( B ) Quantification of SOD1G85R protein levels by western blotting ( A ) . n = 3 ( supernatant ) ; n = 8 ( pellet ) .

(A) Western blots of cell lysates derived from mock (CTRL), single UBE4B or LSD1, or double UBE4B and LSD1 knockdowns. Supernatant (S) and pellet (P) fractions were probed with indicated antibodies. While the LSD1 or UBE4B single-knockdown reduces the SOD1G85R aggregates in both supernatant and pellet fractions, the combined knockdown produces the strongest reduction in the aggregates. (B) Quantification of SOD1G85R protein levels by western blotting (A). n = 3 (supernatant); n = 8 (pellet).


( C ) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half - lives in the double UBE4B and LSD1 knockdown cells versus controls . ( D ) Quantification of SOD1G85R clearance , as analyzed by western blotting in ( C ) . The graph indicates the relative band intensity of SOD1G85R at each chase time point . n = 5 ; Overall p = 0 . 02 ( paired t test , CTRL versus UBE4B and LSD1 double knockdown ) . Individual p = 0 . 03 ( 3 h ) , p = 0 . 003 ( 6 h ) , p = 0 . 06 ( 9 h ) , and p = 0 . 004 ( 12 - 21 h ) .

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(C) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half-lives in the double UBE4B and LSD1 knockdown cells versus controls. (D) Quantification of SOD1G85R clearance, as analyzed by western blotting in (C). The graph indicates the relative band intensity of SOD1G85R at each chase time point. n = 5; Overall p = 0.02 (paired t test, CTRL versus UBE4B and LSD1 double knockdown). Individual p = 0.03 (3 h), p = 0.003 (6 h), p = 0.06 (9 h), and p = 0.004 (12-21 h).


( E ) The half - life of SOD1G85R is reduced from 8 . 5 h to 5 h upon knockdown of UBE4B and LSD1 . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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(E) The half-life of SOD1G85R is reduced from 8.5 h to 5 h upon knockdown of UBE4B and LSD1. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( B ) The volcano scatter plot indicates fold changes in the levels of gene transcripts affected differentially by the UBE4B and LSD1 double knockdown versus control shRNA . Gray spots represent 22 , 148 annotated transcripts . Red spots are predicted p53 - activated targets , and blue spots are predicted p53 - inhibited targets . The enrichment of red spots in the up - regulated genes ( right upper quadrant , > 1 . 2 - fold change , p ≤ 0 . 04 ) and blue spots in the down - regulated genes ( left upper quadrant ) indicates that the p53 - mediated transcription is activated by the UBE4B and LSD1 double knockdown .

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(B) The volcano scatter plot indicates fold changes in the levels of gene transcripts affected differentially by the UBE4B and LSD1 double knockdown versus control shRNA. Gray spots represent 22,148 annotated transcripts. Red spots are predicted p53-activated targets, and blue spots are predicted p53-inhibited targets. The enrichment of red spots in the up-regulated genes (right upper quadrant, >1.2-fold change, p ≤ 0.04) and blue spots in the down-regulated genes (left upper quadrant) indicates that the p53-mediated transcription is activated by the UBE4B and LSD1 double knockdown.


C ) RT - qPCR validation of the expression levels of representative p53 target genes in the UBE4B and LSD1 double - knockdown samples . n = ( 2 to 6 ) , p ≤ 0 . 04 .

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C) RT-qPCR validation of the expression levels of representative p53 target genes in the UBE4B and LSD1 double-knockdown samples. n = (2 to 6), p ≤ 0.04.


( D ) The p53 protein level is significantly increased by UBE4B and LSD1 double knockdown . Left : representative western blots showing p53 levels , the knockdown of UBE4B and LSD1 , and the actin control in HEK293T cells . Right : quantification of the p53 protein levels normalized against actin ( n = 3 ) .

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(D) The p53 protein level is significantly increased by UBE4B and LSD1 double knockdown. Left: representative western blots showing p53 levels, the knockdown of UBE4B and LSD1, and the actin control in HEK293T cells. Right: quantification of the p53 protein levels normalized against actin (n = 3).


E ) The p53 - mediated transcriptional activity is measured by a luciferase reporter under the control of a p53 - response element promoter , which was transfected into HEK293T cells 72 - 96 h after the initiation of the UBE4B and LSD1 single or double knockdowns ( n = 6 ) .

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E) The p53-mediated transcriptional activity is measured by a luciferase reporter under the control of a p53-response element promoter, which was transfected into HEK293T cells 72-96 h after the initiation of the UBE4B and LSD1 single or double knockdowns (n = 6).


( F ) Increased interaction of p53 with its activating partner 53BP1 in response to UBE4B and LSD1 knockdown . Left : representative western blots of 53BP1 co - immunoprecipitation . Right : quantification of the 53BP1 co - immunoprecipitation ( n = 2 ) . Data represent means ± SEM .

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(F) Increased interaction of p53 with its activating partner 53BP1 in response to UBE4B and LSD1 knockdown. Left: representative western blots of 53BP1 co-immunoprecipitation. Right: quantification of the 53BP1 co-immunoprecipitation (n = 2). Data represent means ± SEM.


( G ) RT - qPCR validation of the expression levels of FOXO3a , FOXO4 , and PSMD11 ( n = 6 ) .

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(G) RT-qPCR validation of the expression levels of FOXO3a, FOXO4, and PSMD11 (n = 6).


( H ) The FOXO3a - mediated transcriptional activity was measured with a luciferase reporter under the control of a FOXO - response element promoter . HEK293T cells were transfected with shRNAs for control , LSD1 , UBE4B , or both LSD1 and UBE4B , followed by transfection of the luciferase reporter together with a constitutively active form of FOXO3a ( TM ) 72 - 96 h later ( n = 6 ) . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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(H) The FOXO3a-mediated transcriptional activity was measured with a luciferase reporter under the control of a FOXO-response element promoter. HEK293T cells were transfected with shRNAs for control, LSD1, UBE4B, or both LSD1 and UBE4B, followed by transfection of the luciferase reporter together with a constitutively active form of FOXO3a (TM) 72-96 h later (n = 6). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) Increased protein levels of proteasome subunits upon the UBE4B and LSD1 double knockdown in HEK293T

(A) Increased protein levels of proteasome subunits upon the UBE4B and LSD1 double knockdown in HEK293T cells


. ( B ) The UBE4B and LSD1 single or double knockdowns increase the proteasomal degradation of a reporter substrate , Suc - LLVY - Luciferin , whose degradation is measured in a luciferase release assay . The double knockdown shows synergistic proteasomal activation when compared with the individual knockdowns ( n = 6 ) .

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. (B) The UBE4B and LSD1 single or double knockdowns increase the proteasomal degradation of a reporter substrate, Suc-LLVY-Luciferin, whose degradation is measured in a luciferase release assay. The double knockdown shows synergistic proteasomal activation when compared with the individual knockdowns (n = 6).


( C ) The autophagy activity is significantly increased in cells with the UBE4B and LSD1 double knockdown . The in vivo activity of the ATG4B protease , which cleaves LC3 precursors , was quantified via a Gaussia luciferase release assay ( see S5D Fig . , S5E Fig . , and Materials and Methods for details ) . The HCT116 cells were analyzed 48 h after the initiation of the knockdown .

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(C) The autophagy activity is significantly increased in cells with the UBE4B and LSD1 double knockdown. The in vivo activity of the ATG4B protease, which cleaves LC3 precursors, was quantified via a Gaussia luciferase release assay (see S5D Fig., S5E Fig., and Materials and Methods for details). The HCT116 cells were analyzed 48 h after the initiation of the knockdown.


( D ) Quantification of LC3 - II levels in HEK293T cells with the UBE4B and LSD1 double knockdown or the mock control . The cells were treated with or without 10 mM 3 - methyladenine ( 3 - MA ) for 48h before lysis . Western blots indicate an increase in LC3 levels ( top panels ) in the double UBE4B and LSD1 knockdown cells ( middle panels ) , while the actin control is unchanged ( bottom panels ) . The graph shows the quantification of LC3 - II levels as normalized to actin levels ( n = 3 ) . A one - way ANOVA test with matched multiple comparisons and Tukey correction was used for statistics . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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(D) Quantification of LC3-II levels in HEK293T cells with the UBE4B and LSD1 double knockdown or the mock control. The cells were treated with or without 10 mM 3-methyladenine (3-MA) for 48h before lysis. Western blots indicate an increase in LC3 levels (top panels) in the double UBE4B and LSD1 knockdown cells (middle panels), while the actin control is unchanged (bottom panels). The graph shows the quantification of LC3-II levels as normalized to actin levels (n = 3). A one-way ANOVA test with matched multiple comparisons and Tukey correction was used for statistics. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) p53 small molecule activators Tenovin - 1 and CP - 31398 reduce the levels of misfolded SOD1 proteins , as determined by the SOD1G85R solubility assay in HEK293 cells . Increasing concentrations of the p53 activators significantly decrease the levels of SOD1G85R but not the endogenous WT SOD1 proteins in western blots of both supernatant and pellet fractions .

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(A) p53 small molecule activators Tenovin-1 and CP-31398 reduce the levels of misfolded SOD1 proteins, as determined by the SOD1G85R solubility assay in HEK293 cells. Increasing concentrations of the p53 activators significantly decrease the levels of SOD1G85R but not the endogenous WT SOD1 proteins in western blots of both supernatant and pellet fractions.


( B ) A decrease in p53 as the result of shRNA knockdown increases the levels of SOD1G85R but not WT SOD1 proteins in the SOD1G85R aggregation assay , as shown by western blots of both supernatant ( S ) ( n = 2 ) and pellet ( P ) ( n = 3 ) fractions .

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(B) A decrease in p53 as the result of shRNA knockdown increases the levels of SOD1G85R but not WT SOD1 proteins in the SOD1G85R aggregation assay, as shown by western blots of both supernatant (S) (n = 2) and pellet (P) (n = 3) fractions.


( C ) A complete absence of p53 increases the accumulation of SOD1G85R mutant proteins in p53 - / - HCT116 cells when compared with controls . Representative western blots ( left panels ) and quantification of SOD1G85R levels in the supernatant lysates are shown . The middle graph indicates the ratio of G85R to WT SOD1 proteins in the presence or absence of p53 with varying amounts of transfected mutant SOD1 . The right graph panel shows the same data as shown in the middle panel , but normalized to the average SOD1G85R level for each amount of the transfected plasmid . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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(C) A complete absence of p53 increases the accumulation of SOD1G85R mutant proteins in p53-/- HCT116 cells when compared with controls. Representative western blots (left panels) and quantification of SOD1G85R levels in the supernatant lysates are shown. The middle graph indicates the ratio of G85R to WT SOD1 proteins in the presence or absence of p53 with varying amounts of transfected mutant SOD1. The right graph panel shows the same data as shown in the middle panel, but normalized to the average SOD1G85R level for each amount of the transfected plasmid. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) Left : p53 knockdown reverses the enhanced clearance of SOD1G85R proteins conferred by the UBE4B and LSD1 double knockdown . Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs . Right : quantification of insoluble aggregated SOD1G85R in pellet fractions from HEK293T cells transfected with control , double ( UBE4B / LSD1 ) , or triple ( UBE4B / LSD1 / p53 ) shRNAs ( n = 2 ) .

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(A) Left: p53 knockdown reverses the enhanced clearance of SOD1G85R proteins conferred by the UBE4B and LSD1 double knockdown. Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs. Right: quantification of insoluble aggregated SOD1G85R in pellet fractions from HEK293T cells transfected with control, double (UBE4B/LSD1), or triple (UBE4B/LSD1/p53) shRNAs (n = 2).


( B ) The degenerative TDP - 43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 ( p53 RNAi ) , or by the overexpression of a dominant - negative p53 mutant ( p53 . R155H ) . Expression of p53 RNAi , p53 . R155H , and TDP - 43M337V are driven by GMR - Gal4 .

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(B) The degenerative TDP-43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 (p53 RNAi), or by the overexpression of a dominant-negative p53 mutant (p53.R155H). Expression of p53 RNAi, p53.R155H, and TDP-43M337V are driven by GMR-Gal4.


( C ) The p53 - activating drug Tenovin - 1 ( TEN1 ) protects spinal cord motor neurons from SOD1G85R - induced proteotoxicity . Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0 . 8 μM TEN1 or vehicle ( VEH ) DMSO , followed by infection of HSV - SOD1G85R or the HSV - LacZ control after 24 h . At day 5 post - infection , cells were fixed and stained with a motor neuron - specific anti - neurofilament H ( NF - H ) antibody . Left : representative images of motor neurons in each condition . Scale bar = 50 μm . Right : quantification of motor neuron survival ( one - way ANOVA with multiple comparisons and Tukey correction ) . Data represent means ± SEM .

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(C) The p53-activating drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SOD1G85R-induced proteotoxicity. Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0.8 μM TEN1 or vehicle (VEH) DMSO, followed by infection of HSV-SOD1G85R or the HSV-LacZ control after 24 h. At day 5 post-infection, cells were fixed and stained with a motor neuron-specific anti-neurofilament H (NF-H) antibody. Left: representative images of motor neurons in each condition. Scale bar = 50 μm. Right: quantification of motor neuron survival (one-way ANOVA with multiple comparisons and Tukey correction). Data represent means ± SEM.


( C ) Representative confocal image of long propriospinal neurons expressing FGF22 ( magenta ) in cervical spinal cord .

(C) Representative confocal image of long propriospinal neurons expressing FGF22 (magenta) in cervical spinal cord.


( E ) Schematic illustration of the hCST collateral into the cervical cord and confocal images of hCST collaterals ( green ) , stained for vGlut ( red ) and vGAT ( blue ) 21 days following spinal cord injury . Insets represent 3D views generated in Imaris of the deconvolved confocal image . ( F ) Quantification of bouton density and characterization of bouton between control and FGF22 overexpressing mice ( n = 4 - 5 mice per group ) . (

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

(E) Schematic illustration of the hCST collateral into the cervical cord and confocal images of hCST collaterals (green), stained for vGlut (red) and vGAT (blue) 21 days following spinal cord injury. Insets represent 3D views generated in Imaris of the deconvolved confocal image. (F) Quantification of bouton density and characterization of bouton between control and FGF22 overexpressing mice (n = 4-5 mice per group). (


( G ) Schematic illustration and representative confocal image of LPSN viral labeling ( magenta ) and CST collaterals ( green ) . Insets represent 3D views generated in Imaris of the deconvolved confocal image . ( H ) Quantification of hCST contacts onto LPSNs and characterization of vGlut intensity onto individual LPSNs ( n = 4 - 5 mice per group ) .

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

(G) Schematic illustration and representative confocal image of LPSN viral labeling (magenta) and CST collaterals (green). Insets represent 3D views generated in Imaris of the deconvolved confocal image. (H) Quantification of hCST contacts onto LPSNs and characterization of vGlut intensity onto individual LPSNs (n = 4-5 mice per group).


( C ) Schematic illustration and representative confocal image for vGlut2 - Cre neuronal labeling ( cyan ) and hCST collaterals ( white ) . Insets represent 3D views generated in Imaris of the confocal image . Quantification of hCST contacts onto vGlut neurons ( n = 4 - 6 mice per group ) .

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

(C) Schematic illustration and representative confocal image for vGlut2-Cre neuronal labeling (cyan) and hCST collaterals (white). Insets represent 3D views generated in Imaris of the confocal image. Quantification of hCST contacts onto vGlut neurons (n = 4-6 mice per group).


( C ) Schematic illustration of the hCST collateral exiting into the cervical cord and confocal images showing exiting hCST collaterals in control ( left panel ) and FGF22 treated ( right panel ) mice and quantification of the number of hCST collaterals that enter the cervical grey matter 3 weeks following spinal cord injury . ( n = 6 mice per group ) .

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

(C) Schematic illustration of the hCST collateral exiting into the cervical cord and confocal images showing exiting hCST collaterals in control (left panel) and FGF22 treated (right panel) mice and quantification of the number of hCST collaterals that enter the cervical grey matter 3 weeks following spinal cord injury. (n = 6 mice per group).


( E ) Schematic illustration of the boutons on cervical hCST collateral and confocal images showing putative synaptic boutons ( arrows ) on newly formed cervical hCST collaterals at 3 weeks following spinal cord injury in a FGF22 ( right ) and a control ( left ) mice . Quantification of bouton density in control and FGF22 treated mice . ( n = 6 mice per

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Schematic illustration of the boutons on cervical hCST collateral and confocal images showing putative synaptic boutons (arrows) on newly formed cervical hCST collaterals at 3 weeks following spinal cord injury in a FGF22 (right) and a control (left) mice. Quantification of bouton density in control and FGF22 treated mice. (n = 6 mice per group


( F ) Schematic illustration of the hCST contacts onto relay neurons in the cervical cord and confocal images showing contacts between CST collaterals ( red ) and cervical interneurons ( cyan ) . Insets represent 3D views generated in Imaris of the confocal image . Quantification of the contacts between the hCST collaterals and cervical interneurons neurons . ( n = 6 per group ) .

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

(F) Schematic illustration of the hCST contacts onto relay neurons in the cervical cord and confocal images showing contacts between CST collaterals (red) and cervical interneurons (cyan). Insets represent 3D views generated in Imaris of the confocal image. Quantification of the contacts between the hCST collaterals and cervical interneurons neurons. (n = 6 per group).


( G ) Confocal images showing contacts between CST collaterals ( red ) and long propriospinal neurons ( magenta ) transduced with FGF22 ( cyan ) . Quantification of the contacts between the hCST collaterals and long propriospinal transduced with FGF22 or control virus ( DP refers to double positive cells ) . Insets on the right are magnification of the boxed area on the left . ( n = 6 mice per group ) .

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

(G) Confocal images showing contacts between CST collaterals (red) and long propriospinal neurons (magenta) transduced with FGF22 (cyan). Quantification of the contacts between the hCST collaterals and long propriospinal transduced with FGF22 or control virus (DP refers to double positive cells). Insets on the right are magnification of the boxed area on the left. (n = 6 mice per group).


( C ) Timeline of the experiment with FGF22 injection right after the onset of the spinal cord lesion ( top ) . Quantification of the functional recovery in the regular ( middle ) and irregular ( bottom ) ladder rung test in controls ( grey ) and FGF22 ( orange ) treated mice at baseline , at 3 days post injury ( " 3dpi " ) , 14dpi and 21dpi after spinal cord injury . ( n = 9 - 10 mice per group ) . ( D ) Timeline of the experiment with FGF22 injection 24hrs after the onset of the spinal cord lesion ( top ) . Quantification of the functional recovery in the regular ( middle ) and irregular ( bottom ) ladder rung test in control ( grey ) and FGF22 ( orange ) treated mice at baseline , at 3 days post injury ( " 3dpi " or 2 days " 2dpi for the late treatment ) , 14dpi and 21dpi after spinal cord injury . ( n = 9 - 10 per group ) . ( E ) Timeline of the experiment with FGF22 injection 5 days after the onset of the spinal cord lesion ( top ) . Quantification of the functional recovery in the regular ( middle ) and irregular ( bottom ) ladder rung test in control ( grey ) and FGF22 ( orange ) treated mice at baseline , at 3 days post injury ( " 3dpi " ) , 14dpi and 21dpi after spinal cord injury . ( n = 9 - 10 per group ) . ) . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Timeline of the experiment with FGF22 injection right after the onset of the spinal cord lesion (top). Quantification of the functional recovery in the regular (middle) and irregular (bottom) ladder rung test in controls (grey) and FGF22 (orange) treated mice at baseline, at 3 days post injury ("3dpi"), 14dpi and 21dpi after spinal cord injury. (n = 9-10 mice per group). (D) Timeline of the experiment with FGF22 injection 24hrs after the onset of the spinal cord lesion (top). Quantification of the functional recovery in the regular (middle) and irregular (bottom) ladder rung test in control (grey) and FGF22 (orange) treated mice at baseline, at 3 days post injury ("3dpi" or 2 days "2dpi for the late treatment), 14dpi and 21dpi after spinal cord injury. (n = 9-10 per group). (E) Timeline of the experiment with FGF22 injection 5 days after the onset of the spinal cord lesion (top). Quantification of the functional recovery in the regular (middle) and irregular (bottom) ladder rung test in control (grey) and FGF22 (orange) treated mice at baseline, at 3 days post injury ("3dpi"), 14dpi and 21dpi after spinal cord injury. (n = 9-10 per group).). (F


( F ) Representative confocal image of CST boutons ( green ) and synapsin staining ( red ) and quantification of the percentage of bouton synapsin positive ( n = 6 - 7 mice per group ) . ( G ) Representative confocal images of CST bouton ( green ) and bassoon staining ( magenta ) and quantification of the percentage of boutons that are bassoon positive ( n = 5 - 6 mice per group ) .

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

(F) Representative confocal image of CST boutons (green) and synapsin staining (red) and quantification of the percentage of bouton synapsin positive (n = 6-7 mice per group). (G) Representative confocal images of CST bouton (green) and bassoon staining (magenta) and quantification of the percentage of boutons that are bassoon positive (n = 5-6 mice per group). D


A In silico analysis of Sirt7 mRNA expression in different tissues of the BXD genetic reference population ( www . genenetwork . org ) . Adr : Adrenal Gland mRNA , Bla : Bladder mRNA , Bone : Bone Tibia mRNA , CNS : CNS mRNA , Cec : Cecum mRNA , Epi : Epididymis mRNA , Eso : Esophagus mRNA , Eye : Eye mRNA , Fat : Peritoneal Fat mRNA , GI : GI Track mRNA , Hea : Heart mRNA , Kid : Kidney mRNA , Liv : Liver mRNA , Lung : Lung mRNA , Mus : Gastrocnemius Muscle mRNA , Ova : Ovary mRNA , Pro : Prostate mRNA , Sal : Salivary Gland mRNA , Skin : Skin Back mRNA , Sn : Sciatic Nerve mRNA , Spl : Spleen mRNA , Tes : Testis mRNA , Thy : Thymus mRNA , Ton : Tongue mRNA , Tra : Trachea mRNA , and Ute : Uterus mRNA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In silico analysis of Sirt7 mRNA expression in different tissues of the BXD genetic reference population (www.genenetwork.org). Adr: Adrenal Gland mRNA, Bla: Bladder mRNA, Bone: Bone Tibia mRNA, CNS: CNS mRNA, Cec: Cecum mRNA, Epi: Epididymis mRNA, Eso: Esophagus mRNA, Eye: Eye mRNA, Fat: Peritoneal Fat mRNA, GI: GI Track mRNA, Hea: Heart mRNA, Kid: Kidney mRNA, Liv: Liver mRNA, Lung: Lung mRNA, Mus: Gastrocnemius Muscle mRNA, Ova: Ovary mRNA, Pro: Prostate mRNA, Sal: Salivary Gland mRNA, Skin: Skin Back mRNA, Sn: Sciatic Nerve mRNA, Spl: Spleen mRNA, Tes: Testis mRNA, Thy: Thymus mRNA, Ton: Tongue mRNA, Tra: Trachea mRNA, and Ute: Uterus mRNA.


B Correlation plot for kidney mRNA expression of Sirt7 and Slc12a7 that codifies KCC4 in the mouse BxD genetic reference population ( www . genenetwork . org ) .

O O O O O 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 Correlation plot for kidney mRNA expression of Sirt7 and Slc12a7 that codifies KCC4 in the mouse BxD genetic reference population (www.genenetwork.org).


C Oocytes injected with cRNA encoding KCC4 were incubated with NAD + 200 μM and NAM 10 mM for 4 h . Lysates were immunoprecipitated with an anti - KCC4 antibody and then analyzed by SDS - PAGE / immunoblotting using an anti - acetylated - lysine ( AcK ) or anti - KCC4 antibody . Non - injected oocytes ( UI ) were used as a control .

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C Oocytes injected with cRNA encoding KCC4 were incubated with NAD+ 200 μM and NAM 10 mM for 4 h. Lysates were immunoprecipitated with an anti-KCC4 antibody and then analyzed by SDS-PAGE/immunoblotting using an anti-acetylated-lysine (AcK) or anti-KCC4 antibody. Non-injected oocytes (UI) were used as a control.


D Oocytes expressing KCC4 were incubated with NAM for the indicated times , and KCC4 protein levels ( upper panel ) , and activity ( lower panel ) were analyzed by SDS - PAGE / immunoblotting or Cl - - dependent 86Rb + uptake under hypotonic conditions .

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D Oocytes expressing KCC4 were incubated with NAM for the indicated times, and KCC4 protein levels (upper panel), and activity (lower panel) were analyzed by SDS-PAGE/immunoblotting or Cl--dependent 86Rb+ uptake under hypotonic conditions.


E Oocytes injected with H20 or KCC4 were incubated for the indicated times , and KCC4 protein levels were analyzed by SDS - PAGE / immunoblotting .

O O O 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 Oocytes injected with H20 or KCC4 were incubated for the indicated times, and KCC4 protein levels were analyzed by SDS-PAGE/immunoblotting.


F Oocytes were injected with either WT , K114R or K114Q KCC4 and incubated with vehicle ( V ) , NAD + or NAM for 48 h , and KCC4 protein levels ( upper pannel ) , and activity ( lower pannel ) were analyzed as in D .

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F Oocytes were injected with either WT, K114R or K114Q KCC4 and incubated with vehicle (V), NAD+ or NAM for 48 h, and KCC4 protein levels (upper pannel), and activity (lower pannel) were analyzed as in D.


A , B KCC4 activity ( A ) , and protein levels ( B ) were evaluated in Xenopus oocytes expressing SIRT7 and KCC4 that were incubated for the indicated times with NAD + . Non - injected oocytes ( UI ) were used as a control .

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

A, B KCC4 activity (A), and protein levels (B) were evaluated in Xenopus oocytes expressing SIRT7 and KCC4 that were incubated for the indicated times with NAD+. Non-injected oocytes (UI) were used as a control.


C KCC4 activity was evaluated in Xenopus oocytes injected with cRNA encoding KCC4 and coinjected with either wild - type ( SIRT7 WT ) or the catalytically inactive SIRT7 ( SIRT7 H188Y ) for 48 h .

O O O O O O O O O O O O O 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 KCC4 activity was evaluated in Xenopus oocytes injected with cRNA encoding KCC4 and coinjected with either wild-type (SIRT7 WT) or the catalytically inactive SIRT7 (SIRT7 H188Y) for 48 h.


D Oocytes were injected with either KCC4 WT , K114R or K114Q for 48 h and then incubated with vehicle ( V ) , NAD + or NAM for 24 h , and KCC4 and SIRT7 protein levels were analyzed by SDS - PAGE / immunoblotting .

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D Oocytes were injected with either KCC4 WT, K114R or K114Q for 48 h and then incubated with vehicle (V), NAD+ or NAM for 24 h, and KCC4 and SIRT7 protein levels were analyzed by SDS-PAGE/immunoblotting.


E Oocytes were injected with either KCC4 WT , K114R or K114Q with and without SIRT7 for 48 h and then incubated with vehicle ( V ) or NAM for 4 h . After lysis , KCC4 was immunoprecipitated using an anti - KCC4 antibody , and the acetylation status of KCC4 was determined by SDS - PAGE / Western blotting using and anti - acetyl lysine antibody .

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E Oocytes were injected with either KCC4 WT, K114R or K114Q with and without SIRT7 for 48 h and then incubated with vehicle (V) or NAM for 4 h. After lysis, KCC4 was immunoprecipitated using an anti-KCC4 antibody, and the acetylation status of KCC4 was determined by SDS-PAGE/Western blotting using and anti-acetyl lysine antibody.


F , G FLAG - KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD + ( F ) or NAM ( G ) and then analyzed by SDS - PAGE / immunoblotting using an anti - KCC4 or anti - SIRT7 antibody . Non - transfected cells ( UT ) were used as a control .

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F, G FLAG-KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD+ (F) or NAM (G) and then analyzed by SDS-PAGE/immunoblotting using an anti-KCC4 or anti-SIRT7 antibody. Non-transfected cells (UT) were used as a control.


H FLAG - KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD + and NAM . After lysis , KCC4 immunoprecipitation using an anti - FLAG antibody was performed , and the interaction with SIRT7 was determined by SDS - PAGE / Western blotting .

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H FLAG-KCC4 and SIRT7 were transfected into HEK293 cells and treated for the indicated times with NAD+ and NAM. After lysis, KCC4 immunoprecipitation using an anti-FLAG antibody was performed, and the interaction with SIRT7 was determined by SDS-PAGE/Western blotting.


I HEK293 cells expressing KCC4 were incubated with NAD + 200 μM and cycloheximide ( CHX ) for 0 , 3 , 6 and 9 h . KCC4 protein levels are shown at the indicated times in cells transfected with a scramble shRNA ( + SIRT7 ) or a shRNA against SIRT7 ( - SIRT7 ) .

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I HEK293 cells expressing KCC4 were incubated with NAD+ 200 μM and cycloheximide (CHX) for 0, 3, 6 and 9 h. KCC4 protein levels are shown at the indicated times in cells transfected with a scramble shRNA (+SIRT7) or a shRNA against SIRT7 (-SIRT7).


A KCC4 acetylation at the indicated pH in HEK293 transfected with KCC4 . After lysis , KCC4 was immunoprecipitated using an anti - FLAG antibody , and then analyzed by SDS - PAGE / Western blotting using an anti - acetylated - lysine ( AcK ) or anti - KCC4 antibody . Non - transfected cells ( UT ) were used as a control .

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

A KCC4 acetylation at the indicated pH in HEK293 transfected with KCC4. After lysis, KCC4 was immunoprecipitated using an anti-FLAG antibody, and then analyzed by SDS-PAGE/Western blotting using an anti-acetylated-lysine (AcK) or anti-KCC4 antibody. Non-transfected cells (UT) were used as a control.


B HEK293 cells were transfected with KCC4 together with SIRT7 with a scramble shRNA ( - ) or a shRNA against SIRT7 ( + ) , and KCC4 activity and protein level were determined at the indicated pH . SIRT7 expression in these conditions is presented in C .

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

B HEK293 cells were transfected with KCC4 together with SIRT7 with a scramble shRNA (-) or a shRNA against SIRT7 (+), and KCC4 activity and protein level were determined at the indicated pH. SIRT7 expression in these conditions is presented in C.


C Nuclear and cytoplasmic SIRT7 levels at different pH values in HEK293 cells . At a lower pH , SIRT7 appears to translocate to the cytoplasm .

C Nuclear and cytoplasmic SIRT7 levels at different pH values in HEK293 cells. At a lower pH, SIRT7 appears to translocate to the cytoplasm.


A , B Sirt7 mRNA ( A ) and protein ( B ) abundance in microdissected renal tubules normalized to Rpl26 or Ponceau , respectively . Enrichment for each segment was previously validated by immunoblotting against segment - specific markers as indicated in material and methods ( Spirli et al , 2019 ) .

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

A, B Sirt7 mRNA (A) and protein (B) abundance in microdissected renal tubules normalized to Rpl26 or Ponceau, respectively. Enrichment for each segment was previously validated by immunoblotting against segment-specific markers as indicated in material and methods (Spirli et al, 2019).


C Immunolocalization of SIRT7 and ATPase in the kidneys of C57BL6 mice with metabolic acidosis ( SIRT7 , red ; ATPase , green ; DAPI , Blue ) . Scale bar : 20 μm . Controls of the specificity of antibodies using kidney of SIRT7 - deficient mice are provided in Appendix Fig S4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunolocalization of SIRT7 and ATPase in the kidneys of C57BL6 mice with metabolic acidosis (SIRT7, red; ATPase, green; DAPI, Blue). Scale bar: 20 μm. Controls of the specificity of antibodies using kidney of SIRT7-deficient mice are provided in Appendix Fig S4.


D KCC4 and SIRT7 protein levels in kidneys from control mice or mice with metabolic acidosis . Values are the mean ± SEM .

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D KCC4 and SIRT7 protein levels in kidneys from control mice or mice with metabolic acidosis. Values are the mean ± SEM.


A KCC4 protein levels were determined in the kidneys of wild - type ( SIRT7 + / + ) and SIRT7 - deficient mice ( SIRT7 - / - ) . SIRT7 protein expression are absent in the kidneys of SIRT7 - deficient mice .

O O O O O O O O O O O O O O O O O O O O O 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 KCC4 protein levels were determined in the kidneys of wild-type (SIRT7+/+) and SIRT7-deficient mice (SIRT7-/-). SIRT7 protein expression are absent in the kidneys of SIRT7-deficient mice.


B KCC4 protein levels , acetylation status of KCC4 , and interaction with SIRT7 in kidneys of SIRT7 + / + and SIRT7 - / - mice under normal conditions ( Control ) or with metabolic acidosis .

O O O O O O O O O O O O O O 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 KCC4 protein levels, acetylation status of KCC4, and interaction with SIRT7 in kidneys of SIRT7+/+ and SIRT7-/-mice under normal conditions (Control) or with metabolic acidosis.


C Immunolocalization of KCC4 and H - ATPase in the kidneys of SIRT7 + / + and SIRT7 - / - mice under normal conditions ( Control ) or with metabolic acidosis ( NH4Cl ) . ( KCC4 , red ; H - ATPase , green ) . Scale bar : 20 μm . Cell count was performed automatically using the Gen5 software to report the percentage of KCC4 + cells among H - ATPase + cells ( lower panel ) .

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C Immunolocalization of KCC4 and H-ATPase in the kidneys of SIRT7+/+ and SIRT7-/- mice under normal conditions (Control) or with metabolic acidosis (NH4Cl). (KCC4, red; H-ATPase, green). Scale bar: 20 μm. Cell count was performed automatically using the Gen5 software to report the percentage of KCC4+ cells among H-ATPase+ cells (lower panel).


D NCC and NKCC2 protein levels in the kidney of SIRT7 + / + and SIRT7 - / - mice under basal conditions .

D NCC and NKCC2 protein levels in the kidney of SIRT7+/+ and SIRT7-/- mice under basal conditions.


B Analysis of anti - Bax and anti - Bak antibody specificity using whole cell lysates from HCT116 wild - type , Bax KO , Bak KO and Bax / Bak DKO . After detection of Bax the membrane was redecorated with anti - Bak antibodies ( Bax / Bak ) . Actin is used as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Analysis of anti-Bax and anti-Bak antibody specificity using whole cell lysates from HCT116 wild-type, Bax KO, Bak KO and Bax/Bak DKO. After detection of Bax the membrane was redecorated with anti-Bak antibodies (Bax/Bak). Actin is used as loading control.


C Western blot analysis of Bak in the heavy membrane ( HM ) fraction of human heart , cerebral cortex ( CNS GM ) , kidney , brain white matter ( CNS WM ) , lung , liver , and skeletal muscle tissue . The cytosol fraction obtained from CNS cortex ( C ) , LDH , and Tom20 serve as fractionation and loading controls . n = 3 .

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C Western blot analysis of Bak in the heavy membrane (HM) fraction of human heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue. The cytosol fraction obtained from CNS cortex (C), LDH, and Tom20 serve as fractionation and loading controls. n = 3.


D Presence of Bak in the cytosol of heart , cerebral cortex ( CNS GM ) , kidney , brain white matter ( CNS WM ) , lung , liver , and skeletal muscle tissue analyzed by Western blot in parallel to C . The heavy membrane heart fraction ( HM ) , LDH and Tom20 serve as fractionation and loading controls . n = 3 .

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

D Presence of Bak in the cytosol of heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue analyzed by Western blot in parallel to C. The heavy membrane heart fraction (HM), LDH and Tom20 serve as fractionation and loading controls. n = 3.


E Carbonate extraction of the heavy membrane protein fraction of human brain white matter ( CNS WM ) and lung tissue analyzed for the presence of Bak by Western blot . Membrane - associated protein supernatant ( S ) and membrane - integral protein pellet ( P ) are displayed . Smac is released from the mitochondrial inter membrane space ( IMS ) during carbonate extraction and CoxIV remains in the pellet . n = 2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Carbonate extraction of the heavy membrane protein fraction of human brain white matter (CNS WM) and lung tissue analyzed for the presence of Bak by Western blot. Membrane-associated protein supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the mitochondrial inter membrane space (IMS) during carbonate extraction and CoxIV remains in the pellet. n = 2.


A Confocal images of HCT116 Bax / Bak DKO cells transfected with GFP - Bak in the absence ( left ) or the presence ( right ) of Bcl - xL overexpression . Scale bar 15 µm . n ≥ 10 .

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A Confocal images of HCT116 Bax/Bak DKO cells transfected with GFP-Bak in the absence (left) or the presence (right) of Bcl-xL overexpression. Scale bar 15 µm. n ≥ 10.


B FLIP ( Fluorescence Loss in Photobleaching ) of mitochondrial GFP - Bak in the absence ( ● , straight line ) or the presence ( ○ , broken line ) of overexpressed Bcl - xL . Fluorescence of the neighboring cell ( in Fig . 2C ) is shown as control ( , dotted line ) . Data represent averages ± SEM from 16 ( - Bcl - xL ) and 40 ( + Bcl - xL ) ROI measurements .

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B FLIP (Fluorescence Loss in Photobleaching) of mitochondrial GFP-Bak in the absence (●, straight line) or the presence (○, broken line) of overexpressed Bcl-xL. Fluorescence of the neighboring cell (in Fig. 2C) is shown as control (, dotted line). Data represent averages ± SEM from 16 (-Bcl-xL) and 40 (+Bcl-xL) ROI measurements.


C FLIP of GFP - Bak in the absence ( top ) or the presence ( bottom ) of overexpressed Bcl - xL diminishes GFP - Bak fluorescence in the cytosol of targeted cells ( circled ) completely after 90 s and GFP fluorescence is detected only on the mitochondria ( arrows ) . During FLIP measurements mitochondrial GFP - Bak fluorescence is monitored , while the cytosol is bleached repeatedly . Time points in seconds are displayed above the images .

C FLIP of GFP-Bak in the absence (top) or the presence (bottom) of overexpressed Bcl-xL diminishes GFP-Bak fluorescence in the cytosol of targeted cells (circled) completely after 90 s and GFP fluorescence is detected only on the mitochondria (arrows). During FLIP measurements mitochondrial GFP-Bak fluorescence is monitored, while the cytosol is bleached repeatedly. Time points in seconds are displayed above the images.


A Western blot analysis of GFP - Bak localization in cytosol ( C ) and heavy membrane fraction ( HM ) of HCT116 Bax / Bak DKO cells with or without overexpressed wild - type Bcl - xL or Bcl - xL G138A . GAPDH and Tom20 serve as fractionation controls . n = 10 . Bak shuttling by Bcl - xL is independent of the GFP fusion ( Fig . E2a ) . B Quantification of predominant mitochondrial localization ( light grey bars ) and mixed cytosolic and mitochondrial localization ( dark grey bars ) of Bak in HCT116 Bax / Bak DKO cells expressing GFP - Bak with or without Bcl - xL overexpression . Data represent averages ± SD from 3 independent experiments with n ≥ 100 .

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A Western blot analysis of GFP-Bak localization in cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells with or without overexpressed wild-type Bcl-xL or Bcl-xL G138A. GAPDH and Tom20 serve as fractionation controls. n = 10. Bak shuttling by Bcl-xL is independent of the GFP fusion (Fig. E2a).B Quantification of predominant mitochondrial localization (light grey bars) and mixed cytosolic and mitochondrial localization (dark grey bars) of Bak in HCT116 Bax/Bak DKO cells expressing GFP-Bak with or without Bcl-xL overexpression. Data represent averages ± SD from 3 independent experiments with n ≥ 100.


C Retrotranslocation rates measured for wild - type Bak or Bak D83R in the presence or the absence of Bcl - 2 , Mcl - 1 , wild - type Bcl - xL or Bcl - xL G138A . Data represent averages ± SD .

C Retrotranslocation rates measured for wild-type Bak or Bak D83R in the presence or the absence of Bcl-2, Mcl-1, wild-type Bcl-xL or Bcl-xL G138A. Data represent averages ± SD.


D Analysis of endogenous Bak localization in cytosol ( C ) and heavy membrane fraction ( HM ) of HeLa cells expressing degradation - prone DD - FLAG - Bcl - xL . In the absence of Shield - 1 DD - FLAG - Bcl - xL is readily degraded , resulting in unaltered Bcl - xL levels . Addition of 0 . 5 µM Shield - 1 instantly stabilizes DD - FLAG - Bcl - xL resulting in elevated Bcl - xL levels that increase cytosolic levels of endogenous Bak ( Fig . E3C , D ) . Akt1 and Tom20 serve as fractionation controls . n = 3 .

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D Analysis of endogenous Bak localization in cytosol (C) and heavy membrane fraction (HM) of HeLa cells expressing degradation-prone DD-FLAG-Bcl-xL. In the absence of Shield-1 DD-FLAG-Bcl-xL is readily degraded, resulting in unaltered Bcl-xL levels. Addition of 0.5 µM Shield-1 instantly stabilizes DD-FLAG-Bcl-xL resulting in elevated Bcl-xL levels that increase cytosolic levels of endogenous Bak (Fig. E3C, D). Akt1 and Tom20 serve as fractionation controls. n = 3.


B Quantification of HCT116 Bax / Bak DKO cells expressing Bax , BaxTBak , BakTBax or Bak with the expressed protein being present largely cytosolic ( white bars ) , in a mixed distribution between cytosol and mitochondria ( grey bars ) or largely mitochondrial ( black bars ) . Data represent averages ± SEM from 7 independent experiments with n ≥ 100 cells .

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B Quantification of HCT116 Bax/Bak DKO cells expressing Bax, BaxTBak, BakTBax or Bak with the expressed protein being present largely cytosolic (white bars), in a mixed distribution between cytosol and mitochondria (grey bars) or largely mitochondrial (black bars). Data represent averages ± SEM from 7 independent experiments with n ≥ 100 cells.


C Confocal images of HCT116 Bax / Bak DKO cells expressing Bax , BaxTBak , BakTBax or Bak . The GFP / YFP fluorescence of the expressed protein variants is depicted in the top panels and in green in the merged image on the bottom . The mitochondria were stained by MitoTracker far red depicted in red in the merged images ( bottom row ) . Scale 10 µm . n ≥ 5 .

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C Confocal images of HCT116 Bax/Bak DKO cells expressing Bax, BaxTBak, BakTBax or Bak. The GFP/YFP fluorescence of the expressed protein variants is depicted in the top panels and in green in the merged image on the bottom. The mitochondria were stained by MitoTracker far red depicted in red in the merged images (bottom row). Scale 10 µm. n ≥ 5.


D Western blot analysis of Bax , BaxTBak , Bak and BakTBax localization expressed in HCT116 Bax / Bak DKO cells . Cytosol ( C ) and heavy membrane fraction ( HM ) of HCT116 Bax / Bak DKO cells are displayed . GAPDH and VDAC serve as fractionation controls . n = 3 .

D Western blot analysis of Bax, BaxTBak, Bak and BakTBax localization expressed in HCT116 Bax/Bak DKO cells. Cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells are displayed. GAPDH and VDAC serve as fractionation controls. n = 3.


A Caspase 3 / 7 activity measured in HCT116 Bax / Bak DKO cells overexpressing Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression in the absence of apoptosis stimuli . Caspase activity is displayed in relative fluorescence units ( RFU ) . pcDNA3 . 1 - transfected cells served as control . Data represent averages ± SEM . n ≥ 3 . p - values according to One Way ANOVA are displayed . B Staurosporine ( STS , 1 µM ) - induced caspase 3 / 7 activity of Bax / Bak DKO cells overexpressing Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression displayed in relative fluorescence units ( RFU ) . Data represent averages ± SEM . n ≥ 3 . p - values according to One Way ANOVA . BaxTBak activities with and without Bcl - xL expression revealed no significant difference ( n . s . ) , while in the presence of Bcl - xL overexpression BaxTBak activity is significantly higher than Bax , Bak or BakTBax activities ( p < 0 . 001 ) .

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A Caspase 3/7 activity measured in HCT116 Bax/Bak DKO cells overexpressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression in the absence of apoptosis stimuli. Caspase activity is displayed in relative fluorescence units (RFU). pcDNA3.1-transfected cells served as control. Data represent averages ± SEM. n ≥ 3. p-values according to One Way ANOVA are displayed.B Staurosporine (STS, 1 µM)-induced caspase 3/7 activity of Bax/Bak DKO cells overexpressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression displayed in relative fluorescence units (RFU). Data represent averages ± SEM. n ≥ 3. p-values according to One Way ANOVA. BaxTBak activities with and without Bcl-xL expression revealed no significant difference (n.s.), while in the presence of Bcl-xL overexpression BaxTBak activity is significantly higher than Bax, Bak or BakTBax activities (p < 0.001).


C Analysis of the active Bax conformation in HCT116 Bax / Bak DKO cells expressing wild - type Bax or BaxTBak with ( dark grey bars ) or without Bcl - xL overexpression ( light grey bars ) by the monoclonal antibody 6A7 ( Sigma ) detecting the active Bax protein fold by fluorescence imaging . Cells were analyzed prior or after treatment with 1 µM STS in the presence of the pan - caspase inhibitor qVD . Data are represented as % of the expressing cell population ± SEM . n = 4 .

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C Analysis of the active Bax conformation in HCT116 Bax/Bak DKO cells expressing wild-type Bax or BaxTBak with (dark grey bars) or without Bcl-xL overexpression (light grey bars) by the monoclonal antibody 6A7 (Sigma) detecting the active Bax protein fold by fluorescence imaging. Cells were analyzed prior or after treatment with 1 µM STS in the presence of the pan-caspase inhibitor qVD. Data are represented as % of the expressing cell population ± SEM. n = 4.


D HCT116 Bax / Bak DKO cells ectopically expressing wild - type Bax or BaxTBak with ( dark grey bars ) or without Bcl - xL ( light grey bars ) were analyzed in the presence or the absence of 1 µM STS and qVD for retained mitochondrial cyt c . Data are represented as % of the expressing cell population ± SEM . n = 4 .

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D HCT116 Bax/Bak DKO cells ectopically expressing wild-type Bax or BaxTBak with (dark grey bars) or without Bcl-xL (light grey bars) were analyzed in the presence or the absence of 1 µM STS and qVD for retained mitochondrial cyt c. Data are represented as % of the expressing cell population ± SEM. n = 4.


E Flow cytometry analysis of Annexin V - staining of Bax / Bak DKO cells expressing Bax , BaxTBak , Bak or BakTBax in the absence ( red line ) or the presence of Bcl - xL overexpression ( black line ) , following STS - treatment . The percentage of gated cells is displayed in the color of the corresponding graph . Data represent averages ± SD . n = 4 .

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E Flow cytometry analysis of Annexin V-staining of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax in the absence (red line) or the presence of Bcl-xL overexpression (black line), following STS-treatment. The percentage of gated cells is displayed in the color of the corresponding graph. Data represent averages ± SD. n = 4.


F Colony formation of Bax / Bak DKO cells transfected with pcDNA , Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression . STS ( 1 µM ) was added for 24 hrs before cells were replated and colonies were stained with methylene blue typically 14 days after treatment .

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F Colony formation of Bax/Bak DKO cells transfected with pcDNA, Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression. STS (1 µM) was added for 24 hrs before cells were replated and colonies were stained with methylene blue typically 14 days after treatment.


G Quantification of colony formation of Bax / Bak DKO cells expressing Bax , BaxTBak , Bak or BakTBax with or without Bcl - xL overexpression after STS treatment ( Fig . 5F ) . Data represent averages ± SEM . n = 4 . p - values according to One Way ANOVA . BaxTBak expressing cells with or without Bcl - xL expression showed no significant difference ( n . s . ) . H Quantification of the colony formation of Bax / Bak DKO cells expressing Bax , BaxTBak , Bak or BakTBax in presence of Bcl - xL overexpression without apoptosis stimulation . Data represent averages ± SEM . n = 5 . p - values according to One Way ANOVA .

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G Quantification of colony formation of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression after STS treatment (Fig. 5F). Data represent averages ± SEM. n = 4. p-values according to One Way ANOVA. BaxTBak expressing cells with or without Bcl-xL expression showed no significant difference (n.s.).H Quantification of the colony formation of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax in presence of Bcl-xL overexpression without apoptosis stimulation. Data represent averages ± SEM. n = 5. p-values according to One Way ANOVA.


A FLIP of BaxTBakSS without ( ○ , broken line ) or with ( ● , straight line ) overexpressed Bcl - xL . Fluorescence of a neighboring cell is shown as control ( , dotted line ) . Data represent averages ± SEM from 20 ( - Bcl - xL ) and 16 ( + Bcl - xL ) ROI measurements .

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A FLIP of BaxTBakSS without (○, broken line) or with (●, straight line) overexpressed Bcl-xL. Fluorescence of a neighboring cell is shown as control (, dotted line). Data represent averages ± SEM from 20 (-Bcl-xL) and 16 (+Bcl-xL) ROI measurements.


B Western blot analysis of PARP cleavage in HCT116 Bax / Bak DKO cells overexpressing Bax , Bax S184V , BaxTBak or BaxTBak V197 / 198S . The experiment was carried out in the absence of apoptotic stimuli . Cells transfected with pcDNA3 . 1 vector serve as control for PARP cleavage . Similar sample loading is controlled by actin . n = 4 .

B Western blot analysis of PARP cleavage in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BaxTBak or BaxTBak V197/198S. The experiment was carried out in the absence of apoptotic stimuli. Cells transfected with pcDNA3.1 vector serve as control for PARP cleavage. Similar sample loading is controlled by actin. n = 4.


C Colony formation of Bax / Bak DKO cells transfected with pcDNA , BaxTBak or BaxTBakSS with Bcl - xL overexpression in the absence of apoptotic stimuli . n = 3 .

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C Colony formation of Bax/Bak DKO cells transfected with pcDNA, BaxTBak or BaxTBakSS with Bcl-xL overexpression in the absence of apoptotic stimuli. n = 3.


D Caspase 3 / 7 activity was measured in HCT116 Bax / Bak DKO cells overexpressing Bax , Bax S184V , BaxTBak or BaxTBak V197 / 198S . Caspase activity is displayed in relative fluorescence units ( RFU ) . The experiment was carried out in the absence of apoptotic stimuli and pcDNA3 . 1 - transfected cells served as control . Data represent averages ± SEM . n = 4 . p - values according to One Way ANOVA using the Holm - Sidak method are displayed .

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D Caspase 3/7 activity was measured in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BaxTBak or BaxTBak V197/198S. Caspase activity is displayed in relative fluorescence units (RFU). The experiment was carried out in the absence of apoptotic stimuli and pcDNA3.1-transfected cells served as control. Data represent averages ± SEM. n = 4. p-values according to One Way ANOVA using the Holm-Sidak method are displayed.


F STS - induced caspase 3 / 7 activity of HCT116 Bax / Bak DKO cells expressing Bax , Bax S184V , BakTBax or BakTBax S184V is displayed normalized to Bax activity . Data represent averages ± SEM . n ≥ 3 .

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F STS-induced caspase 3/7 activity of HCT116 Bax/Bak DKO cells expressing Bax, Bax S184V, BakTBax or BakTBax S184V is displayed normalized to Bax activity. Data represent averages ± SEM. n ≥ 3.


G Western blot analysis of PARP cleavage after STS treatment in HCT116 Bax / Bak DKO cells overexpressing Bax , Bax S184V , BakTBax or BakTBax S184V . Empty vector - transfected cells serve as control for PARP cleavage and actin is the loading control . n = 3 .

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G Western blot analysis of PARP cleavage after STS treatment in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BakTBax or BakTBax S184V. Empty vector-transfected cells serve as control for PARP cleavage and actin is the loading control. n = 3.


H Plot of Bax ( ● ) and Bak ( ○ ) retrotranslocation rates vs . hydrophobicity of the MA of the constructs . R2 = 0 . 95 .

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H Plot of Bax (●) and Bak (○) retrotranslocation rates vs. hydrophobicity of the MA of the constructs. R2 = 0.95.


A Carbonate extraction of the HM protein fraction of Bax / Bak DKO cells expressing wild - type Bcl - xL in the presence or the absence of Bak or Bax analyzed by Western blot . Carbonate extractable supernatant ( S ) and membrane - integral protein pellet ( P ) are displayed . Smac is released from the IMS during carbonate extraction and VDAC remains in the pellet . n = 4 . B Western blot analysis of Bcl - xLTBax carbonate extraction from the heavy membrane fraction from Bax / Bak DKO cells expressing the Bcl - xL variant with or without Bak or Bax . Smac and VDAC serve as controls for supernatant ( S ) and pellet ( P ) . n = 4 . C Carbonate extraction of the HM protein fraction of Bax / Bak DKO cells expressing Bcl - xL G138A with or without Bak or Bax . Smac and VDAC serve as controls for supernatant ( S ) and pellet ( P ) . n = 4 .

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A Carbonate extraction of the HM protein fraction of Bax/Bak DKO cells expressing wild-type Bcl-xL in the presence or the absence of Bak or Bax analyzed by Western blot. Carbonate extractable supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the IMS during carbonate extraction and VDAC remains in the pellet. n = 4.B Western blot analysis of Bcl-xLTBax carbonate extraction from the heavy membrane fraction from Bax/Bak DKO cells expressing the Bcl-xL variant with or without Bak or Bax. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.C Carbonate extraction of the HM protein fraction of Bax/Bak DKO cells expressing Bcl-xL G138A with or without Bak or Bax. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.


D HM protein fraction and carbonate extraction of Bax / Bak DKO cells expressing Bax , BaxTBak or Bax S184V . Smac and VDAC serve as controls for supernatant ( S ) and pellet ( P ) . n = 4 .

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D HM protein fraction and carbonate extraction of Bax/Bak DKO cells expressing Bax, BaxTBak or Bax S184V. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.


E Smac release from the heavy membrane fraction ( HM ) into the cytosol ( C ) in HCT116 Bax / Bak DKO cells overexpressing Bax and BaxTBak without apoptotic stimuli analyzed by Western blot . Bax and BaxTBak were expressed differentially to produce similar levels of mitochondrial Bax . GAPDH and Tom20 serve as fractionation controls . n = 3 .

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E Smac release from the heavy membrane fraction (HM) into the cytosol (C) in HCT116 Bax/Bak DKO cells overexpressing Bax and BaxTBak without apoptotic stimuli analyzed by Western blot. Bax and BaxTBak were expressed differentially to produce similar levels of mitochondrial Bax. GAPDH and Tom20 serve as fractionation controls. n = 3.


F PARP cleavage in HCT116 Bax / Bak DKO cells expressing similar levels of mitochondrial Bax or BaxTBak analyzed by Western blot without apoptotic stimuli . Actin is used as loading control . n = 3 .

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F PARP cleavage in HCT116 Bax/Bak DKO cells expressing similar levels of mitochondrial Bax or BaxTBak analyzed by Western blot without apoptotic stimuli. Actin is used as loading control. n = 3.


( A ) NPM1 expression in CD34 + Lin - cells from myelodysplastic syndrome ( MDS ) patients compared to healthy volunteers ( n = 29 ) .

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(A) NPM1 expression in CD34+Lin- cells from myelodysplastic syndrome (MDS) patients compared to healthy volunteers (n = 29).


( B ) Representative staining of NPM1 protein levels in bone marrow samples of MDS or acute myeloid leukemia ( AML ) with multi - lineage dysplasia ( MLD ) . Insets show representative staining in blasts [ B ] and in differentiated myeloid cells [ e . g . , neutrophil , M ] with a high magnification .

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(B) Representative staining of NPM1 protein levels in bone marrow samples of MDS or acute myeloid leukemia (AML) with multi-lineage dysplasia (MLD). Insets show representative staining in blasts [B] and in differentiated myeloid cells [e.g., neutrophil, M] with a high magnification.


( C ) Percentages of patient samples with NPM1 - low and NPM1 - normal in healthy , MDS and MLD in AML groups .

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(C) Percentages of patient samples with NPM1-low and NPM1-normal in healthy, MDS and MLD in AML groups.


( C ) Representative distribution of Tubulin and CDC42 in Ctrl , 4 - months old Npm1 cKO and 2 - years old WT HSCs determined by IF . Scale bars , 5 μm .

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(C) Representative distribution of Tubulin and CDC42 in Ctrl, 4-months old Npm1 cKO and 2-years old WT HSCs determined by IF. Scale bars, 5 μm.


( F ) HSCs of Ctrl , 4 - months old Npm1 cKO and 2 - years old WT mice were sorted and stained for TOM20 . Representative images ( top ) and quantification of mitochondrial volume and number ( bottom ) in single cells ( n = 58 cells in Ctrl , n = 70 cells in cKO and n = 23 cells in Old , from 3 mice per group ) , identified by volume rendering of TOM20 . Scale bars , 2 μm .

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(F ) HSCs of Ctrl, 4-months old Npm1 cKO and 2-years old WT mice were sorted and stained for TOM20. Representative images (top) and quantification of mitochondrial volume and number (bottom) in single cells (n = 58 cells in Ctrl, n = 70 cells in cKO and n = 23 cells in Old, from 3 mice per group), identified by volume rendering of TOM20. Scale bars, 2 μm.


( J ) Representative images ( top ) and quantification of mitochondrial volume and number ( bottom ) , identified by volume rendering of TOM20 , in MDS - L cells transfected with siNPM1 or control ( siCTRL ) ( n = 58 cells per group , from 3 transfected MDS - L cultures ) . Scale bars , 2 μm .

(J) Representative images (top) and quantification of mitochondrial volume and number (bottom), identified by volume rendering of TOM20, in MDS-L cells transfected with siNPM1 or control (siCTRL) (n = 58 cells per group, from 3 transfected MDS-L cultures). Scale bars, 2 μm.


( F ) Representative peripheral blood ( PB ) smears . Insets show the differentiated granulocytes ( a , b ) in control mice , and anisocytosis ( c ) , dysplastic myeloid cells ( d - g ) in Npm1 cKO mice ( Npm1F / FVav1Cre + ) . Npm1 cKO mice show dysplastic erythroid cells ( polychromatophilic , blue arrowhead ; schistocyte , blue - outlined arrowhead ; tear drop , Howell - Jolly bodies , black arrowhead ; black - outlined arrowhead ) , dysplastic monocytes ( black arrow ) , and dysplastic neutrophils ( d - f ) . Scale bars , 10 μm .

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(F) Representative peripheral blood (PB) smears. Insets show the differentiated granulocytes (a, b) in control mice, and anisocytosis (c), dysplastic myeloid cells (d-g) in Npm1 cKO mice (Npm1F/FVav1Cre+). Npm1 cKO mice show dysplastic erythroid cells (polychromatophilic, blue arrowhead; schistocyte, blue-outlined arrowhead; tear drop, Howell-Jolly bodies, black arrowhead; black-outlined arrowhead), dysplastic monocytes (black arrow), and dysplastic neutrophils (d-f). Scale bars, 10 μm.


( C ) Npm1 and p53 protein levels in KSL cells of Ctrl ( Npm1F / FVav1Cre - ) , and cKO ( Npm1F / FVav1Cre + ) mice .

(C) Npm1 and p53 protein levels in KSL cells of Ctrl (Npm1F/FVav1Cre-), and cKO (Npm1F/FVav1Cre+) mice.


( F ) Representative PB smears of DKO mice . Insets show anisocytosis ( a ) , poikilocytosis ( b ) and dysplastic myeloid cell ( c ) . DKO mice show dysplastic erythroid cells ( polychromatophilic , blue arrowhead ) , Howell - Jolly bodies , ( black arrowhead in insert a ) , dysplastic monocytes ( black arrow , and i - v ) , dysplastic neutrophils ( vi ) , a pseudo - Pelger - Huet anomaly ( ii ) , and dysplastic platelets ( giant platelet , iv and vii ) . Scale bars , 10μm .

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(F) Representative PB smears of DKO mice. Insets show anisocytosis (a), poikilocytosis (b) and dysplastic myeloid cell (c). DKO mice show dysplastic erythroid cells (polychromatophilic, blue arrowhead), Howell-Jolly bodies, (black arrowhead in insert a), dysplastic monocytes (black arrow, and i-v), dysplastic neutrophils (vi), a pseudo-Pelger-Huet anomaly (ii), and dysplastic platelets (giant platelet, iv and vii). Scale bars, 10μm.


( I ) Representative H & E staining of sections of the spleen of 2 - month - old DKO mice with myeloid disorders . Arrowheads ( right ) indicate the infiltrating myeloid blasts . Scale bars , 250 μm ( left ) and 10μm ( right ) , respectively .

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(I) Representative H&E staining of sections of the spleen of 2-month-old DKO mice with myeloid disorders. Arrowheads (right) indicate the infiltrating myeloid blasts. Scale bars, 250 μm (left) and 10μm (right), respectively.


A ) Live confocal imaging of liver stage P . berghei expressing UIS4 ‐ mCherry and cytoplasmic GFP reveals different morphologies of the LS ‐ TVN : elongated membrane clusters ( left ) , vesicles in the host cell cytoplasm ( center ) , and a thin tubule protruding from the PVM ( right ) . Live imaging was performed 20 h after infection of hepatoma cells . Features are marked with white arrowheads .

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A) Live confocal imaging of liver stage P. berghei expressing UIS4‐mCherry and cytoplasmic GFP reveals different morphologies of the LS‐TVN: elongated membrane clusters (left), vesicles in the host cell cytoplasm (center), and a thin tubule protruding from the PVM (right). Live imaging was performed 20 h after infection of hepatoma cells. Features are marked with white arrowheads.


B ) The UIS4 ‐ and IBIS1 ‐ positive tubules rapidly extend from and retract to the PVM . Confocal time ‐ lapse imaging of a UIS4 ‐ mCherry ‐ expressing parasite at 22 h ( top ) and an IBIS1 ‐ mCherry ‐ expressing parasite at 26 h ( bottom ) after infection of hepatoma cells . Tubules are indicated with a yellow arrow , and the time is shown in minutes and seconds .

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B) The UIS4‐ and IBIS1‐positive tubules rapidly extend from and retract to the PVM. Confocal time‐lapse imaging of a UIS4‐mCherry‐expressing parasite at 22 h (top) and an IBIS1‐mCherry‐expressing parasite at 26 h (bottom) after infection of hepatoma cells. Tubules are indicated with a yellow arrow, and the time is shown in minutes and seconds.


C ) Imaging of IBIS1 ‐ mCherry ‐ expressing P . berghei reveals similar features of the LS ‐ TVN , i . e . elongated membrane cluster ( left ) , vesicles in the host cell cytoplasm ( center ) , and a membrane tubule ( right ) . Infected cells were fixed 26 h post ‐ infection and imaged by epifluorescence microscopy . Features are indicated with a yellow arrowhead . Scale bars , 10 µm .

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C) Imaging of IBIS1‐mCherry‐expressing P. berghei reveals similar features of the LS‐TVN, i.e. elongated membrane cluster (left), vesicles in the host cell cytoplasm (center), and a membrane tubule (right). Infected cells were fixed 26 h post‐infection and imaged by epifluorescence microscopy. Features are indicated with a yellow arrowhead. Scale bars, 10 µm.


D ) Quantification of the distinct UIS4 ‐ and IBIS1 ‐ positive membranous structures in fixed infected hepatoma cells at three different time points : 6 h after infection ( the period of sporozoite transformation into exo ‐ erythrocytic forms ) , 24 h after infection ( onset of parasite replication and growth ) , and 48 h after infection ( mature liver stages ) . Values are shown as mean ( ± SD ) from three separate wells ( n = 100 per well ) of one experiment and are representative of two experimental repetitions .

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D) Quantification of the distinct UIS4‐ and IBIS1‐positive membranous structures in fixed infected hepatoma cells at three different time points: 6 h after infection (the period of sporozoite transformation into exo‐erythrocytic forms), 24 h after infection (onset of parasite replication and growth), and 48 h after infection (mature liver stages). Values are shown as mean (± SD) from three separate wells (n = 100 per well) of one experiment and are representative of two experimental repetitions.


Hepatoma cells infected with Plasmodium berghei ANKA parasites were fixed and stained with anti - UIS4 and anti - HSP70 A ) Some UIS4 ‐ positive vesicles are tethered to the PVM as ' beads ‐ ona ‐ string ' . Infected cell fixed 24 h post ‐ infection .

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Hepatoma cells infected with Plasmodium berghei ANKA parasites were fixed and stained with anti-UIS4 and anti-HSP70 A) Some UIS4‐positive vesicles are tethered to the PVM as 'beads‐ona‐string'. Infected cell fixed 24 h post‐infection.


B ) The UIS4 ‐ mCherry features we detect in live imaging of UIS4 ‐ mCherry ‐ expressing P . berghei , i . e . elongated membrane clusters ( left ) , vesicles ( center ) and tubules ( right ) are also apparent in fixed wildtype parasites stained with anti ‐ UIS4 antibody . Infected cells 24 h post ‐ infection . Scale bars , 10 µm .

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B) The UIS4‐mCherry features we detect in live imaging of UIS4‐mCherry‐expressing P. berghei, i.e. elongated membrane clusters (left), vesicles (center) and tubules (right) are also apparent in fixed wildtype parasites stained with anti‐UIS4 antibody. Infected cells 24 h post‐infection. Scale bars, 10 µm.


Hepatoma cells infected with Plasmodium berghei ANKA parasites were fixed and stained with anti - UIS4 . C ) Often the LS ‐ TVN is a composite of vesicles , tubules and membrane clusters . Infected cell 22 h post ‐ infection .

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Hepatoma cells infected with Plasmodium berghei ANKA parasites were fixed and stained with anti-UIS4. C) Often the LS‐TVN is a composite of vesicles, tubules and membrane clusters. Infected cell 22 h post‐infection.


D ) The LS ‐ TVN detectable in primary hepatocytes infected with UIS4 ‐ mCherry ‐ expressing P . berghei also consists of elongated membrane clusters ( yellow arrowhead ) , vesicles ( white arrow ) and tubules ( yellow arrow ) . Infected cell 18 h post ‐ infection .

D) The LS‐TVN detectable in primary hepatocytes infected with UIS4‐mCherry‐expressing P. berghei also consists of elongated membrane clusters (yellow arrowhead), vesicles (white arrow) and tubules (yellow arrow). Infected cell 18 h post‐infection.


E ) Dynamic tubules ( yellow arrow ) are detectable with UIS4 ‐ mCherry in infected primary hepatocytes 24 h post ‐ infection . Location of parasite is marked with a yellow asterisk . Scale bars , 10 µm .

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E) Dynamic tubules (yellow arrow) are detectable with UIS4‐mCherry in infected primary hepatocytes 24 h post‐infection. Location of parasite is marked with a yellow asterisk. Scale bars, 10 µm.


A ) Confocal microscopy images show a tubule ( upper panel ) labeled with UIS4 and IBIS1 ( white arrowheads ) , an elongated membrane cluster ( middle panel ) in which both proteins are present , and vesicles ( bottom panel ) that are often labeled with both UIS4 and IBIS1 ( yellow arrow ) and frequently labeled with only one protein ( yellow arrowheads ) .

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A) Confocal microscopy images show a tubule (upper panel) labeled with UIS4 and IBIS1 (white arrowheads), an elongated membrane cluster (middle panel) in which both proteins are present, and vesicles (bottom panel) that are often labeled with both UIS4 and IBIS1 (yellow arrow) and frequently labeled with only one protein (yellow arrowheads).


B ) Epifluorescence microscopy of IBIS1 and UIS4 reveals that while most tubules are labeled with both proteins as above ( white arrowhead ) , some are labeled exclusively with UIS4 ( upper panel ) or IBIS1 ( bottom panel ) . Single ‐ labeled tubules were highlighted with white arrows and their frequency in infected cells with tubules is indicated in percent ( ± SD ) , which was calculated from two separate wells 30 h post ‐ infection ( n > 150 infected cells per well ) .

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B) Epifluorescence microscopy of IBIS1 and UIS4 reveals that while most tubules are labeled with both proteins as above (white arrowhead), some are labeled exclusively with UIS4 (upper panel) or IBIS1 (bottom panel). Single‐labeled tubules were highlighted with white arrows and their frequency in infected cells with tubules is indicated in percent (± SD), which was calculated from two separate wells 30 h post‐infection (n > 150 infected cells per well).


A ‐ C ) Time ‐ lapse imaging of hepatoma cells infected with UIS4 ‐ mCherry ‐ expressing Plasmodium berghei was used to determine the velocities of the UIS4 ‐ positive features , e . g . , a tubule ( A ) , elongated membrane cluster ( B ) and vesicle ( C ) were tracked over time . Measured speeds of the dynamic features indicated with white arrows are depicted graphically to the right . Scale bars , 10 µm .

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A‐C) Time‐lapse imaging of hepatoma cells infected with UIS4‐mCherry‐expressing Plasmodium berghei was used to determine the velocities of the UIS4‐positive features, e.g., a tubule (A), elongated membrane cluster (B) and vesicle (C) were tracked over time. Measured speeds of the dynamic features indicated with white arrows are depicted graphically to the right. Scale bars, 10 µm.


D ) The velocities of several UIS4 ‐ or IBIS1 ‐ positive features [ tubules ( left ) , elongated membrane clusters ( center ) and vesicles ( right ) ] were measured over time , and the peak velocity of each was plotted . Plotted data was derived from multiple imaged infected cells : For IBIS1 ‐ positive tubules data is derived from three infected cells and for UIS4 ‐ positive tubules from 13 infected cells . Elongated membrane cluster speeds were analyzed in at least five infected cells for both UIS4 and IBIS1 , and vesicle speeds were analyzed in at least 12 different cells for both protein markers .

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D) The velocities of several UIS4‐ or IBIS1‐positive features [tubules (left), elongated membrane clusters (center) and vesicles (right)] were measured over time, and the peak velocity of each was plotted. Plotted data was derived from multiple imaged infected cells: For IBIS1‐positive tubules data is derived from three infected cells and for UIS4‐positive tubules from 13 infected cells. Elongated membrane cluster speeds were analyzed in at least five infected cells for both UIS4 and IBIS1, and vesicle speeds were analyzed in at least 12 different cells for both protein markers.


B ) Cells infected with UIS4 ‐ mCherry ‐ expressing parasites were treated with nocodazole for 1 . 5 h ( top ) or cytochalasin D for 30 min ( bottom ) and imaged by confocal microscopy . Imaging occurred 29 and 24 h post ‐ infection , respectively . UIS4 ‐ positive tubules , static ( top ) and dynamic ( bottom ) , are indicated with white arrowheads . Displayed cells are representative of five imaged parasites from at least two different experiments .

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B) Cells infected with UIS4‐mCherry‐expressing parasites were treated with nocodazole for 1.5 h (top) or cytochalasin D for 30 min (bottom) and imaged by confocal microscopy. Imaging occurred 29 and 24 h post‐infection, respectively. UIS4‐positive tubules, static (top) and dynamic (bottom), are indicated with white arrowheads. Displayed cells are representative of five imaged parasites from at least two different experiments.


C ) No colocalization between host microtubules and UIS4 ‐ positive tubules is detectable . Infected cells were fixed 25 h post ‐ infection and stained with antibodies against UIS4 and Tubulin . Scale bars , 10 µm .

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C) No colocalization between host microtubules and UIS4‐positive tubules is detectable. Infected cells were fixed 25 h post‐infection and stained with antibodies against UIS4 and Tubulin. Scale bars, 10 µm.


A and B ) The bleach was applied to elongated membrane clusters of the LS ‐ TVN approximately 27 h post ‐ infection . The UIS4 ‐ mCherry signal typically did not recover in the bleached area ( A ) , although it was found to recover in some elongated membrane clusters ( B ) .

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A and B) The bleach was applied to elongated membrane clusters of the LS‐TVN approximately 27 h post‐infection. The UIS4‐mCherry signal typically did not recover in the bleached area (A), although it was found to recover in some elongated membrane clusters (B).


C ) When the bleach was applied to the PVM at 25 h post ‐ infection , the signal recovered rapidly in the bleached region . Upon the application of additional bleach pulses in the same area , the bleached area ( region # 1 measured graphically ) repeatedly recovered , and the remaining PVM ‐ associated UIS4 ‐ mCherry signal depleted ( region # 2 ) although the LS ‐ TVN ‐ associated signal remained ( region # 3 ) . When a region of the LS ‐ TVN was subsequently bleached ( yellow arrow , region # 4 ) , no recovery was observed . The times are shown in minutes and seconds . Scale bars , 10 µm .

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C) When the bleach was applied to the PVM at 25 h post‐infection, the signal recovered rapidly in the bleached region. Upon the application of additional bleach pulses in the same area, the bleached area (region #1 measured graphically) repeatedly recovered, and the remaining PVM‐associated UIS4‐mCherry signal depleted (region #2) although the LS‐TVN‐associated signal remained (region #3). When a region of the LS‐TVN was subsequently bleached (yellow arrow, region #4), no recovery was observed. The times are shown in minutes and seconds. Scale bars, 10 µm.


A and B ) A phase ‐ contrast image containing the infected cell of interest ( A ) was used for comparison to the EM grid after fixation and preparation of the cells for TEM ( B ) . Green outlining of the cells in the phase ‐ contrast image was used as a tool for comparison and alignment . The insets defined in white are shown in the right panels merged with the fluorescent image of the UIS4 ‐ mCherry signal acquired immediately prior to fixation . Scale bars , 10 µm .

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A and B) A phase‐contrast image containing the infected cell of interest (A) was used for comparison to the EM grid after fixation and preparation of the cells for TEM (B). Green outlining of the cells in the phase‐contrast image was used as a tool for comparison and alignment. The insets defined in white are shown in the right panels merged with the fluorescent image of the UIS4‐mCherry signal acquired immediately prior to fixation. Scale bars, 10 µm.