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 .

O O 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 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 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 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 ) .

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

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


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

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

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


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

O O 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

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


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

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

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


LSK ( Lin - Sca1 + cKit + ) 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 ) .

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

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

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


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

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 I-CONTROLLED_VAR I-CONTROLLED_VAR 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

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


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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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

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


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

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

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

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

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


Terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) 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 .

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

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

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


DAPI - negative viable LK - 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 .

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

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


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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - 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 .

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


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

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

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

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


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

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

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


Chk1fl / fl Hoxb8 - FL progenitor 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 .

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

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


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 ) .

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


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

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

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


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 ) .

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


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

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

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


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

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

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


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 .

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


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

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

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


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

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


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

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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 tumor - bearing mice ( n = 8 ) .

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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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. (A+B) CD31 staining revealed that blocking of EGFL7, VEGF, or a combination of both lead to decreased tumor vessel density (n = 3; one-way ANOVA).


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

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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 (C+D) PDGFRβ (n = 3; one-way ANOVA),


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

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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 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 .

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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 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 ) .

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


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

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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 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 .

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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 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 ) .

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


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

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


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

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


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

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(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 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 .

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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 ) 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 .

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(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 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 ) .

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

(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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 ) .

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(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 - 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 ) .

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

O O O O 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

(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 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 .

O O O 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 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

(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 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 .

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

O O O O O O O O O O O O O O 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 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 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 ) 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 .

O O O O O 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 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

(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 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 .

O O O 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 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 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 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 .

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(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 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 .

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

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

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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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 and GAPDH in whole cell extracts of eEND , HDMEC , HUVEC and MVF .

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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 and CD31 / PDGFR - β in MVF . Cell 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 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 and PI + MVF on days - 5 , - 3 and 0 . Scale bar : 200 µm .

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


G Surface morphology of FI , CI , PI and 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 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 .

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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 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 ) .

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

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

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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and HO - 1 / CD31 in PI + MVF . Cell 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 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 analysis of β - actin and HO - 1 in whole cell extracts from PI and 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 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 .

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

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 analysis of β - actin and insulin in whole cell extracts from FI , CI , PI and 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 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 .

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

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 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.


C Representative Western blot analysis of CD31 and β - actin in whole cell extracts from CI , FI , PI and PI + MVF .

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C Representative Western blot analysis of CD31 and β-actin in whole cell extracts from CI, FI, PI and PI+MVF.


D Quantitative analysis of CD31 expression in FI , CI , PI and PI + MVF in % of FI ( n = 3 ) . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantitative analysis of CD31 expression in FI, CI, PI and PI+MVF in % of FI (n = 3). 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 Confocal microscopic images of CD31 and cell nuclei ( blue ) in PI + MVF , demonstrating endothelial cells ( arrowheads ) . Scale bar : 100 µm .

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E Confocal microscopic images of CD31 and cell nuclei (blue) in PI+MVF, demonstrating endothelial cells (arrowheads). Scale bar: 100 µm.


F Representative immunofluorescence staining of Ki67 and CD31 in PI + MVF . Cell nuclei are stained with Hoechst 33342 ( blue ) . Arrows mark endothelial cells expressing Ki67 . Scale bar : 100 µm .

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F Representative immunofluorescence staining of Ki67 and CD31 in PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Arrows mark endothelial cells expressing Ki67. Scale bar: 100 µm.