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 .

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

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 .

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

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

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 .

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

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


Ultrastructural 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

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

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 .

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

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 .

O O O 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

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 .

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

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 .

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

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 .

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

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 .

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 B-CONTROLLED_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

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 .

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 B-MEASURED_VAR I-MEASURED_VAR O

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 .

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

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 .

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

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 .

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

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 .

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

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

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


Colony formation potential of human CD34 + HSPCs transduced with empty control or BCL2 encoding . 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 .

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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 ) .

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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 .

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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 .

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

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

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

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 .

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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 .

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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 ) .

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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 .

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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 ) .

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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 .

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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 .

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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 .

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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 ) .

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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 ) .

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


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

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


Immunocompetent mice bearing GL261 tumors . Representative MRI 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 ) .

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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 ) .

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 ) .

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


Residual EGFL7 expression in BTPC11 glioma 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 ) .

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


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 .

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


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

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


T2 - weighted magnetic resonance imaging ( MRI ) 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 .

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 .

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


Enhanced maturation of glioma vessels in 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 .

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


Enhanced maturation of glioma vessels in 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 .

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 .

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


Angiogenic sprouting of 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 .

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 ± ) .

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


Measurement of Rho GTPase signaling downstream of integrins . 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 .

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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 ) .

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


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

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


Ectopic expression of murine EGFL7 ( mE7 ) in GL261 mouse glioma 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 .

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 ) .

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 ) ,

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 )

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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 .

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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 ) .

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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 ) .

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


Treatment of glioma - bearing NOD SCID mice with temozolomide ( TMD ) as a chemotherapeutic agent 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 .

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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 .

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


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

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

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


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

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


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

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


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

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

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


( a ) Accumulation of MRs in treated with PI ( 3 ) K inhibitor 3 - MA . Left panels show HeLa treated with 3 - MA ( 4 mM ) for 3 days . were fixed stained . are colour - inverted for better visualization . of this experiment is shown on upper right ( mean ± s . d . of three independent experiments ; n = total number of counted ) . lower right panel shows an enlarged of 3 - MA - treated in true colour . Scale bars are 20 μm .

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(a) Accumulation of MRs in cells treated with the PI(3)K inhibitor 3-MA. Left panels show HeLa cells treated with 3-MA (4 mM) for 3 days. Cells were fixed and stained. Images are colour-inverted for better visualization. The quantification of this experiment is shown on the upper right (mean ± s.d. of three independent experiments; n = total number of cells counted). The lower right panel shows an enlarged area of 3-MA-treated cells in true colour. Scale bars are 20 μm.


( b ) siRNA - mediated depletion of Atg6 ( Beclin - 1 ) Atg7 from HeLa causes MR accumulations . right panel shows of MRs per ( mean ± s . d . of three independent experiments ) ; left panel efficiency of Atg6 Atg7 depletion .

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

(b) siRNA-mediated depletion of Atg6 (Beclin-1) and Atg7 from HeLa cells causes MR accumulations. The right panel shows the quantification of MRs per cell (mean ± s.d. of three independent experiments); the left panel the efficiency of Atg6 and Atg7 depletion.


( c ) Inhibition of lysosomal acidification leads to MR accumulation . Panels on left show HeLa left untreated or treated with chloroquine ( 20 μM ) or ammonium chloride ( 10 mM ) for 3 days . Scale bar are 20 μm . were fixed stained as indicated . A of this experiment is shown on left ( mean ± s . d . of three independent experiments ; n , total number of ; P , probability of a two - paired t - ) .

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

(c) Inhibition of lysosomal acidification leads to MR accumulation. Panels on the left show HeLa cells left untreated or treated with chloroquine (20 μM) or ammonium chloride (10 mM) for 3 days. Scale bar are 20 μm. Cells were fixed and stained as indicated. A quantification of this experiment is shown on the left (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).


( a ) - cycle lengths of different lines correlate well with average amount of MRs . Indicated lines were grown on coverslips , fixed MRs were counted after with anti - MKLP1 antibodies . - cycle lengths were assessed as described in Methods . Data are mean ± s . d . of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell-cycle lengths of different cell lines correlate well with the average amount of MRs. Indicated cell lines were grown on coverslips, fixed and MRs were counted after staining with anti-MKLP1 antibodies. Cell-cycle lengths were assessed as described in the Methods. Data are mean ± s.d. of three independent experiments.


( b ) degree of MR persistence depends on time not on - cycle duration . HeLa were either left untreated or were arrested in S - phase with hydroxyurea ( HU , 5 mM ) . were fixed stained as indicated . Scale bars are 20 μm . right panel shows of MR counting ( mean ± s . d . of three independent experiments ; n , total number of ; P , probability of a two - paired t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The degree of MR persistence depends on the time and not on cell-cycle duration. HeLa cells were either left untreated or were arrested in S-phase with hydroxyurea (HU, 5 mM). Cells were fixed and stained as indicated. Scale bars are 20 μm. The right panel shows quantification of MR counting (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).


( a ) Fibroblasts of individuals with mucolipidosis II ( I - disease ; upper right panel ) or Hurler syndrome ( lower right panel ) were compared with parental control ( left panels ) . alleles are indicated . were fixed stained with indicated antibodies DAPI to visualize . Arrowheads point to accumulated MRs . Scale bars are 20 μm . ( b ) of a ( mean ± s . d . of three independent experiments ; n , total number of ; P , probability of a two - paired t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fibroblasts of individuals with mucolipidosis II (I-cell disease; upper right panel) or Hurler syndrome (lower right panel) were compared with parental control cells (left panels). The alleles are indicated. Cells were fixed and stained with the indicated antibodies and DAPI to visualize DNA. Arrowheads point to the accumulated MRs. Scale bars are 20 μm. (b) Quantification of a (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).


( G ) Representative immunofluorescence of ∆ FosB ( green ) in cultured MSNs exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 DAPI is shown in lower row . Scale bar , 25μM . ( H ) of ∆ FosB + nuclei in cultured MSNs after indicated treatments ( same treatment in both treatment days ) . Results are expressed as average ± of % of MAP2 + displaying ∆ FosB in nucleus . N = 6 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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

(G) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM. (H) Quantification of ∆FosB+ nuclei in cultured MSNs after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( I ) Representative immunofluorescence of ∆ FosB ( green ) in cultured MSNs exposed to consecutive stimulation , with different 1st 2nd treatments with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 DAPI is shown in right column . Scale bar , 25μM . ( J ) of ∆ FosB + nuclei in cultured MSNs after different 1st 2nd treatments with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Results are expressed as average ± of % of MAP2 + displaying ∆ FosB in nucleus . N = 6 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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

(I) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs exposed to consecutive stimulation, with different 1st and 2nd treatments with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the right column. Scale bar, 25μM. (J) Quantification of ∆FosB+ nuclei in cultured MSNs after different 1st and 2nd treatments with activin A (Act), SKF81297 (SKF) or their combination as indicated. Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( K ) Representative immunofluorescence of ∆ FosB ( green ) in cultured MSNs extracted from mutant mice lacking ALK4 in GABAergic neurons ( Gad67Cre ; Alk4fl / fl ) control mice ( Alk4fl / fl ) after exposure to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 DAPI is shown in second fourth rows . Scale bar , 25μM . ( L ) of ∆ FosB + nuclei in cultured MSNs from mutant control mice after indicated treatments ( same treatment in both treatment days ) . Results are expressed as average ± of % of MAP2 + displaying ∆ FosB in nucleus . N = 6 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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

(K) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs extracted from mutant mice lacking ALK4 in GABAergic neurons (Gad67Cre;Alk4fl/fl) and control mice (Alk4fl/fl) after exposure to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the second and fourth rows. Scale bar, 25μM. (L) Quantification of ∆FosB+ nuclei in cultured MSNs from mutant and control mice after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( A ) Western blot of Kinase A phospho - substrates ( PKA Ⓟ - subs ) in PCBP1 immunoprecipitates from HeLa exposed to sequential stimulation ( same treatment in both treatment days ) with forskolin ( Fsk ) in absence or presence of PKA inhibitor PKI14 - 22 ( PKI ) . Reprobing with anti - PCBP1 antibodies is shown in middle blot . Lower blot shows total amount of PCBP1 in lysates . of three independent experiments is shown on right . Results are expressed as average ± of fold increase over vehicle normalized to PCBP1 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

O O O O O O O O O O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of Protein Kinase A phospho-substrates (PKA Ⓟ-subs) in PCBP1 immunoprecipitates from HeLa cells exposed to sequential stimulation (same treatment in both treatment days) with forskolin (Fsk) in the absence or presence of the PKA inhibitor PKI14-22 (PKI). Reprobing with anti-PCBP1 antibodies is shown in the middle blot. Lower blot shows the total amount of PCBP1 in the cell lysates. Quantification of three independent experiments is shown on the right. Results are expressed as average ± SEM of fold increase over vehicle normalized to PCBP1 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( B ) localization of PCBP1 analyzed by Western blotting of extracts from HeLa exposed to sequential stimulation ( same treatment in both treatment days ) with activin A ( Act ) , forskolin ( Fsk ) , PKA inhibitor ( PKI ) ALK4 inhibitor SB431542 ( SB ) as indicated . Blots of fractions were reprobed with anti - PCNA anti - αTubulin ( αTub ) antibodies , respectively . of three independent experiments is shown on right . Results are expressed as average ± of fold increase over vehicle ( first column ) normalized to PCBP1 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Nuclear localization of PCBP1 analyzed by Western blotting of extracts from HeLa cells exposed to sequential stimulation (same treatment in both treatment days) with activin A (Act), forskolin (Fsk), PKA inhibitor (PKI) and the ALK4 inhibitor SB431542 (SB) as indicated. Blots of nuclear and cytosolic fractions were reprobed with anti-PCNA and anti-αTubulin (αTub) antibodies, respectively. Quantification of three independent experiments is shown on the right. Results are expressed as average ± SEM of fold increase over vehicle (first column) normalized to PCBP1 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( C ) Western blot of Smad3 in PCBP1 immunoprecipitates from HeLa exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , forskolin ( Fsk ) or their combination as indicated . Act + Fsk extracts were also immuoprecipitated using control IgG antibodies as additional control . Reprobing with anti - PCBP1 antibodies is shown in middle blot . lower blot shows unaltered Smad3 across conditions . of four independent experiments ( biological replicates ) is shown on right . Results are expressed as average ± of fold increase of Smad3 / PCBP1 co - immunoprecipitation ( coIP ) over vehicle ( first column ) normalized to PCBP1 . ( D ) Western blot of PCBP1 in Smad3 immunoprecipitates from HeLa using conditions identical to panel ( C ) . lower blot shows unaltered PCBP1 across conditions . of four independent experiments ( biological replicates ) is shown on right . Results are expressed as average ± of fold increase of PCBP1 / Smad3 co - immunoprecipitation ( coIP ) over vehicle ( first column ) normalized to Smad3 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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

(C) Western blot analysis of Smad3 in PCBP1 immunoprecipitates from HeLa cells exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), forskolin (Fsk) or their combination as indicated. The Act+Fsk extracts were also immuoprecipitated using control IgG antibodies as additional control. Reprobing with anti-PCBP1 antibodies is shown in the middle blot. The lower blot shows unaltered Smad3 levels across all conditions. Quantification of four independent experiments (biological replicates) is shown on the right. Results are expressed as average ± SEM of fold increase of Smad3/PCBP1 co-immunoprecipitation (coIP) over vehicle (first column) normalized to PCBP1 levels. (D) Western blot analysis of PCBP1 in Smad3 immunoprecipitates from HeLa cells using conditions identical to panel (C). The lower blot shows unaltered PCBP1 levels across all conditions. Quantification of four independent experiments (biological replicates) is shown on the right. Results are expressed as average ± SEM of fold increase of PCBP1/Smad3 co-immunoprecipitation (coIP) over vehicle (first column) normalized to Smad3 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( E ) Representative immunofluorescence of Smad3 ( green ) PCBP1 ( red ) in cultured MSNs exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with MAP2 DAPI is shown in lower row . Scale bar , 25μM .

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(E) Representative immunofluorescence staining of Smad3 (green) and PCBP1 (red) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM.


( F , G ) of PCBP1 + ( F ) or Smad3 + ( G ) nuclei in cultured MSNs after indicated treatments ( same treatment in both treatment days ) . Results are expressed as average ± of % of MAP2 + displaying PCBP1 ( F ) or Smad3 ( G ) in nucleus . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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(F, G) Quantification of PCBP1+ (F) or Smad3+ (G) nuclei in cultured MSNs after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying PCBP1 (F) or Smad3 (G) in the nucleus. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( H ) of PCBP1 / Smad3 co - localization in nuclei of cultured MSNs using conditions identical to panel ( F ) . Results are expressed as average ± ( biological replicates ) of % of MAP2 + displaying co - localization of PCBP1 Smad3 in nucleus . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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(H) Quantification of PCBP1/Smad3 co-localization in nuclei of cultured MSNs using conditions identical to panel (F). Results are expressed as average ± SEM (biological replicates) of % of MAP2+ cells displaying co-localization of PCBP1 and Smad3 in the nucleus. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( I ) RT - qPCR of PCBP1 mRNA in cultured MSNs exposed to consecutive stimulation as indicated ( same treatment in both treatment days ) . Results are expressed as average ± of fold change over vehicle . No significant differences were found by one - way ANOVA followed with Tukey ' s multiple comparisons ( p ≥ 0 . 05 ; N = 4 ) . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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

(I) RT-qPCR analysis of PCBP1 mRNA in cultured MSNs exposed to consecutive stimulation as indicated (same treatment in both treatment days). Results are expressed as average ± SEM of fold change over vehicle. No significant differences were found by one-way ANOVA followed with Tukey's multiple comparisons test (p≥0.05; N=4). Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( J ) Representative of PLA ( red ) between Smad3 PCBP1 ( S3 / PCBP1 ) in cultured MSNs exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) , PKA inhibitor ( PKI ) or SB431542 ( SB ) as indicated . Counterstaining with MAP2 DAPI is shown in lower row . Scale bar , 25μM . ( K ) of PLA between Smad3 PCBP1 in cultured MSNs after indicated treatments . Results are expressed as average ± of number of PLA dots per . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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

(J) Representative images of PLA (red) between Smad3 and PCBP1 (S3/PCBP1) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF), PKA inhibitor (PKI) or SB431542 (SB) as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM. (K) Quantification of PLA between Smad3 and PCBP1 in cultured MSNs after the indicated treatments. Results are expressed as average ± SEM of number of PLA dots per cell. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( D ) Representative of - PLA ( green ) between PCBP1 ( left ) or Smad3 ( right ) sequences in exon 4 ( pE4 ) of FosB mRNA , respectively , in HeLa exposed to consecutive stimulation ( same treatment in both treatment days ) as indicated . Counterstaining with phalloidin DAPI is shown in second fourth columns . Scale bars , 20μM . ( E , F ) of - PLA between PCBP1 ( E ) or Smad3 ( F ) pE4 in HeLa after indicated treatments . Results are expressed as average ± of number of - PLA dots per . Only dots were quantified in this . N = 4 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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

(D) Representative images of RNA-PLA (green) between PCBP1 (left) or Smad3 (right) and sequences in exon 4 (pE4) of FosB mRNA, respectively, in HeLa cells exposed to consecutive stimulation (same treatment in both treatment days) as indicated. Counterstaining with phalloidin and DAPI is shown in the second and fourth columns. Scale bars, 20μM. (E, F) Quantification of RNA-PLA between PCBP1 (E) or Smad3 (F) and pE4 in HeLa cells after the indicated treatments. Results are expressed as average ± SEM of number of RNA-PLA dots per cell. Only nuclear dots were quantified in this analysis. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( G ) Representative of - PLA ( green ) between PCBP1 ( left ) or Smad3 ( right ) sequences in exon 4 ( pE4 ) of FosB mRNA , respectively , in cultured MSNs exposed to consecutive stimulation as indicated . Counterstaining with phalloidin DAPI is shown in second fourth columns . Scale bars , 20μM . ( H , I ) of - PLA between PCBP1 ( E ) or Smad3 ( F ) pE4 in cultured MSNs after indicated treatments . Results are expressed as average ± of number of - PLA dots per . Only dots were quantified in this . N = 4 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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

(G) Representative images of RNA-PLA (green) between PCBP1 (left) or Smad3 (right) and sequences in exon 4 (pE4) of FosB mRNA, respectively, in cultured MSNs exposed to consecutive stimulation as indicated. Counterstaining with phalloidin and DAPI is shown in the second and fourth columns. Scale bars, 20μM. (H, I) Quantification of RNA-PLA between PCBP1 (E) or Smad3 (F) and pE4 in cultured MSNs after the indicated treatments. Results are expressed as average ± SEM of number of RNA-PLA dots per cell. Only nuclear dots were quantified in this analysis. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( D ) Representative immunofluorescence of ∆ FosB ( red ) in cultured MSNs treated with scrambled shPCBP1 lentiviruses exposed to consecutive stimulation ( same treatment in both treatment days ) with activin A ( Act ) , SKF81297 ( SKF ) or their combination as indicated . Counterstaining with green ( GFP , expressed by lentiviruses ) DAPI is shown in second fourth rows . Scale bar , 25μM . ( E ) of ∆ FosB + nuclei in cultured MSNs treated with scrambled shPCBP1 lentiviruses followed by indicated treatments . Results are expressed as average ± of % of GFP + displaying ∆ FosB in nucleus . N = 4 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; two - way ANOVA followed with Tukey ' s multiple comparisons in E ) . N denotes biological replicates .

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

(D) Representative immunofluorescence staining of ∆FosB (red) in cultured MSNs treated with scrambled and shPCBP1 lentiviruses and exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with green fluorescent protein (GFP, expressed by the lentiviruses) and DAPI is shown in the second and fourth rows. Scale bar, 25μM. (E) Quantification of ∆FosB+ nuclei in cultured MSNs treated with scrambled and shPCBP1 lentiviruses followed by the indicated treatments. Results are expressed as average ± SEM of % of GFP+ cells displaying ∆FosB in the nucleus. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; two-way ANOVA followed with Tukey's multiple comparisons test in E). N denotes biological replicates.


( A - F ) RT - qPCR of FosB ( A , D ) , c - Fos ( B , E ) ∆ FosB ( C , F ) mRNAs in NAc of Gad67Cre ; Alk4fl / fl ( A - C ) mutant mice 1 hour ( A - C ) or 24 hour ( D - F ) after second saline ( Sal ) or cocaine ( Coc ) injection of TIPS . Results are expressed as average ± of fold increase over saline group ( Sal / Sal ) of Gad67Cre mice . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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

(A-F) RT-qPCR analysis of FosB (A, D), c-Fos (B, E) and ∆FosB (C, F) mRNAs in NAc of Gad67Cre;Alk4fl/fl (A-C) mutant mice 1 hour (A-C) or 24 hour (D-F) after the second saline (Sal) or cocaine (Coc) injection of TIPS. Results are expressed as average ± SEM of fold increase over the saline group (Sal/Sal) of Gad67Cre mice. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( G ) Representative immunofluorescence of ∆ FosB in Nac of control ( Gad67Cre ) mutant ( Gad67Cre ; Alk4fl / fl ) mice , 24 hours after second injection of TIPS protocol . Scale bars 20 μm . ( H ) of ∆ FosB positive 24 hours after second injection of TIPS protocol in Nac of Gad67Cre Gad67Cre ; Alk4fl / fl mice . Results are expressed as average ± of number of ∆ FosB + per mm2 . At least 10 from both left right hemispheres were analyzed per mouse condition . N = 5 . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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

(G) Representative immunofluorescence staining of ∆FosB in Nac of control (Gad67Cre) and mutant (Gad67Cre;Alk4fl/fl) mice, 24 hours after the second injection of the TIPS protocol. Scale bars 20 μm. (H) Quantification of ∆FosB positive cells 24 hours after the second injection of TIPS protocol in the Nac of Gad67Cre and Gad67Cre;Alk4fl/fl mice. Results are expressed as average ± SEM of the number of ∆FosB+ cells per mm2. At least 10 images from both left and right hemispheres were analyzed per mouse and condition. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


( I ) Western blot of FosB ∆ FosB in in NAc of Gad67Cre Gad67Cre ; Alk4fl / fl mice 24 hour after second injection of TIPS . Blots were probed with an antibody that recognizes both FosB ( upper band , 48kDa ) ∆ FosB ( lower band , 38 kDa ) . Lower blot shows reprobing with anti - β - actin . ( J , K ) of FosB ( J ) ∆ FosB ( K ) in NAc of Gad67Cre Gad67Cre ; Alk4fl / fl mice 24 hour after second injection of TIPS . Results are expressed as average ± of fold increase of FosB ∆ FosB over saline group ( Sal / Sal ) of Gad67Cre mice normalized to β - actin . N = 4s . Data information : * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; one - way ANOVA followed with Tukey ' s multiple comparisons . N denotes biological replicates .

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

(I) Western blot analysis of FosB and ∆FosB in in NAc of Gad67Cre and Gad67Cre;Alk4fl/fl mice 24 hour after the second injection of TIPS. Blots were probed with an antibody that recognizes both FosB (upper band, 48kDa) and ∆FosB (lower band, 38 kDa). Lower blot shows reprobing with anti-β-actin. (J, K) Quantification of FosB (J) and ∆FosB (K) levels in NAc of Gad67Cre and Gad67Cre;Alk4fl/fl mice 24 hour after the second injection of TIPS. Results are expressed as average ± SEM of fold increase of FosB and ∆FosB over the saline group (Sal/Sal) of Gad67Cre mice normalized to β-actin levels. N=4s. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.


A A neutral red - stained mouse pancreas is excised digested with collagenase . Islets are then separated by picking further digested into single islet . Scale bars : 10 mm ( left image ) ; 200 µm ( middle image ) ; 60 µm ( right image ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 A neutral red-stained mouse pancreas is excised and digested with collagenase. Islets are then separated by hand picking and further digested into single islet cells. Scale bars: 10 mm (left image); 200 µm (middle image); 60 µm (right image).


B Epidydimal fat pads are isolated processed mechanically enzymatically . obtained mixture , containing MVF ECM , is filtrated for purification of MVF . Scale bars : 20 mm ( left image ) ; 25 µm ( right image ) .

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B Epidydimal fat pads are isolated and processed mechanically and enzymatically. The obtained mixture, containing MVF and ECM, is filtrated for the purification of MVF. Scale bars: 20 mm (left image); 25 µm (right image).


C Western blots of CD31 , α - SMA GAPDH in whole extracts of eEND , HDMEC , HUVEC MVF .

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

C Western blots of CD31, α-SMA and GAPDH in whole cell extracts of eEND, HDMEC, HUVEC and MVF.


D Immunofluorescence stainings of CD31 / α - SMA CD31 / PDGFR - β in MVF . nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 20 µm .

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

D Immunofluorescence stainings of CD31 / α-SMA and CD31 / PDGFR-β in MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 20 µm.


F PI PI + MVF on days - 5 , - 3 0 . Scale bar : 200 µm .

F PI and PI+MVF on days -5, -3 and 0. Scale bar: 200 µm.


G Surface of FI , CI , PI PI + MVF on day 0 as visualized by scanning electron microscopy . Scale bar : 50 µm .

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G Surface morphology of FI, CI, PI and PI+MVF on day 0 as visualized by scanning electron microscopy. Scale bar: 50 µm.


A Neutral red trypan blue stainings of FI , CI , PI PI + MVF . CI incubated for 24 h with 0 . 2 % H2O2 served as control . Scale bar : 100 µm .

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A Neutral red and trypan blue stainings of FI, CI, PI and PI+MVF. CI incubated for 24 h with 0.2% H2O2 served as control. Scale bar: 100 µm.


B Representative flow cytometric scatterplots of propidium iodide / annexin V - stained from PI + MVF CI + H2O2 ( positive control ) . C Quantitative of propidium iodide / annexin V - stained from FI , CI , PI , PI + MVF CI + H2O2 subdivided in necrotic , necroptotic , apoptotic vital in % of total number ( n = 3 each ) .

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B Representative flow cytometric scatterplots of propidium iodide / annexin V-stained cells from PI+MVF and CI+H2O2 (positive control). C Quantitative analysis of propidium iodide / annexin V-stained cells from FI, CI, PI, PI+MVF and CI+H2O2 subdivided in necrotic, necroptotic, apoptotic and vital cells in % of total cell number (n = 3 each).


D Schematic illustration of mitochondrial ROS - sensor mito - roGFP2 - Orp1 . maximum of this sensor shifts from 488 nm to 405 nm with increasing mitochondrial H2O2 , as shown by representative of FI . Scale bar : 70 µm .

O O O O 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

D Schematic illustration of the mitochondrial ROS-sensor mito-roGFP2-Orp1. The fluorescence maximum of this sensor shifts from 488 nm to 405 nm with increasing mitochondrial H2O2 levels, as shown by representative fluorescence images of FI. Scale bar: 70 µm.


E Quantitative of ROS sensor ( 405 nm / 488 nm ) of FI , CI , PI PI + MVF ( n = 25 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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

E Quantitative analysis of the ROS sensor ratio (405 nm / 488 nm) of FI, CI, PI and PI+MVF (n = 25 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


F Representative Western blot of β - actin HO - 1 in whole extracts from FI , CI , PI PI + MVF . G Quantitative of HO - 1 expression in FI , CI , PI PI + MVF ( n = 3 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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

F Representative Western blot analysis of β-actin and HO-1 in whole cell extracts from FI, CI, PI and PI+MVF. G Quantitative analysis of HO-1 expression in FI, CI, PI and PI+MVF (n = 3 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


H Representative immunofluorescence stainings of HO - 1 / insulin HO - 1 / CD31 in PI + MVF . nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 50 µm .

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

H Representative immunofluorescence stainings of HO-1 / insulin and HO-1 / CD31 in PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 50 µm.


I Representative Western blot of β - actin HO - 1 in whole extracts from PI MVF .

I Representative Western blot analysis of β-actin and HO-1 in whole cell extracts from PI and MVF.


A Representative immunofluorescence stainings of insulin / glucagon insulin / somatostatin in FI , CI , PI PI + MVF . nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 60 µm . B - D Quantitative of insulin - ( B ) , glucagon - ( C ) somatostatin - positive ( D ) in FI , CI , PI PI + MVF in % of islet or organoid ( n = 20 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

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

A Representative immunofluorescence stainings of insulin / glucagon and insulin / somatostatin in FI, CI, PI and PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 60 µm. B-D Quantitative analysis of insulin- (B), glucagon- (C) and somatostatin-positive cells (D) in FI, CI, PI and PI+MVF in % of all islet or organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.


E Western blot of β - actin insulin in whole extracts from FI , CI , PI PI + MVF .

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E Western blot analysis of β-actin and insulin in whole cell extracts from FI, CI, PI and PI+MVF.


Insulin secretion ( µU / mL ) ( normalized to content ) of FI , CI , PI PI + MVF exposed to 1 . 1 mM 16 . 5 mM glucose ( n = 3 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc as well as an unpaired Student ' s t - were used for statistical . * P < 0 . 05 vs . insulin secretion at 1 . 1 mM glucose within each group ; + P < 0 . 05 vs . insulin secretion of PI at 16 . 5 mM glucose .

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

Insulin secretion (µU/mL) (normalized to DNA content) of FI, CI, PI and PI+MVF exposed to 1.1 mM and 16.5 mM glucose (n = 3 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test as well as an unpaired Student's t-test were used for statistical analysis. *P < 0.05 vs. insulin secretion at 1.1 mM glucose within each individual group; +P < 0.05 vs. insulin secretion of PI at 16.5 mM glucose.


A Representative immunofluorescence stainings of insulin CD31 in CI , FI , PI PI + MVF . nuclei are stained with Hoechst 33342 ( blue ) . Scale bar : 100 µm . B Quantitative of CD31 - positive in FI , CI , PI PI + MVF in % of islet or islet organoid ( n = 20 each ) . Mean ± SD . One - way ANOVA Tukey ' s multiple comparisons post hoc were used for statistical . * P < 0 . 05 vs . FI ; # P < 0 . 05 vs . CI ; + P < 0 . 05 vs . PI .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative immunofluorescence stainings of insulin and CD31 in CI, FI, PI and PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 100 µm. B Quantitative analysis of CD31-positive cells in FI, CI, PI and PI+MVF in % of all islet or islet organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.