( B ) The mouse Reno1 locus . Expression levels during neuronal differentiation of ES cells , in different cellular fractions of ES cells ( data from ( Engreitz et al , 2016 ) ) and motor neurons ( GSE90913 ) , and in forebrain during different stages of embryonic development ( data from the ENCODE project ) . Each set of tracks was normalized separately .

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(B) The mouse Reno1 locus. Expression levels during neuronal differentiation of ES cells, in different cellular fractions of ES cells (data from (Engreitz et al, 2016)) and motor neurons (GSE90913), and in forebrain during different stages of embryonic development (data from the ENCODE project). Each set of tracks was normalized separately.


( C ) RENO1 locus in human . Expression levels in dorsolateral prefrontal cortex across six age groups , and in different cellular fractions in fetal and adult cells . Each set of tracks was normalized separately .

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(C) RENO1 locus in human. Expression levels in dorsolateral prefrontal cortex across six age groups, and in different cellular fractions in fetal and adult cells. Each set of tracks was normalized separately.


( D ) Reno1 smFISH signal ( red ) and DAPI staining ( blue ) in WT and Reno1m / m cells at day eight of neuronal differentiation , imaged using 100x objective .

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(D) Reno1 smFISH signal (red) and DAPI staining (blue) in WT and Reno1m/m cells at day eight of neuronal differentiation, imaged using 100x objective.


( E ) Expression levels of Reno1 and Bahcc1 in 30 adult and embryonic tissues . Data from the ENCODE project , quantification was done using RSEM ( Li & Dewey , 2011 ) .

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(E) Expression levels of Reno1 and Bahcc1 in 30 adult and embryonic tissues. Data from the ENCODE project, quantification was done using RSEM (Li & Dewey, 2011).


( F ) Targeted Chromosome Conformation Capture ( 4C ) using the Reno1 promoter as bait . Top , smoothed trend lines and raw counts of the contact profile in ES cells ( red ) or differentiated neurons ( black ) ; bottom , domainogram showing mean contact per fragment end for a series of window sizes .

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(F) Targeted Chromosome Conformation Capture (4C) using the Reno1 promoter as bait. Top, smoothed trend lines and raw counts of the contact profile in ES cells (red) or differentiated neurons (black); bottom, domainogram showing mean contact per fragment end for a series of window sizes.


( B ) Expression levels of Reno1 ( both the spliced and unspliced isoforms ) and Bahcc1 following four days of differentiation of the indicated cells . Mean ± SEM is shown for 3 - 4 independent experiments , * P < 0 . 05 , * * P < 0 . 01 ( unpaired two sample t - test ) .

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(B) Expression levels of Reno1 (both the spliced and unspliced isoforms) and Bahcc1 following four days of differentiation of the indicated cells. Mean ± SEM is shown for 3-4 independent experiments, * P<0.05, ** P<0.01 (unpaired two sample t-test).


( C ) Left : Oct4 staining of the indicated cells . Scale bar : 200 µm . Right : Cell count of the indicated ES cells grown in ES medium . Cells were harvested from one well of a 6 - well plate , and counted using Orflo MOXI Z Mini Automated Cell Counter . Mean ± SEM is shown for three independent experiments .

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(C) Left: Oct4 staining of the indicated cells. Scale bar: 200 µm. Right: Cell count of the indicated ES cells grown in ES medium. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments.


( D ) Left : Nestin staining of the indicated cells following four days of differentiation . Scale bar : 200 µm . Right : Cell count of the indicated cells following four days of differentiation . Cells were harvested from one well of a 6 - well plate , and counted using Orflo MOXI Z Mini Automated Cell Counter . Mean ± SEM is shown for three independent experiments . * * P < 0 . 01 ( unpaired two sample t - test ) .

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(D) Left: Nestin staining of the indicated cells following four days of differentiation. Scale bar: 200 µm. Right: Cell count of the indicated cells following four days of differentiation. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments. ** P<0.01 (unpaired two sample t-test).


( E ) Flow cytometry analyses of dead and live cells in the indicated ES cells following four days of differentiation . Y axis : forward scatter ; X axis : PI fluorescence intensity .

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(E) Flow cytometry analyses of dead and live cells in the indicated ES cells following four days of differentiation. Y axis: forward scatter; X axis: PI fluorescence intensity.


( A ) . Heatmap showing log2 - transformed fold changes of Reno1 depleted cells compared to their respective controls . All genes which were significantly differentially expressed ( P < 0 . 05 ) in any of the treatments are shown . ( B ) Boxplots indicating the median , quartiles , and 5th and 95th percentiles of changes in expression levels of Reno1m / m cells compared to WT cells , for genes with significant ( P < 0 . 05 ) change in cells where Reno1 was perturbed with the indicated method . Number of genes in each group is indicated in the x axis . * * P < 0 . 01 ( two - sided Wilcoxon rank sum test ) .

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(A). Heatmap showing log2-transformed fold changes of Reno1 depleted cells compared to their respective controls. All genes which were significantly differentially expressed (P<0.05) in any of the treatments are shown. (B) Boxplots indicating the median, quartiles, and 5th and 95th percentiles of changes in expression levels of Reno1m/m cells compared to WT cells, for genes with significant (P<0.05) change in cells where Reno1 was perturbed with the indicated method. Number of genes in each group is indicated in the x axis. ** P<0.01 (two-sided Wilcoxon rank sum test).


( C ) Mean expression levels of genes in genes which were significantly ( P < 0 . 05 ) dysregulated by at least two out of three Reno1 perturbations at day 2 in fig . 5A ( n = 168 down - regulated and 94 up - regulated ) , during an eight time point neuronal differentiation and maturation of mouse ES cells : Neurons1 corresponds to day in vitro ( DIV ) 1 ; Neurons2 - DIV7 ; Neurons3 - DIV16 ; Neurons4 - DIV21 ; and Neurons5 - DIV28 ( Hubbard et al . , 2013 ) .

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(C) Mean expression levels of genes in genes which were significantly (P<0.05) dysregulated by at least two out of three Reno1 perturbations at day 2 in fig. 5A (n= 168 down-regulated and 94 up-regulated), during an eight time point neuronal differentiation and maturation of mouse ES cells: Neurons1 corresponds to day in vitro (DIV)1; Neurons2 - DIV7; Neurons3 - DIV16; Neurons4 - DIV21; and Neurons5 - DIV28 (Hubbard et al., 2013).


( D ) Expression levels of Reno1 following Dox addition in WT and Reno1m / m cells . Mean ± SEM is shown for three independent experiments , * P < 0 . 05 ( unpaired two sample t - test ) .

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(D) Expression levels of Reno1 following Dox addition in WT and Reno1m/m cells. Mean ± SEM is shown for three independent experiments, * P<0.05 (unpaired two sample t-test).


( E ) Cell count of WT and Reno1m / m cells following four days of differentiation , with or without Dox addition . Cells were harvested from one well of a 6 - well plate , and counted using Orflo MOXI Z Mini Automated Cell Counter . Mean ± SEM is shown for three independent experiments . * P < 0 . 05 ( unpaired two sample t - test ) .

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(E) Cell count of WT and Reno1m/m cells following four days of differentiation, with or without Dox addition. Cells were harvested from one well of a 6-well plate, and counted using Orflo MOXI Z Mini Automated Cell Counter. Mean ± SEM is shown for three independent experiments. * P<0.05 (unpaired two sample t-test).


( F - G ) Boxplots indicating the median , quartiles , and 5th and 95th percentiles of changes in expression levels of WT ( F ) or Reno1m / m ( G ) cell treated with Dox for the first two days of differentiation compared to untreated cells , in genes which were significantly ( P < 0 . 05 ) downregulated ( n = 252 ) or upregulated ( n = 223 ) by at least two out of three Reno1 perturbations at day 2 in fig . 5A , compared to all genes ( n = 16 , 511 ) . * * P < 0 . 01 ( two - sided Wilcoxon rank sum test ) .

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(F-G) Boxplots indicating the median, quartiles, and 5th and 95th percentiles of changes in expression levels of WT (F) or Reno1m/m (G) cell treated with Dox for the first two days of differentiation compared to untreated cells, in genes which were significantly (P<0.05) downregulated (n=252) or upregulated (n=223) by at least two out of three Reno1 perturbations at day 2 in fig. 5A, compared to all genes (n=16,511). ** P<0.01 (two-sided Wilcoxon rank sum test).


( A ) Boxplots indicating the median , quartiles and 1 . 5 time the interquartile range of ratios of ATAC - seq signal for the indicated group of peaks , comparing the indicated genotypes or treatments . Core promoter peaks are < 300 nt from a TSS ; Extended promoter are more than 300 nt but < 2 Kb for a TSS ; Gene body peaks overlap transcription units , and Intergenic do not . Each group of peaks was compared to the intergenic peaks . In addition , for each group of peaks , the peaks within 1 Mb of Reno1 where compared to peaks further away . P - values computed using two - sided Wilcoxon rank sum test , and shown only for cases where P < 0 . 05 . Number of data points ( from left to right ) : 39 ; 10 , 777 ; 11 ; 3 , 550 ; 17 ; 14 , 380 ; 18 ; 20 , 930 . ( B ) Changes in ATAC - seq signal for peaks assigned as falling within core or extended promoters , of genes in the indicated group ( up / down - regulated by at least 30 % and with adjusted P < 0 . 05 ) in cells with Reno1 ( left ) or Bahcc1 ( right ) KD . P - values computed using two sided Wilcoxon rank sum test .

O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

(A) Boxplots indicating the median, quartiles and 1.5 time the interquartile range of ratios of ATAC-seq signal for the indicated group of peaks, comparing the indicated genotypes or treatments. Core promoter peaks are <300 nt from a TSS; Extended promoter are more than 300 nt but <2 Kb for a TSS; Gene body peaks overlap transcription units, and Intergenic do not. Each group of peaks was compared to the intergenic peaks. In addition, for each group of peaks, the peaks within 1 Mb of Reno1 where compared to peaks further away. P-values computed using two-sided Wilcoxon rank sum test, and shown only for cases where P<0.05. Number of data points (from left to right): 39; 10,777; 11; 3,550; 17; 14,380; 18; 20,930. (B) Changes in ATAC-seq signal for peaks assigned as falling within core or extended promoters, of genes in the indicated group (up/down-regulated by at least 30% and with adjusted P<0.05) in cells with Reno1 (left) or Bahcc1 (right) KD. P-values computed using two sided Wilcoxon rank sum test.


( C ) Top : Boxplots indicating the median , quartiles and 1 . 5 time the interquartile range of changes in ATAC - seq signal on day 2 of neuronal differentiation of peaks from indicated Reno1 perturbation , classified according to the underlying chromatin state in mouse ES cells ; Bottom : Boxplots indicating the median , quartiles and 1 . 5 time the interquartile range of ChIP - seq coverage of the indicated chromatin marks in ENCODE mouse ES data in the peak regions . Number of peaks in each group is indicated at the bottom .

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(C) Top: Boxplots indicating the median, quartiles and 1.5 time the interquartile range of changes in ATAC-seq signal on day 2 of neuronal differentiation of peaks from indicated Reno1 perturbation, classified according to the underlying chromatin state in mouse ES cells; Bottom: Boxplots indicating the median, quartiles and 1.5 time the interquartile range of ChIP-seq coverage of the indicated chromatin marks in ENCODE mouse ES data in the peak regions. Number of peaks in each group is indicated at the bottom.


( D ) Correlation between changes in chromatin accessibility on day 2 of neuronal differentiation in the indicated perturbations and H3K4me3 or Fam60a ChIP coverage from mouse ES cells ( H3K4me3 is from ENCODE project and Fam60a from ( Streubel et al . , 2017 ) ) . Coefficient and P - value computed using Spearman ' s correlation . n = 49 , 722 peaks .

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(D) Correlation between changes in chromatin accessibility on day 2 of neuronal differentiation in the indicated perturbations and H3K4me3 or Fam60a ChIP coverage from mouse ES cells (H3K4me3 is from ENCODE project and Fam60a from (Streubel et al., 2017)). Coefficient and P-value computed using Spearman's correlation. n=49,722 peaks.


Quantitative RT - PCR shows that Lpin1 expression is depleted in the skeletal muscles Gastrocnemius ( GC ) and Tibialis Anterior ( TA ) , but not in White Adipose Tissue ( WAT ) from 3 - month - old HSACre / + / Lpin1fEx2 - 3 / fEx2 - 3 mice ( HSA - Cre ) compared to control mice . Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice .

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Quantitative RT-PCR shows that Lpin1 expression is depleted in the skeletal muscles Gastrocnemius (GC) and Tibialis Anterior (TA), but not in White Adipose Tissue (WAT) from 3-month-old HSACre/+/Lpin1fEx2-3/fEx2-3 mice (HSA-Cre) compared to control mice. Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice.


PAP1 and PAP2 activities were respectively substantially decreased and increased in TA , of HSA - Cre mice as compared to control mice , respectively ( n = 6 ; * P < 0 . 05 ) .

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PAP1 and PAP2 activities were respectively substantially decreased and increased in TA, of HSA-Cre mice as compared to control mice, respectively (n = 6; *P<0.05).


Measurements of muscle / body weight ratio of GC and TA of 3 - month - old HSA - Cre and control mice .

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Measurements of muscle/body weight ratio of GC and TA of 3-month-old HSA-Cre and control mice.


Hematoxylin and Eosin ( HE ) staining of representative transverse sections of GC muscles from 3 - month - old HSA - Cre and control mice . The black arrows indicate fibers with centrally located nuclei and yellow arrows indicate mononuclear cell infiltration . Scale bars : 40

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Hematoxylin and Eosin (HE) staining of representative transverse sections of GC muscles from 3-month-old HSA-Cre and control mice. The black arrows indicate fibers with centrally located nuclei and yellow arrows indicate mononuclear cell infiltration. Scale bars: 40 μm


Relative force measurements after normalization of absolute tetanic force to GC muscle weight from 3 - month - old HSA - Cre and control male mice .

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Relative force measurements after normalization of absolute tetanic force to GC muscle weight from 3-month-old HSA-Cre and control male mice.


Phosphatidic Acid ( PA ) levels in HSA - Cre and control GC muscles .

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Phosphatidic Acid (PA) levels in HSA-Cre and control GC muscles.


Phospholipidome in HSA - Cre and control GC muscles . Proportions of indicated phospholipid classes in total phospholipids are represented . PC ; Phosphatidylcholine , PE ; Phosphatidylethanolamine , PI ; Phosphatidylinositol , PS ; Phoshpatidylserine , PA ; Phosphatidic Acid , PG ; Phosphatidylglycerol .

Phospholipidome in HSA-Cre and control GC muscles. Proportions of indicated phospholipid classes in total phospholipids are represented. PC; Phosphatidylcholine, PE; Phosphatidylethanolamine, PI; Phosphatidylinositol, PS; Phoshpatidylserine, PA; Phosphatidic Acid, PG; Phosphatidylglycerol.


Amounts of indicated phospholipid classes normalized by protein content in HSA - Cre and control GC muscles . ( D ) Total phospholipid ( PL ) amount normalized by protein content in HSA - Cre and control GC muscles .

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Amounts of indicated phospholipid classes normalized by protein content in HSA-Cre and control GC muscles. (D) Total phospholipid (PL) amount normalized by protein content in HSA-Cre and control GC muscles.


Distribution of the total carbon number in acyl chains of PC in HSA - Cre and control GC muscles .

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Distribution of the total carbon number in acyl chains of PC in HSA-Cre and control GC muscles.


Distribution of the number of double bonds in acyl chains of PC in HSA - Cre and control GC muscles .

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Distribution of the number of double bonds in acyl chains of PC in HSA-Cre and control GC muscles.


Distribution of the total carbon number in acyl chains of PE in HSA - Cre and control GC muscles .

Distribution of the total carbon number in acyl chains of PE in HSA-Cre and control GC muscles.


Distribution of the number of double bonds in acyl chains of PE in HSA - Cre and control GC muscles .

Distribution of the number of double bonds in acyl chains of PE in HSA-Cre and control GC muscles.


HE , Oil Red O ( ORO ) , Cytochrome c Oxidase ( COX ) and Succinate Deshydrogenase ( SDH ) staining of representative frozen sections of TA muscle from 3 - month - old HSA - Cre and control mice .

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HE, Oil Red O (ORO), Cytochrome c Oxidase (COX) and Succinate Deshydrogenase (SDH) staining of representative frozen sections of TA muscle from 3-month-old HSA-Cre and control mice.


Quantification of triacylglycerol ( TAG ) levels in GC muscles from 3 - month - old HSA - Cre and and control mice .

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Quantification of triacylglycerol (TAG) levels in GC muscles from 3-month-old HSA-Cre and and control mice.


Content of TAG assessed by MS in HSA - Cre and control GC muscles .

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Content of TAG assessed by MS in HSA-Cre and control GC muscles.


Cholesterol , ( E ) Cholesteryl ester ( CE ) , ( F ) DAG was assessed by MS in HSA - Cre and control GC muscles .

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Cholesterol, (E) Cholesteryl ester (CE), (F) DAG was assessed by MS in HSA-Cre and control GC muscles.


Quantitative RT - PCR detection of the level of expression of Fatty acid synthase ( Fasn ) , ATP citrate lyase ( Acly ) , Thyroid hormone - inducible hepatic protein 1 ( Thrsp1 ) , Angiopoietin - like protein 4 / fast - induced adipose factor ( ANGPTL4 / Fiaf ) , Insulin - induced gene 1 ( Insig1 ) and Peroxisome proliferator - activated receptor gamma ( PPARγ ) in TA from 3 - month - old HSA - Cre and control mice . Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice .

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Quantitative RT-PCR detection of the level of expression of Fatty acid synthase (Fasn), ATP citrate lyase (Acly), Thyroid hormone-inducible hepatic protein 1 (Thrsp1), Angiopoietin-like protein 4/ fast-induced adipose factor (ANGPTL4/Fiaf), Insulin-induced gene 1 (Insig1) and Peroxisome proliferator-activated receptor gamma (PPARγ) in TA from 3-month-old HSA-Cre and control mice. Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice.


Immunoblot analysis of Fasn in TA muscle of 3 - month - old HSA - Cre and control mice .

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Immunoblot analysis of Fasn in TA muscle of 3-month-old HSA-Cre and control mice.


Quantitative RT - PCR measurement of Carnitine palmitoyltransferase 1B ( Cpt1b ) and Medium - chain acyl - CoA dehydrogenase ( Mcad ) in TA from 3 - month - old HSA - Cre and control mice .

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Quantitative RT-PCR measurement of Carnitine palmitoyltransferase 1B (Cpt1b) and Medium-chain acyl-CoA dehydrogenase (Mcad) in TA from 3-month-old HSA-Cre and control mice.


Immunoblot analysis of full length and N - terminal SREBP1c and SREBP2 protein levels in GC muscle of 4 - month - old HSA - Cre and control mice ( n = 7 - 4 ) . The ratio of the densitometric assay of the N - term and Full length forms is presented .

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Immunoblot analysis of full length and N-terminal SREBP1c and SREBP2 protein levels in GC muscle of 4-month-old HSA-Cre and control mice (n=7-4). The ratio of the densitometric assay of the N-term and Full length forms is presented.


Metabolomics profile of GC muscle of 4 - month - old HSA - Cre and control mice presented as heatmap visualization and hierarchical clustering analysis of

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Metabolomics profile of GC muscle of 4-month-old HSA-Cre and control mice presented as heatmap visualization and hierarchical clustering analysis of compounds


Fold change of phospholipid precursors in GC muscles of 4 - month - old HSA - Cre and control mice measured by liquid - chromatography ( LC ) tandem mass spectrometry ( MS / MS ) ( n = 7 - 4 , * P < 0 . 05 ) .

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Fold change of phospholipid precursors in GC muscles of 4-month-old HSA-Cre and control mice measured by liquid-chromatography (LC) tandem mass spectrometry (MS/MS) (n=7-4, *P<0.05).


Fold change of Lactate / Pyruvate , Pyruvate / AcetylCoA , Pyruvate / Citrate , ATP / AMP , and ATP / ADP ratios in GC muscles of 4 month - old HSA - Cre and control mice ( n = 7 - 4 , * P < 0 . 05 ) .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O

Fold change of Lactate/Pyruvate, Pyruvate/AcetylCoA, Pyruvate/Citrate, ATP/AMP, and ATP/ADP ratios in GC muscles of 4 month-old HSA-Cre and control mice (n=7-4, *P<0.05).


UPR activation was analysed by immunoblotting with indicated antibodies in GC muscles of 4 - month - old HSA - Cre and control mice ( n = 7 - 4 ) . Quantification by densitometric analyses of Fgf21 , Bip , sXbp1 Atf6 cleaved form , p - Eif2a and Chop protein levels is presented as a graph . Data are normalized to Gapdh and expressed as a fold change relative to the control mice .

O O O O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O B-ORGANISM O

UPR activation was analysed by immunoblotting with indicated antibodies in GC muscles of 4-month-old HSA-Cre and control mice (n=7-4). Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, p-Eif2a and Chop protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice.


Quantitative RT - PCR measurement of Fgf21 , Gdf15 , Bip / Grp78 , hsp90b1 , and Edem1 in GC muscles from 4 - month - old HSA - Cre and control mice .

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Quantitative RT-PCR measurement of Fgf21, Gdf15, Bip/Grp78, hsp90b1, and Edem1 in GC muscles from 4-month-old HSA-Cre and control mice.


EM analysis of EDL fibers of HSA - Cre mice revealed the presence of areas in which the non - junctional SR is more vesiculated and fragmented compared to control ( black arrows ) . Remodeling of the SR is also accompanied by modification in the positioning of mitochondria ( white arrows ) . A representative Calcium Release Unit ( CRU ) is indicated ( empty arrow ) . Scale bars : 0 . 50 μm .

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EM analysis of EDL fibers of HSA-Cre mice revealed the presence of areas in which the non-junctional SR is more vesiculated and fragmented compared to control (black arrows). Remodeling of the SR is also accompanied by modification in the positioning of mitochondria (white arrows). A representative Calcium Release Unit (CRU) is indicated (empty arrow). Scale bars: 0.50 μm.


The relative mtDNA copy number was determined by the ratio of mitochondrial DNA - encoded Cytochrome B ( CytB ) and nuclear - encoded NADH dehydrogenase ( ubiquinone ) flavoprotein 1 ( Ndufv1 ) genes in GC and TA from HSA - Cre and control mice .

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The relative mtDNA copy number was determined by the ratio of mitochondrial DNA-encoded Cytochrome B (CytB) and nuclear-encoded NADH dehydrogenase (ubiquinone) flavoprotein 1 (Ndufv1) genes in GC and TA from HSA-Cre and control mice.


Mitochondria - encoded gene and Nuclear - encoded gene expression levels by quantitative RT - PCR in GC muscles from 4 - month - old HSA - Cre and control mice .

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Mitochondria-encoded gene and Nuclear-encoded gene expression levels by quantitative RT-PCR in GC muscles from 4-month-old HSA-Cre and control mice.


Electron micrographs of HSA - Cre EDL muscles of 3 - month - old mice showed that mitochondria are almost exclusively positioned at the two sides of Z lines in EDL fibers from control mice ( black arrows ) . On the other hand , in Lipin1 deficient EDL fibers mitochondria are more often clustered and display electron - dense contact sites ( white arrows ) . Scale bars : 0 . 20 μm .

B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD B-TISSUE I-TISSUE O O O O O O B-ORGANISM O O B-SUBCELLULAR O O O O O O O O O O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O O O O O O O B-GENEPROD O B-TISSUE I-TISSUE B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O

Electron micrographs of HSA-Cre EDL muscles of 3-month-old mice showed that mitochondria are almost exclusively positioned at the two sides of Z lines in EDL fibers from control mice (black arrows). On the other hand, in Lipin1 deficient EDL fibers mitochondria are more often clustered and display electron-dense contact sites (white arrows). Scale bars: 0.20 μm.


Ultrastructural analysis of HSA - Cre EDL muscles revealed the presence of numerous autophagic vacuoles of different morphologies ( autophagosomes and lysosomes , black arrows ; multilamellar bodies , white arrow ) containing amorphous material and possibly degraded mitochondria ( M ) . Scale bars : 0 . 50

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Ultrastructural analysis of HSA-Cre EDL muscles revealed the presence of numerous autophagic vacuoles of different morphologies (autophagosomes and lysosomes, black arrows; multilamellar bodies, white arrow) containing amorphous material and possibly degraded mitochondria (M). Scale bars: 0.50 μm


Representative immunoblots from protein extract of GC muscles of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice , with the indicated antibodies .

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Representative immunoblots from protein extract of GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice, with the indicated antibodies.


Quantitative RT - PCR measurement of Fgf21 and GDF15 expression in GC muscle of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice .

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Quantitative RT-PCR measurement of Fgf21 and GDF15 expression in GC muscle of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.


Histological analysis of TA muscles of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice by Oil Red O staining to detect the neutral lipids . Scale bars : 40 μm .

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Histological analysis of TA muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice by Oil Red O staining to detect the neutral lipids. Scale bars: 40 μm.


TAG level measurement in GC muscles of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice .

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TAG level measurement in GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.


In vivo force measurements performed on GC muscles of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice .

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In vivo force measurements performed on GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.


Representative staining of IgG positive fibers on frozen sections of TA of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice . The sections were stained with a secondary fluorescent anti - mouse IgG antibody . Quantification of the number of necrotic fibers over the total number of fibers .

O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD B-SMALL_MOLECULE O O B-ORGANISM O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O B-ORGANISM B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O

Representative staining of IgG positive fibers on frozen sections of TA of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice. The sections were stained with a secondary fluorescent anti-mouse IgG antibody. Quantification of the number of necrotic fibers over the total number of fibers.


Representative immunoblots from protein extract of GC muscles of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice , with the indicated antibodies .

Representative immunoblots from protein extract of GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice, with the indicated antibodies.


Quantitative RT - PCR measurement of Fgf21 and GDF15 expression in GC muscle of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice .

Quantitative RT-PCR measurement of Fgf21 and GDF15 expression in GC muscle of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.


Histological analysis of TA muscles of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice by Oil Red O staining to detect the neutral lipids .

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Histological analysis of TA muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice by Oil Red O staining to detect the neutral lipids.


TAG level measurement in GC muscles of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice .

TAG level measurement in GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.


In vivo force measurements performed on GC muscles of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice .

In vivo force measurements performed on GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.


Representative staining of IgG positive fibers on frozen sections of TA of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice . The sections were stained with a secondary fluorescent anti - mouse IgG antibody . Quantification of the number of necrotic fibers over the total number of fibers .

Representative staining of IgG positive fibers on frozen sections of TA of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice. The sections were stained with a secondary fluorescent anti-mouse IgG antibody. Quantification of the number of necrotic fibers over the total number of fibers.


Hoxb8 - FL cells generated from bone marrow of mice of the indicated genotypes were treated for 24h with graded doses of the CHK1 - inhibitors PF - 477736 ( PF ) or CHIR - 124 ( CHIR ) . Representative histograms of CHK1i - treated ( grey ) or DMSO - treated ( black ) cells , analyzed for cell cycle distribution and cell death using Nicoletti staining and flow cytometry are shown .

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Hoxb8-FL cells generated from bone marrow of mice of the indicated genotypes were treated for 24h with graded doses of the CHK1-inhibitors PF-477736 (PF) or CHIR-124 (CHIR). Representative histograms of CHK1i-treated (grey) or DMSO-treated (black) cells, analyzed for cell cycle distribution and cell death using Nicoletti staining and flow cytometry are shown.


WT Hoxb8 - FL cells were treated with CHIR - 124 in the presence or absence of the pan - caspase inhibitor QVD . Cell death was assessed after 24h using flow cytometry as in ( A ) .

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WT Hoxb8-FL cells were treated with CHIR-124 in the presence or absence of the pan-caspase inhibitor QVD. Cell death was assessed after 24h using flow cytometry as in (A).


Western blot of CHIR - treated Hoxb8 - FL WT and Bax - / - Bak - / - cells using the indicated antibodies .

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Western blot of CHIR-treated Hoxb8-FL WT and Bax-/-Bak-/- cells using the indicated antibodies.


WT and Bax - / - Bak - / - Hoxb8 - FL cells were treated for up to 8h with 500 nM CHIR and p21 mRNA levels were compared to Hprt in response to CHK1 - inhibition by real - time RT - qPCR . Bars represent means ± S . D . of independent experiments ( n = 4 - 6 ) , performed in duplicates .

O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

WT and Bax-/-Bak-/- Hoxb8-FL cells were treated for up to 8h with 500 nM CHIR and p21 mRNA levels were compared to Hprt in response to CHK1-inhibition by real-time RT-qPCR. Bars represent means ± S.D. of independent experiments (n=4-6), performed in duplicates.


50 . 000 Hoxb8 - FL cells of the indicated genotypes were seeded to assess cell growth in response to CHK1 - inhibition . Cells were counted 48h post seeding .

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50.000 Hoxb8-FL cells of the indicated genotypes were seeded to assess cell growth in response to CHK1-inhibition. Cells were counted 48h post seeding.


LSK ( Lin - Sca1 + cKit + ) cells of the indicated genotypes were isolated by cell sorting from the fetal liver at embryonic day E13 . 5 or the bone marrow of adult mice and treated for 48h with PF - 477736 [ 1 . 5 μM ] . Survival was assessed using AnnexinV - staining and flow cytometry . Bars represent means ± S . E . M . from n = 6 wild type and n = 3 Vav - BCL2 fetal liver LSK cells ( left ) and n = 3 bone marrow LSK cells ( right ) .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY 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-TISSUE I-TISSUE B-CELL_TYPE O O O O O O O O B-TISSUE I-TISSUE B-CELL_TYPE O O O O O

LSK (Lin- Sca1+ cKit+) cells of the indicated genotypes were isolated by cell sorting from the fetal liver at embryonic day E13.5 or the bone marrow of adult mice and treated for 48h with PF-477736 [1.5 μM]. Survival was assessed using AnnexinV-staining and flow cytometry. Bars represent means ± S.E.M. from n=6 wild type and n=3 Vav-BCL2 fetal liver LSK cells (left) and n=3 bone marrow LSK cells (right).


WT and Vav - BCL2 derived E14 . 5 total fetal liver cells ( B ) were treated for 72h ( B ) with different doses of CHK1i . Survival was assessed using Nicoletti staining and flow cytometry . Bars represent means ± S . E . M . ( n = 3 biological replicates per genotype ) .

O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

WT and Vav-BCL2 derived E14.5 total fetal liver cells (B) were treated for 72h (B) with different doses of CHK1i. Survival was assessed using Nicoletti staining and flow cytometry. Bars represent means ± S.E.M. (n=3 biological replicates per genotype).


WT and Vav - BCL2 derived E14 . 5 total bone marrow cells ( C ) were treated for up to 48h with different doses of CHK1i . Survival was assessed using Nicoletti staining and flow cytometry . Bars represent means ± S . E . M . ( n = 3 biological replicates per genotype ) .

O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

WT and Vav-BCL2 derived E14.5 total bone marrow cells (C) were treated for up to 48h with different doses of CHK1i. Survival was assessed using Nicoletti staining and flow cytometry. Bars represent means ± S.E.M. (n=3 biological replicates per genotype).


MACS - purified CD34 + human cord - blood derived HSPC were treated with PF - 477736 or CHIR - 124 ± the caspase - inhibitor QVD ( 50 μM ) . Cell death was assessed using AnnexinV / 7 - AAD staining and flow cytometry .

MACS-purified CD34+ human cord-blood derived HSPC were treated with PF-477736 or CHIR-124 ± the caspase-inhibitor QVD (50 μM). Cell death was assessed using AnnexinV/7-AAD staining and flow cytometry.


CD34 + cells were transduced with a BCL2 - encoding lentivirus or a control virus , along with IRES - GFP with a MOI of 10 for two consecutive days , followed by CHK1i application for 48 h . Survival was assessed using Nicoletti staining and flow cytometry .

B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O B-ORGANISM O O O B-ORGANISM O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O

CD34+ cells were transduced with a BCL2-encoding lentivirus or a control virus, along with IRES-GFP with a MOI of 10 for two consecutive days, followed by CHK1i application for 48 h. Survival was assessed using Nicoletti staining and flow cytometry.


Percentage of living GFP + transduced human CD34 + HSPCs , cultured in the absence or presence of CHK1i for 48 hours .

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Percentage of living GFP+ transduced human CD34+ HSPCs, cultured in the absence or presence of CHK1i for 48 hours.


Colony formation potential of human CD34 + HSPCs transduced with empty control or BCL2 encoding virus . Colonies were counted 10 days post seeding of 1000 CD34 + cells per plate / sample . " Myeloid colonies " include granulocyte / monocyte , monocyte , granulocyte and GEMM ( granulocyte , erythroid , monocyte / macrophage and megakaryocyte ) colonies .

Colony formation potential of human CD34+ HSPCs transduced with empty control or BCL2 encoding virus. Colonies were counted 10 days post seeding of 1000 CD34+ cells per plate/sample. "Myeloid colonies" include granulocyte/monocyte, monocyte, granulocyte and GEMM (granulocyte, erythroid, monocyte/macrophage and megakaryocyte) colonies.


Representative pictures of E13 . 5 and E18 . 5 embryos of the indicated genotypes . Scale bar E13 . 5 = 1 mm , E18 . 5 = 5 mm . Quantification of fetal liver cell number at E13 . 5 . Bars represent means ± S . E . M . ( N = 4 for Chk1fl / + , N = 3 for Chk1fl / + Vav - Cre and N = 4 for Chk1fl / - Vav - Cre ) .

O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O

Representative pictures of E13.5 and E18.5 embryos of the indicated genotypes. Scale bar E13.5 = 1 mm, E18.5 = 5 mm. Quantification of fetal liver cell number at E13.5. Bars represent means ± S.E.M. (N=4 for Chk1fl/+, N=3 for Chk1fl/+ Vav-Cre and N=4 for Chk1fl/- Vav-Cre).


Representative pictures of isolated fetal livers at E13 . 5 . Scale bar = 1 mm .

O O O O B-TISSUE I-TISSUE O O O O O O O O O O O

Representative pictures of isolated fetal livers at E13.5. Scale bar = 1 mm.


Representative H & E - staining of fetal liver sections from E13 . 5 embryos . Black arrows indicate lack of erythroid cells in the blood vessels of Chk1fl / - Vav - Cre fetal livers . Scale bar = 500 µM .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O B-ORGANISM O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD B-TISSUE I-TISSUE O O O O O O O

Representative H&E-staining of fetal liver sections from E13.5 embryos. Black arrows indicate lack of erythroid cells in the blood vessels of Chk1fl/- Vav-Cre fetal livers. Scale bar = 500 µM.


Single cell suspensions of fetal livers from Chk1fl / + , Chk1fl / + Vav - Cre and Chk1fl / - Vav - Cre embryos were stained with antibodies recognizing ckit , CD71 , Ter119 or CD45 to assess primitive and definitive erythropoiesis by flow cytometry . Large ' EryA ' erythroblasts ( CD71high Ter119high FSChigh ) , smaller , more mature ' EryB ' erythroblasts ( CD71high Ter119high FSClow ) ; most mature erythroblast subset is EryC ( CD71low Ter119high FSClow ) . Bars represent means ± S . E . M . ctrl N = 5 ( pooled N = 4 Chk1fl / + and N = 1 Chk1fl / + Vav - Cre ) , N = 3 for Chk1fl / - Vav - Cre .

O O O O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD B-ORGANISM O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O B-CELL_TYPE O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O

Single cell suspensions of fetal livers from Chk1fl/+, Chk1fl/+ Vav-Cre and Chk1fl/- Vav-Cre embryos were stained with antibodies recognizing ckit, CD71, Ter119 or CD45 to assess primitive and definitive erythropoiesis by flow cytometry. Large 'EryA' erythroblasts (CD71high Ter119high FSChigh), smaller, more mature 'EryB' erythroblasts (CD71high Ter119high FSClow); most mature erythroblast subset is EryC (CD71low Ter119high FSClow). Bars represent means ± S.E.M. ctrl N=5 (pooled N=4 Chk1fl/+ and N=1 Chk1fl/+ Vav-Cre), N=3 for Chk1fl/- Vav-Cre.


Representative dot - plots of the LSK cell phenotype of Chk1fl / + and Chk1fl / - Vav - Cre embryos observed in E13 . 5 fetal livers and relative distribution / absolute numbers of LKhi and LSKhi cells , as well as LKlow and LSKlow cells , found in the fetal liver on E13 . 5 . Bars represent means ± S . E . M . N = 4 for Chk1fl / + and N = 4 for Chk1fl / - Vav - Cre .

O O O O O O B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD B-ORGANISM O O O O O B-TISSUE I-TISSUE O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-CELL_TYPE O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O

Representative dot-plots of the LSK cell phenotype of Chk1fl/+ and Chk1fl/- Vav-Cre embryos observed in E13.5 fetal livers and relative distribution / absolute numbers of LKhi and LSKhi cells, as well as LKlow and LSKlow cells, found in the fetal liver on E13.5. Bars represent means ± S.E.M. N=4 for Chk1fl/+ and N=4 for Chk1fl/- Vav-Cre.


Colony formation potential of FACS - sorted LSK cells ( left panel , N = 4 for Chk1fl / + and N = 5 for Chk1fl / - Vav - Cre ) and total fetal liver cells ( right panel , N = 3 for Chk1fl / + and N = 6 for Chk1fl / - Vav - Cre ) in methyl - cellulose assays . Bars represent means ± S . E . M .

B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

Colony formation potential of FACS-sorted LSK cells (left panel, N=4 for Chk1fl/+ and N=5 for Chk1fl/- Vav-Cre) and total fetal liver cells (right panel, N=3 for Chk1fl/+ and N=6 for Chk1fl/- Vav-Cre) in methyl-cellulose assays. Bars represent means ± S.E.M.


Flow cytometric analysis of LSKhi or LSKlow cells stained with antibodies specific for CD34 and Flt3 ( N = 4 for Chk1fl / + and N = 4 for Chk1fl / - Vav - Cre ) , to discriminate LT - and ST - HSC from MPP within the LSK - subset . Bars represent means ± S . E . M .

B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O B-CELL_TYPE O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O O O

Flow cytometric analysis of LSKhi or LSKlow cells stained with antibodies specific for CD34 and Flt3 (N=4 for Chk1fl/+ and N=4 for Chk1fl/- Vav-Cre), to discriminate LT- and ST-HSC from MPP within the LSK-subset. Bars represent means ± S.E.M.


Terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) staining of fetal liver cryosections of the indicated genotypes at E13 . 5 and quantification . Scale bar 100x = 200 µM , 400x = 50 µM . The percentage of TUNEL + area / field was assessed across three random fields from two control ( Chk1fl / + ) and two knock - out embryos ( Chk1fl / - Vav - Cre ) using ImageJ , as reported by us before ( 60 ) . Bars represent means ± S . D .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O 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-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of fetal liver cryosections of the indicated genotypes at E13.5 and quantification. Scale bar 100x = 200 µM, 400x = 50 µM. The percentage of TUNEL+ area/field was assessed across three random fields from two control (Chk1fl/+) and two knock-out embryos (Chk1fl/- Vav-Cre) using ImageJ, as reported by us before (60). Bars represent means ± S.D.


Total fetal liver cell suspensions of Chk1fl / + ( + / + ) , Chk1fl / + Vav - Cre ( + / - ) and Chk1fl / - Vav - Cre ( - / - ) E13 . 5 embryos were processed for Western blot analysis using the indicated antibodies . Each lane represents samples isolated from an individual littermate embryo .

O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O

Total fetal liver cell suspensions of Chk1fl/+ (+/+), Chk1fl/+ Vav-Cre (+/-) and Chk1fl/- Vav-Cre (-/-) E13.5 embryos were processed for Western blot analysis using the indicated antibodies. Each lane represents samples isolated from an individual littermate embryo.


DAPI - negative viable LK - and LSK - cells of the indicated genotypes ( littermates ) were sorted from E13 . 5 fetal livers and immediately fixed in 70 % EtOH . Fixed cell suspensions were stained for ɣH2A . X and DAPI intracellularly . Shown here are representative dot - plots and quantification of N = 4 embroys per genotype ; Bars represent means ± S . E . M .

B-SMALL_MOLECULE O O O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

DAPI-negative viable LK- and LSK-cells of the indicated genotypes (littermates) were sorted from E13.5 fetal livers and immediately fixed in 70% EtOH. Fixed cell suspensions were stained for ɣH2A.X and DAPI intracellularly. Shown here are representative dot-plots and quantification of N=4 embroys per genotype; Bars represent means ± S.E.M.


Total E13 . 5 fetal liver cells were processed for intracellular Ki67 staining in combination with cell surface antibody staining to discriminate LK ( top panel ) from LSK ( lower panel ) cells . Shown here are representative dot - plots of Ki67 and DAPI staining in LK and LSK cells , quantified in ( E ) . Bars represent means ± S . E . M . N = 4 for Chk1fl / + and N = 5 for Chk1fl / - Vav - Cre .

O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O

Total E13.5 fetal liver cells were processed for intracellular Ki67 staining in combination with cell surface antibody staining to discriminate LK (top panel) from LSK (lower panel) cells. Shown here are representative dot-plots of Ki67 and DAPI staining in LK and LSK cells, quantified in (E). Bars represent means ± S.E.M. N=4 for Chk1fl/+ and N=5 for Chk1fl/- Vav-Cre.


Representative dot - plots and quantification of E13 . 5 fetal liver LSKhi / LSKlow cell phenotypes observed in embryos of the indicated genotypes . N = 21 for Chk1fl / + , N = 5 for Chk1fl / - Vav - Cre , N = 16 for Chk1fl / + Vav - BCL2 , N = 5 for Chk1fl / - Vav - Cre Vav - BCL2 , N = 2 for p53 - / - Chk1fl / + and N = 4 for p53 - / - Chk1fl / - Vav - Cre .

O O O O O O O O O O B-TISSUE I-TISSUE B-CELL_TYPE O B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O

Representative dot-plots and quantification of E13.5 fetal liver LSKhi/LSKlow cell phenotypes observed in embryos of the indicated genotypes. N=21 for Chk1fl/+, N=5 for Chk1fl/- Vav-Cre, N=16 for Chk1fl/+ Vav-BCL2, N=5 for Chk1fl/- Vav-Cre Vav-BCL2, N=2 for p53-/- Chk1fl/+ and N=4 for p53-/- Chk1fl/- Vav-Cre.


Flow cytometry - based analysis of LSK cells stained with antibodies specific for CD34 and Flt3 , to discriminate LT - and ST - HSC from MPP within the LSK subset in fetal livers of embryos of the indicated genotypes . Left : N = 25 for Chk1fl / + , N = 7 for Chk1fl / + Vav - Cre and N = 7 for Chk1fl / - Vav - Cre . Right : N = 16 for Chk1fl / + Vav - BCL2 , N = 6 for Chk1fl / + Vav - Cre Vav - BCL2 and N = 5 for Chk1fl / - Vav - Cre Vav - BCL2 .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE O O B-TISSUE I-TISSUE O B-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O

Flow cytometry-based analysis of LSK cells stained with antibodies specific for CD34 and Flt3, to discriminate LT- and ST-HSC from MPP within the LSK subset in fetal livers of embryos of the indicated genotypes. Left: N=25 for Chk1fl/+, N=7 for Chk1fl/+ Vav-Cre and N=7 for Chk1fl/- Vav-Cre. Right: N=16 for Chk1fl/+ Vav-BCL2, N=6 for Chk1fl/+ Vav-Cre Vav-BCL2 and N=5 for Chk1fl/- Vav-Cre Vav-BCL2.


Analysis of the colony formation potential of E14 . 5 total fetal liver cells of the indicated genotypes in methylcellulose . N = 3 for controls ( pooled ; 1 Chk1fl / + , 2 Chk1fl / + Vav - Cre ) , N = 3 for Chk1fl / - Vav - Cre and N = 3 for Chk1fl / - Vav - Cre Vav - BCL2 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O

Analysis of the colony formation potential of E14.5 total fetal liver cells of the indicated genotypes in methylcellulose. N=3 for controls (pooled; 1 Chk1fl/+, 2 Chk1fl/+ Vav-Cre), N=3 for Chk1fl/- Vav-Cre and N=3 for Chk1fl/- Vav-Cre Vav-BCL2.


Western blot analysis of fetal liver cells of the indicated genotypes ( each lane represents an embryo ) at E14 . 5 using the indicated antibodies .

B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-ORGANISM O O O O O O O O O O

Western blot analysis of fetal liver cells of the indicated genotypes (each lane represents an embryo) at E14.5 using the indicated antibodies.


Three independent Chk1fl / fl mT / mG ( coloured ) and mT / mG ( grey ) Hoxb8 - FL progenitor cell lines were transduced with a retroviral construct carrying Cre ( pMIP - CRE - IRES - PURO ) and monitored over time for GFP - expression . Shown here are three technical replicates per genotype . Additionally , Chk1fl / fl mT / mG Hoxb8 - FL progenitor cells were FACS - sorted for GFP + and dsRED + cells 48h after transduction with the Cre - construct . Sorted cells were processed for western blotting using the indicated antibodies .

O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O O O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O

Three independent Chk1fl/fl mT/mG (coloured) and mT/mG (grey) Hoxb8-FL progenitor cell lines were transduced with a retroviral construct carrying Cre (pMIP-CRE-IRES-PURO) and monitored over time for GFP-expression. Shown here are three technical replicates per genotype. Additionally, Chk1fl/fl mT/mG Hoxb8-FL progenitor cells were FACS-sorted for GFP+ and dsRED+ cells 48h after transduction with the Cre-construct. Sorted cells were processed for western blotting using the indicated antibodies.


Chk1fl / fl Hoxb8 - FL progenitor cells were transduced with a retroviral construct carrying Cre and GFP ( pMIG - CRE - IRES - GFP ) . Cells were FACS - sorted 72h after transduction with the retroviral construct and processed for western blotting using the indicated antibodies .

B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O

Chk1fl/fl Hoxb8-FL progenitor cells were transduced with a retroviral construct carrying Cre and GFP (pMIG-CRE-IRES-GFP). Cells were FACS-sorted 72h after transduction with the retroviral construct and processed for western blotting using the indicated antibodies.


Percentage of GFP + cells indicative for CRE expression and recombination in the peripheral blood after tamoxifen administration by gavage ( 5 x 2 mg , 200 μl / mouse ; daily ) . Bars represent means ± S . E . M . ( Day 0 N = 9 / 10 , WT / Chk1fl / fl , Day 7 N = 8 / 7 , Day 14 N = 3 / 3 ) .

O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-TISSUE O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O

Percentage of GFP+ cells indicative for CRE expression and recombination in the peripheral blood after tamoxifen administration by gavage (5 x 2 mg, 200 μl/mouse; daily). Bars represent means ± S.E.M. (Day 0 N=9/10, WT/Chk1fl/fl, Day 7 N=8/7, Day 14 N=3/3).


Quantification of Chk1 - del PCR - product enrichment in DNA from GFP + vs . Tomato + bone marrow cells sorted on day 8 after first TAM administration . Transgenic Cre levels were quantified in parallel for reference . Bars represent means ± S . E . M . from n = 5 mT / mG Vav - CreERT2 Chk1fl / fl mice .

O O B-GENEPROD O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-ORGANISM O

Quantification of Chk1-del PCR-product enrichment in DNA from GFP+ vs. Tomato+ bone marrow cells sorted on day 8 after first TAM administration. Transgenic Cre levels were quantified in parallel for reference. Bars represent means ± S.E.M. from n=5 mT/mG Vav-CreERT2 Chk1fl/fl mice.


Percentage of living LK or LSK - cells and ( F ) percentage of GFP + LK or LSK - cells found in the bone marrow of mT / mG Vav - CreERT2 ( WT ) and mT / mG Vav - CreERT2 Chk1fl / fl ( Chk1fl / fl ) mice on day 8 or 15 post first TAM administration . Bars in ( E ) / ( F ) represent means ± S . E . M . ( Day 8 N = 8 / 8 , WT / Chk1fl / fl , Day 15 N = 7 / 8 ) .

O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-CELL_TYPE O B-CELL_TYPE O O O O O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O 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-GENEPROD O O O O O O O O O O O O

Percentage of living LK or LSK-cells and (F) percentage of GFP+ LK or LSK-cells found in the bone marrow of mT/mG Vav-CreERT2 (WT) and mT/mG Vav-CreERT2 Chk1fl/fl (Chk1fl/fl) mice on day 8 or 15 post first TAM administration. Bars in (E)/(F) represent means ± S.E.M. (Day 8 N=8/8, WT/Chk1fl/fl, Day 15 N=7/8).


GFP + ( CRE ) and Tomato + ( CRE - ) total bone marrow cells were sorted on day 8 after first TAM - administration and processed for Western Blot using the indicated antibodies . Sorted cells from three animals - mT / mG Vav - CreERT2 ( WT ) and mT / mG Vav - CreERT2 Chk1fl / fl ( Chk1fl / fl ) - were pooled per lane .

B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O

GFP+ (CRE) and Tomato+ (CRE-) total bone marrow cells were sorted on day 8 after first TAM-administration and processed for Western Blot using the indicated antibodies. Sorted cells from three animals - mT/mG Vav-CreERT2 (WT) and mT/mG Vav-CreERT2 Chk1fl/fl (Chk1fl/fl) - were pooled per lane.


Thymocytes of the indicated genotypes were sorted based on GFP + and Tomato + on day 15 after the first TAM administration for western analysis using the indicated antibodies .

B-CELL_TYPE O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O

Thymocytes of the indicated genotypes were sorted based on GFP+ and Tomato+ on day 15 after the first TAM administration for western analysis using the indicated antibodies.


Quantification of human EGFL7 by qRT - PCR in primary GBM biopsies and corresponding PDX xenografts upon implantation of GBM - derived organotypic spheroids ( n = 3 ; Mann - Whitney U - test ; mean ± SEM ) ; B - GBM biopsy ; G - generation .

O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-DISEASE O O O O B-EXP_ASSAY O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O

Quantification of human EGFL7 by qRT-PCR in primary GBM biopsies and corresponding PDX xenografts upon implantation of GBM-derived organotypic spheroids (n = 3; Mann-Whitney U-test; mean ± SEM); B - GBM biopsy; G - generation.


Alignment of EGFL7 expression along with stromal ( VWF , VEGFR1 , VEGFR2 , ACTA2 , CD248 , ITGAX , AIF1 , CD68 , CD4 , and PTPRC ) or tumor markers ( EGFR , PDGFRA , VEGFA , VIM , and NES ) in primary GBM biopsies or GBM - derived organotypic spheroids ( PDX xenografts ) .

O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-DISEASE O O B-DISEASE O O O O O O B-EXP_ASSAY O O

Alignment of EGFL7 expression along with stromal (VWF, VEGFR1, VEGFR2, ACTA2, CD248, ITGAX, AIF1, CD68, CD4, and PTPRC) or tumor markers (EGFR, PDGFRA, VEGFA, VIM, and NES) in primary GBM biopsies or GBM-derived organotypic spheroids (PDX xenografts).


Immunohisto chemical ( IHC ) analyses revealed EGFL7 expression in blood vessels ( arrow ) and neurons ( arrowheads ) of healthy gray matter . Expression in malignant brain tumor specimens was restricted to blood vessels , in particular with a large diameter ( arrows ) . Scale bar represents 50 µm . Quantitative scoring of EGFL7 staining in glioma specimens yielded about 25 - 40 % EGFL7 - positive blood vessels ( n = 13 astrocytoma WHO ° grade II , n = 25 astrocytoma WHO ° grade III , n = 24 GBMs WHO ° grade IV ) .

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Immunohistochemical (IHC) analyses revealed EGFL7 expression in blood vessels (arrow) and neurons (arrowheads) of healthy gray matter. Expression in malignant brain tumor specimens was restricted to blood vessels, in particular with a large diameter (arrows). Scale bar represents 50 µm. Quantitative scoring of EGFL7 staining in glioma specimens yielded about 25-40% EGFL7-positive blood vessels (n = 13 astrocytoma WHO° grade II, n = 25 astrocytoma WHO° grade III, n = 24 GBMs WHO° grade IV).


Overexpression of human EGFL7 ( hE7 ) or murine EGFL7 ( mE7 ) increased tumor growth ( n = 9 ; one - way ANOVA ) .

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Overexpression of human EGFL7 (hE7) or murine EGFL7 (mE7) increased tumor growth (n = 9; one-way ANOVA).


Immunocompetent mice bearing GL261 tumors . Representative MRI images confirmed tumor implantation in all experimental groups ( left ) . Kaplan - Meier curves revealed decreased survival of mice bearing EGFL7 expressing tumors ( right ; 34 d ( control ) vs . 29 . 5 d ( hE7 ) vs . 31 . 5 d ( mE7 ) ; n = 8 ; log - rank test ) . Immunodeficient mice bearing U87 tumors . Representative magnetic resonance images confirmed tumor implantation in all experimental groups ( left ) . Kaplan - Meier curves revealed decreased survival of mice bearing EGFL7 expressing tumors ( right ; 70 d ( control ) vs . 42 . 5 d ( hE7 ) vs . 40 d ( mE7 ) ; n = 8 ; log - rank test ) .

O B-ORGANISM O B-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O B-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O

Immunocompetent mice bearing GL261 tumors. Representative MRI images confirmed tumor implantation in all experimental groups (left). Kaplan-Meier curves revealed decreased survival of mice bearing EGFL7 expressing tumors (right; 34 d (control) vs. 29.5 d (hE7) vs. 31.5 d (mE7); n = 8; log-rank test). Immunodeficient mice bearing U87 tumors. Representative magnetic resonance images confirmed tumor implantation in all experimental groups (left). Kaplan-Meier curves revealed decreased survival of mice bearing EGFL7 expressing tumors (right; 70 d (control) vs. 42.5 d (hE7) vs. 40 d (mE7); n = 8; log-rank test).


miR - 126 KO did not affect overall survival of GL261 tumor - bearing mice ( n = 8 ) .

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miR-126 KO did not affect overall survival of GL261 tumor-bearing mice (n = 8).


EGFL7 KO prolonged overall survival of GL261 tumor - bearing mice as compared to wild - type ( WT ) littermates ( 44 d in KO vs . 35 . 5 d in WT ; n = 8 each ; log - rank test ) .

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EGFL7 KO prolonged overall survival of GL261 tumor-bearing mice as compared to wild-type (WT) littermates (44 d in KO vs. 35.5 d in WT; n = 8 each; log-rank test).


Residual EGFL7 expression in BTPC11 glioma cells was reduced by a lentivirus - based approach . Knock - down of EGFL7 ( shE7 _ 1 or shE7 _ 2 ) prolonged the median survival time of glioma - bearing mice ( 68 d or 69 d ) as compared to scrambled control ( shScr ; 56 d ; n = 6 ; log - rank test ) .

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Residual EGFL7 expression in BTPC11 glioma cells was reduced by a lentivirus-based approach. Knock-down of EGFL7 (shE7_1 or shE7_2) prolonged the median survival time of glioma-bearing mice (68 d or 69 d) as compared to scrambled control (shScr; 56 d; n = 6; log-rank test).


The blood vessel - specific and miR126 - independent KO of EGFL7 in EGFL7fl / fl ; Cdh5 - CreERT2 mice prolonged the overall survival of GL261 tumor - bearing mice as compared to WT littermates ( 38 d in KO vs . 33 d in WT ; n = 7 for KO ; n = 5 for WT ; log - rank test ) . All data are presented as mean ± SEM . Scale bars represent 5 mm .

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The blood vessel-specific and miR126-independent KO of EGFL7 in EGFL7fl/fl;Cdh5-CreERT2 mice prolonged the overall survival of GL261 tumor-bearing mice as compared to WT littermates (38 d in KO vs. 33 d in WT; n = 7 for KO; n = 5 for WT; log-rank test). All data are presented as mean ± SEM. Scale bars represent 5 mm.


CD31 staining for endothelial cells revealed increased tumor vascularization in mice bearing human EGFL7 ( hE7 ) - or murine EGFL7 ( mE7 ) - positive tumors ( n = 3 ; one - way ANOVA ) .

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CD31 staining for endothelial cells revealed increased tumor vascularization in mice bearing human EGFL7 (hE7)- or murine EGFL7 (mE7)-positive tumors (n = 3; one-way ANOVA).


T2 - weighted magnetic resonance imaging ( MRI ) analysis of these brains confirmed that all mice developed tumors of similar size . T1 - weighted MRI images showed decreased vessel permeability as measured by Gadovist extravasation in tumors expressing hE7 or mE7 ( red arrows ; n = 6 ; one - way ANOVA ) . Scale bar represents 2 . 5 mm .

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T2-weighted magnetic resonance imaging (MRI) analysis of these brains confirmed that all mice developed tumors of similar size. T1-weighted MRI images showed decreased vessel permeability as measured by Gadovist extravasation in tumors expressing hE7 or mE7 (red arrows; n = 6; one-way ANOVA). Scale bar represents 2.5 mm.


EGFL7 promoted density and maturation state of glioma vessels Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co - localization of PDGFRβ ( pericytes ) with CD31 ( n = 3 ; one - way ANOVA ; quantifications normalized to CD31 ) . Data presented as mean ± SEM , AU - arbitrary units . Scale bars represent 60 µm .

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EGFL7 promoted density and maturation state of glioma vessels Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co-localization of PDGFRβ (pericytes) with CD31 (n = 3; one-way ANOVA; quantifications normalized to CD31). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm.


Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co - localization of SMA ( smooth muscle cells ) , with CD31 ( n = 3 ; one - way ANOVA ; quantifications normalized to CD31 ) . Data presented as mean ± SEM , AU - arbitrary units . Scale bars represent 60 µm .

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Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co-localization of SMA (smooth muscle cells), with CD31 (n = 3; one-way ANOVA; quantifications normalized to CD31). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm.


Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co - localization of ( I + J ) Col IV ( basement membrane ) with CD31 ( n = 3 ; one - way ANOVA ; quantifications normalized to CD31 ) . Data presented as mean ± SEM , AU - arbitrary units . Scale bars represent 60 µm .

O O O B-DISEASE B-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co-localization of (I+J) Col IV (basement membrane) with CD31 (n = 3; one-way ANOVA; quantifications normalized to CD31). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm.


Structural analysis of recombinant EGFL7 by transmission electron microscopy ( TEM ) showed aggregates with a fibrillar structure of varying sizes . Black scale bar left represents 50 nm . Higher magnifications depict rod - like EGFL7 particles . Black scale bar right represents 15 nm . White scale bar represents 5 nm .

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Structural analysis of recombinant EGFL7 by transmission electron microscopy (TEM) showed aggregates with a fibrillar structure of varying sizes. Black scale bar left represents 50 nm. Higher magnifications depict rod-like EGFL7 particles. Black scale bar right represents 15 nm. White scale bar represents 5 nm.


Angiogenic sprouting of primary human umbilical vein endothelial cell ( HUVEC ) spheroids embedded in a collagen - based matrix was induced by the application of EGFL7 ( E7 ) , fibronectin ( Fn ) , or a combination of both ( upper row ) . Co - application of an integrin α5β1 blocking antibody ( anti - α5β1 ) reduced sprouting in all cases but control ( lower row ) . Scale bar represents 100 µm .

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Angiogenic sprouting of primary human umbilical vein endothelial cell (HUVEC) spheroids embedded in a collagen-based matrix was induced by the application of EGFL7 (E7), fibronectin (Fn), or a combination of both (upper row). Co-application of an integrin α5β1 blocking antibody (anti-α5β1) reduced sprouting in all cases but control (lower row). Scale bar represents 100 µm.


E7 , Fn and a combination of both significantly increased the mean cumulative sprout length per spheroid , an effect blocked by anti - α5β1 ( n = 3 ; one - way ANOVA ( inter - group differences ) and Mann - Whitney U - test ( intra - group differences ) ; mean ± SEM ) .

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E7, Fn and a combination of both significantly increased the mean cumulative sprout length per spheroid, an effect blocked by anti-α5β1 (n = 3; one-way ANOVA (inter-group differences) and Mann-Whitney U-test (intra-group differences); mean ± SEM).


Measurement of Rho GTPase signaling downstream of integrins . Quantification of ( G ) Cdc42 , ( H ) Rac1 and ( I ) RhoA activation by immunoblotting subsequent to seeding HUVECs on E7 , Fn or a combination of both . Preferentially , E7 activated Cdc42 and Fn activated Rac1 , but both proteins antagonized each other . None of the treatments affected RhoA signaling ( n = 3 ; one - way ANOVA ) . IP - immunoprecipitation , AU - arbitrary units .

O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O

Measurement of Rho GTPase signaling downstream of integrins. Quantification of (G) Cdc42, (H) Rac1 and (I) RhoA activation by immunoblotting subsequent to seeding HUVECs on E7, Fn or a combination of both. Preferentially, E7 activated Cdc42 and Fn activated Rac1, but both proteins antagonized each other. None of the treatments affected RhoA signaling (n = 3; one-way ANOVA). IP-immunoprecipitation, AU-arbitrary units.


Surface biotinylation assays verified increased surface expression of αV and β3 upon appli cation of E7 but not Fn ( n = 3 ; one - way ANOVA ) .

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Surface biotinylation assays verified increased surface expression of αV and β3 upon application of E7 but not Fn (n = 3; one-way ANOVA).


Surface expression of α5 and β1 was found increased upon treatment with E7 or Fn and was further enhanced by combination of both ( n = 3 ; one - way ANOVA ) .

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Surface expression of α5 and β1 was found increased upon treatment with E7 or Fn and was further enhanced by combination of both (n = 3; one-way ANOVA).


Ectopic expression of murine EGFL7 ( mE7 ) in GL261 mouse glioma cells followed by intracranial implantation into the striatum of immune - competent mice reduced overall survival but was rescued upon treatment with an integrin α5β1 - inhibiting antibody ( n = 6 ; log - rank test ) . Data presented as mean ± SEM . TCL - total cell lysate , AU - arbitrary units .

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Ectopic expression of murine EGFL7 (mE7) in GL261 mouse glioma cells followed by intracranial implantation into the striatum of immune-competent mice reduced overall survival but was rescued upon treatment with an integrin α5β1-inhibiting antibody (n = 6; log-rank test). Data presented as mean ± SEM. TCL-total cell lysate, AU-arbitrary units.


Glioma - bearing mice were injected with anti - EGFL7 or anti - VEGF antibodies , a combination of both ( Combo ) , or isotype negative controls . Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging ( MRI ) . Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry . ( A + B ) CD31 staining revealed that blocking of EGFL7 , VEGF , or a combination of both lead to decreased tumor vessel density ( n = 3 ; one - way ANOVA ) .

B-DISEASE O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-DISEASE I-DISEASE O O O O B-TISSUE I-TISSUE O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O O O O O O O O O

Glioma-bearing mice were injected with anti-EGFL7 or anti-VEGF antibodies, a combination of both (Combo), or isotype negative controls. Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging (MRI). Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry. (A+B) CD31 staining revealed that blocking of EGFL7, VEGF, or a combination of both lead to decreased tumor vessel density (n = 3; one-way ANOVA).


Glioma - bearing mice were injected with anti - EGFL7 or anti - VEGF antibodies , a combination of both ( Combo ) , or isotype negative controls . Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging ( MRI ) . Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry . Staining for ( C + D ) PDGFRβ ( n = 3 ; one - way ANOVA ) ,

B-DISEASE O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-DISEASE I-DISEASE O O O O B-TISSUE I-TISSUE O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O

Glioma-bearing mice were injected with anti-EGFL7 or anti-VEGF antibodies, a combination of both (Combo), or isotype negative controls. Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging (MRI). Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry. Staining for (C+D) PDGFRβ (n = 3; one-way ANOVA),


Glioma - bearing mice were injected with anti - EGFL7 or anti - VEGF antibodies , a combination of both ( Combo ) , or isotype negative controls . Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging ( MRI ) . Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry . Staining for SMA ( n = 3 ; one - way ANOVA )

B-DISEASE O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-DISEASE I-DISEASE O O O O B-TISSUE I-TISSUE O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O

Glioma-bearing mice were injected with anti-EGFL7 or anti-VEGF antibodies, a combination of both (Combo), or isotype negative controls. Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging (MRI). Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry. Staining for SMA (n = 3; one-way ANOVA)


Glioma - bearing mice were injected with anti - EGFL7 or anti - VEGF antibodies , a combination of both ( Combo ) , or isotype negative controls . Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging ( MRI ) . Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry . Staining for Col IV ( n = 3 ; one - way ANOVA ) revealed decreased amounts of blood vessel - associated pericytes , smooth muscle cells and Col IV in the basement membrane of blood vessels upon treatment with anti - EGFL7 , anti - VEGF and most significantly , a combination of both antibodies .

B-DISEASE O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-DISEASE I-DISEASE O O O O B-TISSUE I-TISSUE O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O

Glioma-bearing mice were injected with anti-EGFL7 or anti-VEGF antibodies, a combination of both (Combo), or isotype negative controls. Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging (MRI). Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry. Staining for Col IV (n = 3; one-way ANOVA) revealed decreased amounts of blood vessel-associated pericytes, smooth muscle cells and Col IV in the basement membrane of blood vessels upon treatment with anti-EGFL7, anti-VEGF and most significantly, a combination of both antibodies.


Glioma - bearing mice were injected with anti - EGFL7 or anti - VEGF antibodies , a combination of both ( Combo ) , or isotype negative controls . Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging ( MRI ) . Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry . This resulted in an increased intratumoral vessel permeability as measured by T1 - weightened MRI analyses of extravasating Gadovist ( n = 6 ; one - way ANOVA ) .

B-DISEASE O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-DISEASE I-DISEASE O O O O B-TISSUE I-TISSUE O O O O O B-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

Glioma-bearing mice were injected with anti-EGFL7 or anti-VEGF antibodies, a combination of both (Combo), or isotype negative controls. Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging (MRI). Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry. This resulted in an increased intratumoral vessel permeability as measured by T1-weightened MRI analyses of extravasating Gadovist (n = 6; one-way ANOVA).


Treatment with anti - EGFL7 ( 47 d ) , anti - VEGF ( 51 d ) , or a combination of both antibodies increased the median survival time of glioma - bearing Rag1 - / - mice ( 58 d ) as compared to isotype - treated controls ( 37 d ; n = 6 ; log - rank test ) .

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Treatment with anti-EGFL7 (47 d), anti-VEGF (51 d), or a combination of both antibodies increased the median survival time of glioma-bearing Rag1-/- mice (58 d) as compared to isotype-treated controls (37 d; n = 6; log-rank test).


Treatment of glioma - bearing NOD SCID mice with temozolomide ( TMD ) as a chemotherapeutic agent and a combination of anti - EGFL7 and anti - VEGF antibody increased the median survival time ( Combo ; 87 d ; n = 5 ; log - rank test ) as compared to anti - VEGF alone ( 80 d ; n = 5 ; log - rank test ) or isotype - treated controls ( 69 d ; n = 5 ; log - rank test ) . Data presented as mean ± SEM , AU - arbitrary units . Scale bars represent 60 µm .

O O B-DISEASE O O O O B-ORGANISM O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Treatment of glioma-bearing NOD SCID mice with temozolomide (TMD) as a chemotherapeutic agent and a combination of anti-EGFL7 and anti-VEGF antibody increased the median survival time (Combo; 87 d; n = 5; log-rank test) as compared to anti-VEGF alone (80 d; n = 5; log-rank test) or isotype-treated controls (69 d; n = 5; log-rank test). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm.


( a ) p62 localizes to the MR during cytokinesis and abscission , and is taken up together with the MR by one daughter cell . U2OS cells were fixed and stained with anti - p62 ( red ) and anti - MKLP1 ( green ) antibodies . Scale bars are 10 μm . Colocalization of p62 with MKLP1 of the MR was regularly observed in cells at this stage of cell division . The arrowheads point to MRs , which are shown enlarged in the insets . The quantification is presented in Supplementary Information Fig . S1a .

O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

(a) p62 localizes to the MR during cytokinesis and abscission, and is taken up together with the MR by one daughter cell. U2OS cells were fixed and stained with anti-p62 (red) and anti-MKLP1 (green) antibodies. Scale bars are 10 μm. Colocalization of p62 with MKLP1 of the MR was regularly observed in cells at this stage of cell division. The arrowheads point to MRs, which are shown enlarged in the insets. The quantification is presented in Supplementary Information Fig. S1a.


( b ) siRNA - mediated depletion of p62 from U2OS cells results in moderate MR accumulation , compared with a control depletion ( lamin A / C ) . For quantification , MRs were counted in control cells and in p62 - depleted cells that were negative for p62 staining . Data are means ± s . d . of three independent experiments ( n , total number of cells counted ; P , probability of a two - tailed paired t - test ) .

O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O 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) siRNA-mediated depletion of p62 from U2OS cells results in moderate MR accumulation, compared with a control depletion (lamin A/C). For quantification, MRs were counted in control cells and in p62-depleted cells that were negative for p62 staining. Data are means ± s.d. of three independent experiments (n, total number of cells counted; P, probability of a two-tailed paired t-test).


( a ) Atg8 ( LC3 ) localizes to the MR during abscission . The upper panels show stills from time - lapse microscopy of GFP - Atg8 - expressing cells . The arrowhead points to the accumulation of Atg8 at the site of the MR during abscission . The enlarged clipping on the right shows the midbody area at time - point 68 min . Scale bar is 10 μm .

O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

(a) Atg8 (LC3) localizes to the MR during abscission. The upper panels show stills from time-lapse microscopy of GFP-Atg8-expressing cells. The arrowhead points to the accumulation of Atg8 at the site of the MR during abscission. The enlarged clipping on the right shows the midbody area at time-point 68 min. Scale bar is 10 μm.


( b ) Cytosolic MRs are found inside Atg8 - positive vesicles . U2OS cells stably expressing GFP - Atg8 were fixed and stained for MKLP1 ( red ) and α - tubulin ( blue ) . Scale bars are 10 μm and 1 μm for the enlarged boxed areas . Putative different stages of autophagosomes ( left ) and lysosomes ( right ) are shown .

O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O

(b) Cytosolic MRs are found inside Atg8-positive vesicles. U2OS cells stably expressing GFP-Atg8 were fixed and stained for MKLP1 (red) and α-tubulin (blue). Scale bars are 10 μm and 1 μm for the enlarged boxed areas. Putative different stages of autophagosomes (left) and lysosomes (right) are shown.


( c ) Juxtanuclear MRs are found in Rab7 - and LAMP2 - positive lysosomes . U2OS cells stably expressing DsRed - Rab7 were fixed and stained for MKLP1 α - tubulin and DAPI ( left panels ) ; wild - type U2OS were stained for MKLP1 and LAMP2 ( right panel ) . Scale bars are 10 μm . The insets are enlargements of the engulfed MRs . Scale bars are 1 μm .

O O O O B-SUBCELLULAR O O O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O

(c) Juxtanuclear MRs are found in Rab7- and LAMP2-positive lysosomes. U2OS cells stably expressing DsRed-Rab7 were fixed and stained for MKLP1 α-tubulin and DAPI (left panels); wild-type U2OS were stained for MKLP1 and LAMP2 (right panel). Scale bars are 10 μm. The insets are enlargements of the engulfed MRs. Scale bars are 1 μm.