( d ) qRT - PCR assays of Miz1 target genes in control Miz1ΔPOZ MEFs after incubation in DMEM or in EBSS . Error bars depict s . d . of technical triplicates from one experiment .

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(d) qRT-PCR assays of Miz1 target genes in control and Miz1ΔPOZ MEFs after incubation in DMEM or in EBSS. Error bars depict s.d. of technical triplicates from one experiment.


( e ) qRT - PCR assays documenting expression of indicated Miz1 target genes in control Miz1ΔPOZ neurospheres ; assays were performed at passage three . s . e . m . values are derived from six biological replicates , each with technical triplicates .

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(e) qRT-PCR assays documenting expression of the indicated Miz1 target genes in control and Miz1ΔPOZ neurospheres; assays were performed at passage three. s.e.m. values are derived from six biological replicates, each with technical triplicates.


( f ) Recombination of Miz1flox / floxCre - ER + neurospheres was tested by genomic PCR . 4 - OHT was added to media for 3 days .

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(f) Recombination of Miz1flox/floxCre-ER+ neurospheres was tested by genomic PCR. 4-OHT was added to the media for 3 days.


( g ) Immunoblots using an α - Lc3 antibody detecting Lc3 - I Lc3 - II . 4 - OHT - treated non - treated Miz1flox / floxCre - ER + neurospheres were incubated with leupeptin ammonium chloride ( lys PI ) for 30 min . expression of α - tubulin was used as loading control .

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(g) Immunoblots using an α-Lc3 antibody detecting Lc3-I and Lc3-II. 4-OHT-treated and non-treated Miz1flox/floxCre-ER+ neurospheres were incubated with leupeptin and ammonium chloride (lys PI) for 30 min. The expression of α-tubulin was used as loading control.


( h ) microscopy of SHEP stably infected with lentiviruses expressing Lc3 - GFP Lamp1 - RFP ( to mark lysosomes ) . Vps28 was depleted with siRNAs a non - targeting pool ( siNTP ) was used as a control . Note accumulation of Lc3 in vesicles that do not overlap with lysosomes upon depletion of Vps28 . Scale bar , 25 μm .

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(h) Fluorescent microscopy of SHEP cells stably infected with lentiviruses expressing Lc3-GFP and Lamp1-RFP (to mark lysosomes). Vps28 was depleted with siRNAs and a non-targeting pool (siNTP) was used as a control. Note the accumulation of Lc3 in vesicles that do not overlap with lysosomes upon depletion of Vps28. Scale bar, 25 μm.


A : Brightfield of a NTH / GFP zygote at 4h post - fertilization , showing dispersed extranuclear . Hoechst ( blue ) marks nucleus . Scale bar = 5μm . B : Image of an NTH / GFP ookinete , showing spots co - localizing with pigment clusters ( arrows ) . Hoechst ( blue ) marks nucleus . Scale bar = 5μm .

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A: Brightfield and fluorescence images of a NTH/GFP zygote at 4h post-fertilization, showing dispersed extranuclear fluorescence. Hoechst DNA staining (blue) marks nucleus. Scale bar = 5μm. B: Image of an NTH/GFP ookinete, showing fluorescent spots co-localizing with pigment clusters (arrows). Hoechst DNA staining (blue) marks nucleus. Scale bar = 5μm.


D : Brightfield of a zygote at 4h post - fertilization simultaneously expressing NTH : : GFP ( green ) LAP3 : : mCherry ( red ) . Scale bar = 5μm . E : An ookinete simultaneously expressing NTH : : GFP ( green ) LAP3 : : mCherry ( red ) , showing co - localization in crystalloid . Scale bar = 5μm .

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D: Brightfield and fluorescence images of a zygote at 4h post-fertilization simultaneously expressing NTH::GFP (green) and LAP3::mCherry (red). Scale bar = 5μm. E: An ookinete simultaneously expressing NTH::GFP (green) and LAP3::mCherry (red), showing co-localization in the crystalloid. Scale bar = 5μm.


F : Western blot of purified NTH / GFP ookinete samples with ( + ) without ( - ) prior in vivo crosslinking . Mr markers ( kDa ) are indicated on right - side .

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F: Western blot of purified NTH/GFP ookinete samples with (+) and without (-) prior in vivo crosslinking. Mr markers (kDa) are indicated on the right-hand side.


A : Brightfield of a NTH - KO NTH / GFP ookinete , showing normal ookinete , but lack of pigment clusters associated with crystalloids ( arrows ) in NTH null mutant . Scale bar = 5μm .

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A: Brightfield images of a NTH-KO and NTH/GFP ookinete, showing normal ookinete morphology, but lack of pigment clusters associated with crystalloids (arrows) in the NTH null mutant. Scale bar = 5μm.


B : Aligned dot plot of oocyst numbers in Anopheles stephensi infected with lines NTH / GFP , NTH - KO NTH ∆ PP . Horizontal lines mark mean values ( n = 20 ) ; ns , not significantly different ( Mann - Whitney ) .

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B: Aligned dot plot of oocyst numbers in Anopheles stephensi infected with parasite lines NTH/GFP, NTH-KO and NTH∆PP. Horizontal lines mark mean values (n=20); ns, not significantly different (Mann-Whitney).


C : Brightfield of oocysts of lines NTH / GFP NTH - KO at 15 days post - infection , showing lack of sporulation in NTH null mutant . Hoechst ( artificially depicted red ) labels nuclei . large nuclei of epithelial midgut also show . Scale bar = 20μm .

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C: Brightfield and fluorescence images of oocysts of parasite lines NTH/GFP and NTH-KO at 15 days post-infection, showing lack of sporulation in the NTH null mutant. Hoechst DNA staining (artificially depicted red) labels parasite nuclei. The large nuclei of the epithelial midgut cells also show. Scale bar = 20μm.


D : Aligned dot plot of oocyst diameters in Anopheles stephensi at 15 days post - infection with lines NTH / GFP , NTH - KO NTH ∆ PP . Horizontal lines mark mean values ( n = 20 ) . * * mark statistically significant differences ( P < 0 . 0001 , Mann - Whitney ) ; ns , not significant .

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D: Aligned dot plot of oocyst diameters in Anopheles stephensi at 15 days post-infection with parasite lines NTH/GFP, NTH-KO and NTH∆PP. Horizontal lines mark mean values (n=20). ** mark statistically significant differences (P<0.0001, Mann-Whitney); ns, not significant.


E : of ookinetes of lines NTH ∆ PP showing dispersed NTH ∆ PP : : GFP ; LAP3 / mCherry showing focal LAP3 : : mCherry in crystalloids ; Cross ( carrying modified alleles encoding NTH ∆ PP : : GFP LAP3 : : mCherry ) , showing dispersed LAP3 : : mCherry . Arrow marks pigment cluster associated with crystalloids . Scale bar = 5μm .

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E: Images of ookinetes of parasite lines NTH∆PP showing dispersed NTH∆PP::GFP fluorescence; LAP3/mCherry showing focal LAP3::mCherry fluorescence in crystalloids; Cross (carrying modified alleles encoding NTH∆PP::GFP and LAP3::mCherry), showing dispersed LAP3::mCherry fluorescence. Arrow marks pigment cluster associated with crystalloids. Scale bar = 5μm.


G : PCR diagnostic for presence of modified nth alleles in blood stage infections obtained after sporozoite transmission of a NTH ∆ PP x LAP3 / mCherry genetic cross ( lane NTH ∆ PP ) , amplifying an approximately 4 . 4kb fragment . NTH / GFP are used as a PCR control , amplifying approximately 4 . 6kb . Parental provide a negative control .

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G: PCR diagnostic for the presence of modified nth alleles in blood stage infections obtained after sporozoite transmission of a NTH∆PP x LAP3/mCherry genetic cross (lane NTH∆PP), amplifying an approximately 4.4kb fragment. NTH/GFP parasites are used as a PCR control, amplifying approximately 4.6kb. Parental parasites provide a negative control.


H : Image of a NTH / ND500LK ookinete , showing spots co - localizing with pigment clusters ( arrows ) indicative of normal crystalloid formation . Scale bar = 5μm .

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H: Image of a NTH/ND500LK ookinete, showing fluorescent spots co-localizing with pigment clusters (arrows) indicative of normal crystalloid formation. Scale bar = 5μm.


A : Brightfield image of an unsporulated NTH / GFP oocyst at 15 days post - infection . Scale bar = 20μm . B : Image of a sporulated NTH / GFP oocyst at 15 days post - infection . Scale bar = 20μm .

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A: Brightfield and fluorescence image of an unsporulated NTH/GFP oocyst at 15 days post-infection. Scale bar = 20μm. B: Image of a sporulated NTH/GFP oocyst at 15 days post-infection. Scale bar = 20μm.


C : Image of a NTH / GFP sporozoite , showing NTH : : GFP localisation in a tubular structure . Scale bar = 10μm .

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C: Image of a NTH/GFP sporozoite, showing NTH::GFP localisation in a tubular structure. Scale bar = 10μm.


D : Image of a sporulated oocyst simultaneously expressing NTH : : GFP ( green ) ACP : : mCherry ( red ) , showing co - localization in apicoplast . Scale bar = 20μm 10μm ( inset ) .

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D: Image of a sporulated oocyst simultaneously expressing NTH::GFP (green) and ACP::mCherry (red), showing co-localization in the apicoplast. Scale bar = 20μm and 10μm (inset).


E : Image of sporozoites expressing NTH : : GFP ( green ) ACP : : mCherry ( red ) , showing co - localization in apicoplast ( arrows ) . Sporozoites expressing only ACP : : mCherry or NTH : : GFP are also present . Scale bar = 10μm .

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E: Image of sporozoites expressing NTH::GFP (green) and ACP::mCherry (red), showing co-localization in the apicoplast (arrows). Sporozoites expressing only ACP::mCherry or NTH::GFP are also present. Scale bar = 10μm.


F : Image of group of NTH / GFP sporozoites stained with MitoTracker Red CMXRos , showing distinct subcellular positions of apicoplasts ( green ) mitochondria ( red ) . Scale bar = 10μm .

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F: Image of group of NTH/GFP sporozoites stained with MitoTracker Red CMXRos, showing distinct subcellular positions of apicoplasts (green) and mitochondria (red). Scale bar = 10μm.


( A ) Immunostaining of ES ( Oct4 ) , NPCs ( day4 , Nestin ) , neurons ( day 8 , Tuj1 ) . Scale bar : 200µm .

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(A) Immunostaining of ES cells (Oct4), NPCs (day4, Nestin), and neurons (day 8, Tuj1). Scale bar: 200µm.


( B ) Expression of candidate lncRNAs during three stages of differentiation , measured by - seq . Gene models of lncRNAs appear in green , adjacent - coding genes appear in grey . Blue depicts transcripts from ' + ' strand , orange represents transcripts from ' - ' strand . three - seq tracks were scaled separately to maximum coverage in each locus .

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(B) Expression levels of candidate lncRNAs during three stages of differentiation, measured by RNA-seq. Gene models of lncRNAs appear in green, adjacent protein-coding genes appear in grey. Blue depicts transcripts from the '+' strand, orange represents transcripts from the '-' strand. All three RNA-seq tracks were scaled separately to the maximum coverage in each locus.


( C ) qRT - PCR of Reno1 , Cox10as1 , lnc - Nr2f1 in ES ( top ) following eight days of neuronal differentiation ( bottom ) , following knockdown using two different shRNAs , normalized to sh - NT . Mean ± is shown for 3 - 6 independent experiments , * P < 0 . 05 , * * P < 0 . 01 ( unpaired two sample t - ) .

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(C) qRT-PCR of Reno1, Cox10as1, and lnc-Nr2f1 in ES cells (top) and following eight days of neuronal differentiation (bottom), following knockdown using two different shRNAs, normalized to sh-NT. Mean ± SEM is shown for 3-6 independent experiments, * P<0.05, ** P<0.01 (unpaired two sample t-test).


( A ) Immunostaining using anti - TUJ1 antibody of ES - - derived neurons following infection with either non - targeting shRNA ( sh - NT ) , or two different shRNAs targeting Reno1 . Scale bar : 200µm . ( B ) of numbers neurite lengths of ten of non - overlapping fields for each shRNA . Mean ± is shown , * * P < 0 . 01 ( unpaired two sample t - ) .

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(A) Immunostaining using anti-TUJ1 antibody of ES-cell-derived neurons following infection with either non-targeting shRNA (sh-NT), or two different shRNAs targeting Reno1. Scale bar: 200µm. (B) Quantification of cell numbers and neurite lengths of ten images of non-overlapping fields for each shRNA. Mean ± SEM is shown, ** P<0.01 (unpaired two sample t-test).


( C ) Mean expression of genes which were down - or upregulated following knockdown ( KD ) of Reno1 or lnc - Nr2f1 during an eight time point neuronal differentiation maturation of mouse ES : Neurons1 corresponds to day in vitro ( DIV ) 1 ; Neurons2 - DIV7 ; Neurons3 - DIV16 ; Neurons4 - DIV21 ; Neurons5 - DIV28 ( Hubbard et al . , 2013 ) .

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(C) Mean expression levels of genes which were down- or upregulated following knockdown (KD) of Reno1 or lnc-Nr2f1 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 ) Transcriptional response of cycle neuron - related genes . Heatmaps show log2FC of infected with shRNAs targeting Reno1 or lnc - Nr2f1 compared to infected with sh - NT , following eight days of neuronal differentiation , of day eight of differentiation compared to ES during differentiation of WT .

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(D) Transcriptional response of cell cycle and neuron-related genes. Heatmaps show log2FC of cells infected with shRNAs targeting Reno1 or lnc-Nr2f1 compared to cells infected with sh-NT, following eight days of neuronal differentiation, and of day eight of differentiation compared to ES cells during differentiation of WT cells.


( A ) Reno1 locus in six vertebrate species . Shaded region indicates Reno1 locus . Blue gene models are from RefSeq or Ensembl , red gene models are on PLAR transcript reconstructions ( Hezroni et al . , 2015 ) . - seq datasets from indicated tissues were taken from publicly - available datasets : BodyMap ( Li et al , 2017a ) ( mouse ) , Reference Epigenome ( human ) , FAANG ( sheep ) , SRP011985 ( opossum ) , SRP016501 ( chicken ) , DRP000627 ( coelacanth ) . - seq data was scaled separately in each species to highest coverage in window , except for opossum chicken , where a custom upper threshold was set as indicated .

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(A) Reno1 locus in six vertebrate species. Shaded region indicates the Reno1 locus. Blue gene models are from RefSeq or Ensembl, red gene models are based on PLAR transcript reconstructions (Hezroni et al., 2015). RNA-seq datasets from indicated tissues were taken from publicly-available datasets: BodyMap (Li et al, 2017a) (mouse), Reference Epigenome (human), FAANG (sheep), SRP011985 (opossum), SRP016501 (chicken), DRP000627 (coelacanth). RNA-seq data was scaled separately in each species to the highest coverage in the window, except for opossum and chicken, where a custom upper threshold was set as indicated.


( B ) mouse Reno1 locus . Expression during neuronal differentiation of ES , in different fractions of ES ( data from ( Engreitz et al , 2016 ) ) motor neurons ( GSE90913 ) , in forebrain during different stages of embryonic development ( data from 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 in dorsolateral prefrontal cortex across six groups , in different fractions in adult . 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 ) DAPI ( blue ) in WT Reno1m / m 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 of Reno1 Bahcc1 in 30 adult embryonic tissues . Data from ENCODE project , 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 Reno1 promoter as bait . Top , smoothed trend lines raw of contact profile in ES ( 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 of Reno1 ( both spliced unspliced isoforms ) Bahcc1 following four days of differentiation of indicated . Mean ± is shown for 3 - 4 independent experiments , * P < 0 . 05 , * * P < 0 . 01 ( unpaired two sample t - ) .

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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 of indicated . Scale bar : 200 µm . Right : of indicated ES grown in ES medium . were harvested from one well of a 6 - well plate , counted using Orflo MOXI Z Mini Automated Counter . Mean ± 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 of indicated following four days of differentiation . Scale bar : 200 µm . Right : of indicated following four days of differentiation . were harvested from one well of a 6 - well plate , counted using Orflo MOXI Z Mini Automated Counter . Mean ± is shown for three independent experiments . * * P < 0 . 01 ( unpaired two sample t - ) .

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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 live in indicated ES following four days of differentiation . Y axis : forward scatter ; X axis : PI .

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

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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 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 94 up - regulated ) , during an eight time point neuronal differentiation maturation of mouse ES : Neurons1 corresponds to day in vitro ( DIV ) 1 ; Neurons2 - DIV7 ; Neurons3 - DIV16 ; Neurons4 - DIV21 ; 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 of Reno1 following Dox addition in WT Reno1m / m . Mean ± is shown for three independent experiments , * P < 0 . 05 ( unpaired two sample t - ) .

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

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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 median , quartiles , 5th 95th percentiles of changes in expression of WT ( F ) or Reno1m / m ( G ) treated with Dox for first two days of differentiation compared to untreated , 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 genes ( n = 16 , 511 ) . * * P < 0 . 01 ( two - sided Wilcoxon rank sum ) .

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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 median , quartiles 1 . 5 time interquartile range of ratios of ATAC - seq signal for indicated group of peaks , comparing indicated genotypes or treatments . promoter peaks are < 300 nt from a TSS ; Extended promoter are more than 300 nt but < 2 Kb for a TSS ; Gene peaks overlap transcription units , Intergenic do not . Each group of peaks was compared to intergenic peaks . In addition , for each group of peaks , peaks within 1 Mb of Reno1 where compared to peaks further away . P - values computed using two - sided Wilcoxon rank sum , 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 or extended promoters , of genes in indicated group ( up / down - regulated by at least 30 % with adjusted P < 0 . 05 ) in with Reno1 ( left ) or Bahcc1 ( right ) KD . P - values computed using two sided Wilcoxon rank sum .

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(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 median , quartiles 1 . 5 time interquartile range of changes in ATAC - seq signal on day 2 of neuronal differentiation of peaks from indicated Reno1 perturbation , classified according to underlying state in mouse ES ; Bottom : Boxplots indicating median , quartiles 1 . 5 time interquartile range of ChIP - seq coverage of indicated marks in ENCODE mouse ES data in peak regions . Number of peaks in each group is indicated at 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 accessibility on day 2 of neuronal differentiation in indicated perturbations H3K4me3 or Fam60a ChIP coverage from mouse ES ( H3K4me3 is from ENCODE project Fam60a from ( Streubel et al . , 2017 ) ) . Coefficient 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 skeletal Gastrocnemius ( GC ) Tibialis Anterior ( TA ) , but not in Adipose Tissue ( WAT ) from 3 - month - old HSACre / + / Lpin1fEx2 - 3 / fEx2 - 3 mice ( HSA - Cre ) compared to control mice . Expression are corrected for expression of control gene Pinin presented in 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 PAP2 activities were respectively substantially decreased 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 / of GC TA of 3 - month - old HSA - Cre control mice .

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


Hematoxylin Eosin ( HE ) of representative transverse sections of GC from 3 - month - old HSA - Cre control mice . black arrows indicate with centrally located nuclei yellow arrows indicate mononuclear 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 from 3 - month - old HSA - Cre control 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 ) in HSA - Cre control GC .

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


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

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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 content in HSA - Cre control GC . ( D ) Total phospholipid ( PL ) amount normalized by content in HSA - Cre control GC .

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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 total carbon number in acyl chains of PC in HSA - Cre control GC .

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


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

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


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

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


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

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 ) Succinate Deshydrogenase ( SDH ) of representative frozen sections of TA muscle from 3 - month - old HSA - Cre 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.


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

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 control GC .

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 control GC .

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

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 of Fasn in TA muscle of 3 - month - old HSA - Cre 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 ) Medium - chain acyl - CoA dehydrogenase ( Mcad ) in TA from 3 - month - old HSA - Cre 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 of full N - terminal SREBP1c SREBP2 in GC muscle of 4 - month - old HSA - Cre control mice ( n = 7 - 4 ) . of densitometric of N - term Full 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 control mice presented as visualization hierarchical clustering 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 of 4 - month - old HSA - Cre 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 , ATP / ADP ratios in GC of 4 month - old HSA - Cre control mice ( n = 7 - 4 , * P < 0 . 05 ) .

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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 of 4 - month - old HSA - Cre control mice ( n = 7 - 4 ) . by densitometric analyses of Fgf21 , Bip , sXbp1 Atf6 form , p - Eif2a Chop is presented as a graph . Data are normalized to Gapdh expressed as a fold change relative to control mice .

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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 , Edem1 in GC from 4 - month - old HSA - Cre 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 of EDL of HSA - Cre mice revealed presence of areas in which non - junctional SR is more vesiculated fragmented compared to control ( black arrows ) . Remodeling of SR is also accompanied by modification in positioning of mitochondria ( 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.


relative mtDNA copy number was determined by of mitochondrial - encoded Cytochrome B ( CytB ) - encoded NADH dehydrogenase ( ubiquinone ) flavoprotein 1 ( Ndufv1 ) genes in GC TA from HSA - Cre 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 - encoded gene expression by quantitative RT - PCR in GC from 4 - month - old HSA - Cre 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 of HSA - Cre EDL of 3 - month - old mice showed that mitochondria are almost exclusively positioned at two sides of Z lines in EDL from control mice ( black arrows ) . On other , in Lipin1 deficient EDL mitochondria are more often clustered display electron - dense contact sites ( arrows ) . Scale bars : 0 . 20 μm .

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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 of HSA - Cre EDL revealed presence of numerous autophagic vacuoles of different morphologies ( autophagosomes lysosomes , black arrows ; multilamellar bodies , arrow ) containing amorphous material 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 extract of GC of 6 - month - old control , HSA - Cre untreated HSA - Cre TUDCA - treated mice , with 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 GDF15 expression in GC muscle of 6 - month - old control , HSA - Cre untreated 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 of TA of 6 - month - old control , HSA - Cre untreated HSA - Cre TUDCA - treated mice by Oil Red O to detect 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 of 6 - month - old control , HSA - Cre untreated 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 of 6 - month - old control , HSA - Cre untreated 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.


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

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 extract of GC of 4 - month - old control , HSA - Cre untreated HSA - Cre Bezafibrate - treated mice , with 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 GDF15 expression in GC muscle of 4 - month - old control , HSA - Cre untreated 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 of TA of 4 - month - old control , HSA - Cre untreated HSA - Cre Bezafibrate - treated mice by Oil Red O to detect 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 of 4 - month - old control , HSA - Cre untreated 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 of 4 - month - old control , HSA - Cre untreated 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 of IgG positive on frozen sections of TA of 4 - month - old control , HSA - Cre untreated HSA - Cre Bezafibrate - treated mice . sections were stained with a secondary anti - mouse IgG antibody . of number of necrotic over total number of .

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 generated from bone marrow of mice of indicated genotypes were treated for 24h with graded doses of CHK1 - inhibitors PF - 477736 ( PF ) or CHIR - 124 ( CHIR ) . Representative histograms of CHK1i - treated ( grey ) or DMSO - treated ( black ) , analyzed for cycle distribution using Nicoletti 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 were treated with CHIR - 124 in presence or absence of pan - caspase inhibitor QVD . 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 Bax - / - Bak - / - using indicated antibodies .

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


WT Bax - / - Bak - / - Hoxb8 - FL were treated for up to 8h with 500 nM CHIR p21 mRNA 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 .

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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 of indicated genotypes were seeded to assess growth in response to CHK1 - inhibition . were counted 48h post seeding .

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

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

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

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

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

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 Vav - BCL2 derived E14 . 5 total bone marrow ( C ) were treated for up to 48h with different doses of CHK1i . Survival was assessed using Nicoletti 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).


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

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

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 + were transduced with a BCL2 - encoding lentivirus or a control , 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 flow cytometry .

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


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

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

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 . Colonies were counted 10 days post seeding of 1000 CD34 + per plate / sample . " Myeloid colonies " include granulocyte / monocyte , monocyte , granulocyte GEMM ( granulocyte , erythroid , monocyte / macrophage megakaryocyte ) colonies .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O 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 livers at E13 . 5 . Scale bar = 1 mm .

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


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

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

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 suspensions of livers from Chk1fl / + , Chk1fl / + Vav - Cre Chk1fl / - Vav - Cre were stained with antibodies recognizing ckit , CD71 , Ter119 or CD45 to assess primitive 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 / + N = 1 Chk1fl / + Vav - Cre ) , N = 3 for Chk1fl / - Vav - Cre .

O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O 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 LSK phenotype of Chk1fl / + Chk1fl / - Vav - Cre observed in E13 . 5 livers relative distribution / absolute numbers of LKhi LSKhi , as well as LKlow LSKlow , found in liver on E13 . 5 . Bars represent means ± S . E . M . N = 4 for Chk1fl / + N = 4 for Chk1fl / - Vav - Cre .

O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O 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 ( left panel , N = 4 for Chk1fl / + N = 5 for Chk1fl / - Vav - Cre ) total liver ( right panel , N = 3 for Chk1fl / + N = 6 for Chk1fl / - Vav - Cre ) in methyl - cellulose assays . Bars represent means ± S . E . M .

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

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

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

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 ) of liver cryosections of indicated genotypes at E13 . 5 . Scale bar 100x = 200 µM , 400x = 50 µM . of TUNEL + / field was assessed across three random fields from two control ( Chk1fl / + ) two knock - out ( Chk1fl / - Vav - Cre ) using ImageJ , as reported by us before ( 60 ) . Bars represent means ± S . D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O 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 liver suspensions of Chk1fl / + ( + / + ) , Chk1fl / + Vav - Cre ( + / - ) Chk1fl / - Vav - Cre ( - / - ) E13 . 5 were processed for Western blot using indicated antibodies . Each lane represents samples isolated from an littermate .

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

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

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

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O 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 of E13 . 5 liver LSKhi / LSKlow observed in of 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 / + N = 4 for p53 - / - Chk1fl / - Vav - Cre .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O 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 - of LSK stained with antibodies specific for CD34 Flt3 , to discriminate LT - ST - HSC from MPP within LSK subset in livers of of indicated genotypes . Left : N = 25 for Chk1fl / + , N = 7 for Chk1fl / + Vav - Cre N = 7 for Chk1fl / - Vav - Cre . Right : N = 16 for Chk1fl / + Vav - BCL2 , N = 6 for Chk1fl / + Vav - Cre Vav - BCL2 N = 5 for Chk1fl / - Vav - Cre Vav - BCL2 .

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR 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 of liver of indicated genotypes ( each lane represents an ) at E14 . 5 using indicated antibodies .

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

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

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 were transduced with a retroviral construct carrying Cre GFP ( pMIG - CRE - IRES - GFP ) . were FACS - sorted 72h after transduction with retroviral construct processed for western blotting using indicated antibodies .

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

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.


of GFP + indicative for CRE expression recombination in 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

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).


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

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


of living LK or LSK - ( F ) of GFP + LK or LSK - found in bone marrow of mT / mG Vav - CreERT2 ( WT ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 ) Tomato + ( CRE - ) total bone marrow were sorted on day 8 after first TAM - administration processed for Western Blot using indicated antibodies . Sorted from three - mT / mG Vav - CreERT2 ( WT ) mT / mG Vav - CreERT2 Chk1fl / fl ( Chk1fl / fl ) - were pooled per lane .

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

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 indicated genotypes were sorted on GFP + Tomato + on day 15 after first TAM administration for western using indicated antibodies .

O O O O O O O O O O O O O O O O O O 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.


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

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

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 , PTPRC ) or markers ( EGFR , PDGFRA , VEGFA , VIM , NES ) in GBM biopsies or GBM - derived organotypic spheroids ( PDX xenografts ) .

O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O 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 ( ) analyses revealed EGFL7 expression in blood vessels ( arrow ) neurons ( arrowheads ) of healthy gray matter . Expression in malignant brain specimens was restricted to blood vessels , in particular with a large diameter ( arrows ) . Scale bar represents 50 µm . Quantitative scoring of EGFL7 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 ) .

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

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 growth ( n = 9 ; one - way ANOVA ) .

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

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


Immunocompetent mice bearing GL261 tumors . Representative MRI confirmed implantation in 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 ) . Immunodeficient mice bearing U87 tumors . Representative magnetic resonance confirmed implantation in 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 ) .

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

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 - bearing mice ( n = 8 ) .

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

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


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

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

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

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

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).


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

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

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

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

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 ) of these brains confirmed that mice developed tumors of similar . T1 - weighted MRI 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 .

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

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 maturation state of glioma vessels Enhanced maturation of glioma vessels in 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 ± , AU - arbitrary units . Scale bars represent 60 µm .

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

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

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR 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 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 presence of hE7 or mE7 was verified by increased co - localization of ( I + J ) Col IV ( basement ) with CD31 ( n = 3 ; one - way ANOVA ; quantifications normalized to CD31 ) . Data presented as mean ± , AU - arbitrary units . Scale bars represent 60 µm .

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O 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 of recombinant EGFL7 by transmission electron microscopy ( ) showed 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 . scale bar represents 5 nm .

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

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 human umbilical vein endothelial ( HUVEC ) spheroids embedded in a collagen - matrix was induced by 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 cases but control ( lower row ) . Scale bar represents 100 µm .

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

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

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

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 . of ( G ) Cdc42 , ( H ) Rac1 ( I ) RhoA activation by immunoblotting subsequent to seeding HUVECs on E7 , Fn or a combination of both . Preferentially , E7 activated Cdc42 Fn activated Rac1 , but both antagonized each other . None of 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 B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

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 β3 upon appli cation of E7 but not Fn ( n = 3 ; one - way ANOVA ) .

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

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

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

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 followed by intracranial implantation into striatum of immune - competent mice reduced overall survival but was rescued upon treatment with an integrin α5β1 - inhibiting antibody ( n = 6 ; log - rank ) . Data presented as mean ± . TCL - total lysate , AU - arbitrary units .

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

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 brains were analyzed by magnetic resonance imaging ( MRI ) . Resulting brain sections were analyzed for blood vessel density maturation state by immunohistochemistry . ( A + B ) CD31 revealed that blocking of EGFL7 , VEGF , or a combination of both lead to decreased vessel density ( n = 3 ; one - way ANOVA ) .

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O 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 brains were analyzed by magnetic resonance imaging ( MRI ) . Resulting brain sections were analyzed for blood vessel density maturation state by immunohistochemistry . for ( C + D ) PDGFRβ ( n = 3 ; one - way ANOVA ) ,

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 brains were analyzed by magnetic resonance imaging ( MRI ) . Resulting brain sections were analyzed for blood vessel density maturation state by immunohistochemistry . for SMA ( n = 3 ; one - way ANOVA )

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 brains were analyzed by magnetic resonance imaging ( MRI ) . Resulting brain sections were analyzed for blood vessel density maturation state by immunohistochemistry . for Col IV ( n = 3 ; one - way ANOVA ) revealed decreased amounts of blood vessel - associated pericytes , smooth muscle Col IV in basement of blood vessels upon treatment with anti - EGFL7 , anti - VEGF most significantly , a combination of both antibodies .

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

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 brains were analyzed by magnetic resonance imaging ( MRI ) . Resulting brain sections were analyzed for blood vessel density 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 ) .

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 median survival time of glioma - bearing Rag1 - / - mice ( 58 d ) as compared to isotype - treated controls ( 37 d ; n = 6 ; log - rank ) .

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

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 a combination of anti - EGFL7 anti - VEGF antibody increased median survival time ( Combo ; 87 d ; n = 5 ; log - rank ) as compared to anti - VEGF alone ( 80 d ; n = 5 ; log - rank ) or isotype - treated controls ( 69 d ; n = 5 ; log - rank ) . Data presented as mean ± , AU - arbitrary units . Scale bars represent 60 µm .

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

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

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

(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.