Left : Representative time - lapse showing migration of single hRPE on surface of polyacrylamide gels with uniform stiffness ( top ) stiffness gradient ( bottom ) , referring as random migration durotaxis migration respectively . Yellow is outline blue is migration trajectory . Scale bar : 50 μm . Middle : Angular displacement of hRPE on uniform stiffness gels ( top ) stiffness gradient gels ( bottom ) . Right : y / x FMI ( forward migration index ) , velocity persistence of hRPE with random migration durotaxis .

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Left: Representative time-lapse images showing the migration of single hRPE cells on the surface of polyacrylamide gels with uniform stiffness (top) and stiffness gradient (bottom), referring as random migration and durotaxis migration respectively. Yellow line is the cell outline and blue line is the cell migration trajectory. Scale bar: 50 μm. Middle: Angular displacement of hRPE cells on the uniform stiffness gels (top) and stiffness gradient gels (bottom). Right: y/x FMI (forward migration index), velocity and persistence of hRPE cells with random migration and durotaxis.


Left : Representative immunofluorescent of F - actin in control CK - 666 ( 100 μM , 3 h ) treated hRPEs . Scale bar : 20 μm . Right : Y FMI of control CK - 666 ( 100 μM , pre - treated for 1h incubated for whole experiment ) treated hRPEs moving on stiffness gradient gel .

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Left: Representative immunofluorescent staining of F-actin in control and CK-666 (100 μM, 3 h) treated hRPEs. Scale bar: 20 μm. Right: Y FMI of control and CK-666 (100 μM, pre-treated for 1h and incubated for the whole experiment) treated hRPEs moving on stiffness gradient gel.


Left : Representative immunofluorescent of α - tubulin in control nocodazole ( 8 μM , 30 min ) treated hRPEs . Scale bar : 20 μm . Right : Y FMI of control nocodazole ( 8 μM , pre - treated for 10 min incubated for whole experiment ) treated hRPEs moving on stiffness gradient gel .

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Left: Representative immunofluorescent staining of α-tubulin in control and nocodazole (8 μM, 30 min) treated hRPEs. Scale bar: 20 μm. Right: Y FMI of control and nocodazole (8 μM, pre-treated for 10 min and incubated for the whole experiment) treated hRPEs moving on stiffness gradient gel.


Representative immunofluorescent of γ - tubulin ( yellow ) DAPI ( blue ) in control centrinone - B ( 500 nM , 7 days ) treated hRPEs .

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Representative immunofluorescent staining of γ-tubulin (yellow) and DAPI (blue) in control and centrinone-B (500 nM, 7 days) treated hRPEs.


Angular displacement y FMI of control centrinone - B ( 500 nM , 7 days ) treated hRPEs moving on stiffness gradient gels .

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Angular displacement and y FMI of control and centrinone-B (500 nM, 7 days) treated hRPEs moving on stiffness gradient gels.


Western blot showing level of CAMSAP2 in negative control CAMSAP2 siRNA transfected hRPEs . GAPDH was used as loading control . Bar chart shows of normalized to GAPDH in each condition .

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Western blot showing the protein level of CAMSAP2 in negative control and CAMSAP2 siRNA transfected hRPEs. GAPDH was used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition.


Angular displacement y FMI of negative control CAMSAP2 siRNA transfected hRPEs moving on stiffness gradient gels .

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Angular displacement and y FMI of negative control and CAMSAP2 siRNA transfected hRPEs moving on stiffness gradient gels.


Western blot showing level of AKAP450 in negative control AKAP450 siRNA transfected hRPEs . α - tubulin was used as loading control . Bar chart shows of normalized to α - tubulin in each condition .

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Western blot showing the protein level of AKAP450 in negative control and AKAP450 siRNA transfected hRPEs. α-tubulin was used as loading control. Bar chart shows quantification of protein levels normalized to α-tubulin in each condition.


Angular displacement y FMI of control AKAP450 siRNA transfected hRPEs moving on stiffness gradient gels .

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Angular displacement and y FMI of control and AKAP450 siRNA transfected hRPEs moving on stiffness gradient gels.


Left : Representative immunofluorescent of α - tubulin in control , CAMSAP2 siRNA transfected centrinone - B ( 500 nM , 7 days ) treated hRPEs . Scale bar : 20 μm . Right : of microtubule .

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Left: Representative immunofluorescent staining of α-tubulin in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Scale bar: 20 μm. Right: Quantification of microtubule fluorescence intensity.


Western blot showing level of α - tubulin in control , CAMSAP2 siRNA transfected centrinone - B ( 500 nM , 7 days ) treated hRPEs . GAPDH is used as loading control . Bar chart shows of normalized to GAPDH in each condition .

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Western blot showing the protein level of α-tubulin in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. GAPDH is used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition.


Left : Representative live - imaging of EGFP - EB1 in control , CAMSAP2 siRNA transfected centrinone - B ( 500 nM , 7 days ) treated hRPEs . Scale bar : 20 μm . Right : of EB1 growth speed obtained by plusTipTracker . Vinlin plot center lines denote median , dashed lines represent 25th 75th percentiles .

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Left: Representative live-cell fluorescence imaging of EGFP-EB1 in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Scale bar: 20 μm. Right: Quantification of EB1 growth speed obtained by plusTipTracker. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.


Western blot showing level of active RhoA , total RhoA in negative control CAMSAP2 siRNA transfected hRPEs . GAPDH is used as loading control . Bar chart shows of normalized to GAPDH in each condition .

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Western blot showing the protein level of active RhoA, total RhoA in negative control and CAMSAP2 siRNA transfected hRPEs. GAPDH is used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition.


Left : Representative live - imaging of GFP - AHPH in negative control CAMSAP2 siRNA transfected hRPEs . Scale bar : 20 μm . Right : of GFP - AHPH .

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Left: Representative live-cell fluorescence imaging of GFP-AHPH in negative control and CAMSAP2 siRNA transfected hRPEs. Scale bar: 20 μm. Right: Quantification of GFP-AHPH fluorescence intensity.


Left : Representative immunofluorescent of pMLC ( yellow ) DAPI ( blue ) in negative control CAMSAP2 siRNA transfected hRPEs . Scale bar : 20 μm . Right : of pMLC . Below : Western blot showing level of pMLC in negative control CAMSAP2 siRNA transfected hRPEs . α - tubulin is used as loading

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Left: Representative immunofluorescent staining of pMLC (yellow) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs. Scale bar: 20 μm. Right: Quantification of pMLC fluorescence intensity. Below: Western blot showing the protein level of pMLC in negative control and CAMSAP2 siRNA transfected hRPEs. α-tubulin is used as loading control


Left : showing collagen gel deformation at 0 h , 8 h 24 h . HRPEs transfected with negative control , CAMSAP2 AKAP450 siRNA were seeded in 1 % collagen gel ( yellow dashed lines ) . Scale bar : 5 mm . Right : of gel .

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Left: Images showing collagen gel deformation at 0 h, 8 h and 24 h. HRPEs transfected with negative control, CAMSAP2 and AKAP450 siRNA were seeded in 1% collagen gel (yellow dashed lines). Scale bar: 5 mm. Right: Quantification of gel area.


Left : Representative live - imaging of RFP - paxillin in control nocodazole ( 8 μM , 30 min ) treated hRPEs . Scale bar : 20 μm . Right : of FA obtained by FAAS .

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Left: Representative live-cell fluorescence imaging of RFP-paxillin in control and nocodazole (8 μM, 30 min) treated hRPEs. Scale bar: 20 μm. Right: Quantification of FA area obtained by FAAS.


Top : Representative live - imaging of RFP - paxillin in negative control CAMSAP2 siRNA transfected hRPEs . Time - lapse imaging zoomed from yellow box showed assembly disassembly stage of a single FA . Scale bar : 20 μm . Bottom : of FA , assembly rate disassembly rate obtained by FAAS . Vinlin plot center lines denote median , dashed lines represent 25th 75th percentiles .

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Top: Representative live-cell fluorescence imaging of RFP-paxillin in negative control and CAMSAP2 siRNA transfected hRPEs. Time-lapse fluorescence imaging zoomed from the yellow box showed the assembly and disassembly stage of a single FA. Scale bar: 20 μm. Bottom: Quantification of FA area, assembly rate and disassembly rate obtained by FAAS. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.


Top : Schematic diagram of FA - MT contact . Position of EB1 paxillin were extracted from live - , followed by automated co - localization identification . Scale bar : 20 μm . Bottom : Representative time - lapse imaging of EGFP - EB1 RFP - paxillin zoomed from yellow box above showed targeting of MT to FA . FA - MT contact was calculated by of co - localization events to total EB1 number . FA - MT contact of negative control , CAMSAP2 , AKAP450 siRNA transfected centrinone - B ( 500 nM , 7 days ) treated hRPEs . Vinlin plot center lines denote median , dashed lines represent 25th 75th percentiles .

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Top: Schematic diagram of FA-MT contact analysis. Position of EB1 and paxillin were extracted from live-cell images, followed by automated co-localization identification. Scale bar: 20 μm. Bottom: Representative time-lapse fluorescence imaging of EGFP-EB1 and RFP-paxillin zoomed from the yellow box above showed the targeting of MT to FA. FA-MT contact ratio was calculated by the ratio of co-localization events to the total EB1 number.   FA-MT contact ratio of negative control, CAMSAP2, AKAP450 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.


Western blot showing level of ELKS in negative control ELKS siRNA transfected hRPEs . α - tubulin was used as loading control . Bar chart shows of normalized to α - tubulin in each condition .

Western blot showing the protein level of ELKS in negative control and ELKS siRNA transfected hRPEs. α-tubulin was used as loading control. Bar chart shows quantification of protein levels normalized to α-tubulin in each condition.


FA - MT contact of negative control ELKS siRNA transfected hRPEs . Vinlin plot center lines denote median , dashed lines represent 25th 75th percentiles .

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FA-MT contact ratio of negative control and ELKS siRNA transfected hRPEs. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles.


Y FMI of negative control ELKS siRNA transfected hRPEs moving on stiffness gradient gels .

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Y FMI of negative control and ELKS siRNA transfected hRPEs moving on stiffness gradient gels.


Left : Representative immunofluorescent of GM130 ( yellow ) , F - actin ( gray ) DAPI ( blue ) in negative control CAMSAP2 siRNA transfected hRPEs at edge of wound after 8 h . Scale bar : 50 μm . Middle : Schematic diagram of Golgi - nucleus orientation towards front ( 45 ° - 135 ° ) or rear ( - 45 ° - - 135 ° ) . Right : of of Golgi - nucleus orientation towards front or rear of .

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Left: Representative immunofluorescent staining of GM130 (yellow), F-actin (gray) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs at the edge of the wound after 8 h. Scale bar: 50 μm. Middle: Schematic diagram of Golgi-nucleus orientation towards the front (45°-135°) or the rear (-45°- -135°). Right: Quantification of the percentage of Golgi-nucleus orientation towards the front or rear of the cell.


Left : Representative immunofluorescent of GM130 ( yellow ) DAPI ( blue ) in negative control CAMSAP2 siRNA transfected hRPEs on stiffness gradient gel . Scale bar : 20 μm . Middle : Schematic diagram of Golgi - nucleus orientation towards stiff side ( 45 ° - 135 ° ) or soft side ( - 45 ° - - 135 ° ) . Right : of of Golgi - nucleus orientation towards stiff or soft side .

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Left: Representative immunofluorescent staining of GM130 (yellow) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs on the stiffness gradient gel. Scale bar: 20 μm. Middle: Schematic diagram of Golgi-nucleus orientation towards the stiff side (45°-135°) or the soft side (-45°- -135°). Right: Quantification of the percentage of Golgi-nucleus orientation towards the stiff or soft side.


Left : Schematic diagram of manufacturing polyacrylamide gels with islands of higher stiffness by 405 nm irradiation . Middle : Modulus of polyacrylamide gels with stiff islands measured by atomic force microscope . Right : Representative live - imaging of marked with Golgi ( green ) nucleus ( blue ) on soft region stiff island .

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Left: Schematic diagram of manufacturing polyacrylamide gels with islands of higher stiffness by 405 nm laser irradiation. Middle: Modulus of polyacrylamide gels with stiff islands measured by atomic force microscope. Right: Representative live-cell fluorescence imaging of cells marked with the Golgi (green) and nucleus (blue) on the soft region and the stiff island.


Representative time - lapse showing migration of hRPE to stiff island reorientation of Golgi in this process . Scale bar : 20 μm . of of having a stiff - end biased Golgi positioning . N = 29 , replicates are biological .

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Representative time-lapse fluorescence images showing the migration of hRPE cell to the stiff island and the reorientation of Golgi in this process. Scale bar: 20 μm.   Quantification of the percentage of cells having a stiff-end biased Golgi positioning. N=29 cells, replicates are biological.


b , MEFs were generated from day 12 . 5embryos of Atg16l1 mutant mice , western blot was used to detect a b isoforms of ATG16L1 from whole -

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b, MEFs were generated from day 12.5embryos of Atg16l1 mutant mice, and western blot analysis was used to detect the a and b isoforms of ATG16L1 from whole-cell lysates


c , d , Atg5 - / - ATG16L1HM MEFs were grown in presence of chemical inducer of autophagy rapamycin ( Rap , 50 µg ml - 1 ) cyclohexamide ( CHX , 5 µg ml - 1 ) for 0 , 4 , 8 12 h . lysates were analysed by western blot for loss of P62 expression ( c ) . P62 were quantified by densitometry normalized to actin ( n = 3 ; d ) . * P 0 . 04 ; * * P 0 . 008 ; * * * P = 0 .

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c, d, Atg5-/- and ATG16L1HM MEFs were grown in the presence of the chemical inducer of autophagy rapamycin (Rap, 50 µg ml-1) and cyclohexamide (CHX, 5 µg ml-1) for 0, 4, 8 and 12 h. Cell lysates were analysed by western blot for loss of P62 expression (c). P62 levels were quantified by densitometry normalized to actin (n = 3; d). *P 0.04; **P 0.008; ***P = 0.0003


e , f , Western blot of LC3 expression in MEFs grown in presence of rapamycin ( 50 µg ml - 1 ) or dimethylsulphoxide ( DMSO ) control for 4 h ( e ) . LC3 - II expression in presence of rapamycin was quantified normalized to actin ( n = 6 ; f ) .

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e, f, Western blot analysis of LC3 expression in MEFs grown in the presence of rapamycin (50 µg ml-1) or dimethylsulphoxide (DMSO) control for 4 h (e). LC3-II expression in the presence of rapamycin was quantified and normalized to actin (n = 6; f).


g , Detection of ATG16L1 , LC3 P62 by western blot of ileal lysates from ATG16L1HM mice . ATG16L1 can be detected in both mutant lines on longer exposures . h , i , of ATG16L1 ( h ) P62 ( i ) normalized to actin detected in ileal lysates ( n = 3 ) . P values were calculated using two - Student ' s t - . Error bars , s . e . m .

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g, Detection of ATG16L1, LC3 and P62 by western blot of ileal lysates from ATG16L1HM mice. ATG16L1 can be detected in both mutant lines on longer exposures. h, i, Quantification of ATG16L1 (h) and P62 (i) normalized to actin detected in ileal lysates (n = 3). P values were calculated using two-tailed Student's t-test. Error bars, s.e.m.


a , b , Whole - mount taken immediately above ileal mucosal surface from WT ( a ) ATG16L1HM ( b ) littermate mice stained with Helix pomatia lectin that labels mucus ( green ) antisera directed against lysozyme ( red ) . are representative of 3 WT 3 ATG16L1HM mice .

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a, b, Whole-mount images taken immediately above the ileal mucosal surface from WT (a) and ATG16L1HM (b) littermate mice stained with Helix pomatia lectin that labels mucus (green) and antisera directed against lysozyme (red). Images are representative of 3 WT and 3 ATG16L1HM mice.


c , d , Ileal sections from WT ( c ) ATG16L1HM ( d ) mice were stained with PAS / alcian blue to detect Paneth granules by microscopy ( dashed denotes crypt unit arrowheads indicate Paneth ) . are representative of 7 WT , 7 ATG16L1HM1 5 ATG16L1HM2 mice ; more than 100 crypts were analysed for

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c, d, Ileal sections from WT (c) and ATG16L1HM (d) mice were stained with PAS/alcian blue to detect Paneth granules by light microscopy (dashed line denotes the crypt unit and arrowheads indicate Paneth cells). Images are representative of 7 WT, 7 ATG16L1HM1 and 5 ATG16L1HM2 mice; more than 100 crypts were analysed for each


e , f , Representative of indirect immunofluorescence of sections stained for lysozyme ( red ) in WT ( e ) ATG16L1HM ( f ) mouse ileal crypts .

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e, f, Representative images of indirect immunofluorescence of sections stained for lysozyme (red) in WT (e) and ATG16L1HM (f) mouse ileal crypts.


g , Paneth display one of four patterns of lysozyme expression ( represented in red ; represents areas that exclude lysozyme ) : normal ( D0 ) , disordered ( D1 ) , depleted ( D2 ) diffuse ( D3 ) .

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g, Paneth cells display one of four patterns of lysozyme expression (represented in red; white represents areas that exclude lysozyme): normal (D0), disordered (D1), depleted (D2) and diffuse (D3).


h , i , Number of Paneth from ATG16L1HM ( h ) Atg5flox / floxvillin - Cre ( i ) mice displaying each pattern of lysozyme expression ( n = 6 , 660 from 5 ATG16L1HM mice 5 , 634 from 5 WT mice ; n = 1 , 756 from 2 Atg5flox / floxvillin - Cre mice 1 , 649 from 2 Atg5flox / flox mice ) . Scale bars : a , b , 200 µm ; c - f , 10 µm . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 . P values were calculated using two - Student ' s t - . Error bars , s . e . m .

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h, i, Number of Paneth cells from ATG16L1HM (h) and Atg5flox/floxvillin-Cre (i) mice displaying each pattern of lysozyme expression (n = 6,660 cells from 5 ATG16L1HM mice and 5,634 cells from 5 WT mice; n = 1,756 cells from 2 Atg5flox/floxvillin-Cre mice and 1,649 from 2 Atg5flox/flox mice). Scale bars: a, b, 200 µm; c-f, 10 µm. *P 0.05, **P 0.01, ***P 0.001. P values were calculated using two-tailed Student's t-test. Error bars, s.e.m.


a - d , Electron micrograph of littermate WT ( a , c ) ATG16L1HM ( b , d ) Paneth ( for a , b , dashed lines denote , asterisk indicates normal progenitor ; for c , d , blue stars indicate normal mitochondria in WT Paneth ATG16L1HM progenitor , red diamonds indicate degenerating mitochondria , dashed box shows normal Golgi compartments , yellow asterisks indicate granules ; n = 3 mice per genotype ) . e , Mitochondrial integrity was quantified on basis of of visible mitochondrial section displaying intact cisternae ( n = 49 WT 71 ATG16L1HM ) .

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a-d, Electron micrograph of littermate WT (a, c) and ATG16L1HM (b, d) Paneth cells (for a, b, dashed lines denote individual cells, asterisk indicates normal progenitor cell; for c, d, blue stars indicate normal mitochondria in WT Paneth cells and ATG16L1HM progenitor cells, red diamonds indicate degenerating mitochondria, the dashed box shows normal Golgi compartments, and yellow asterisks indicate granules; n = 3 mice per genotype). e, Mitochondrial integrity was quantified on the basis of the percentage of the visible mitochondrial section displaying intact cisternae (n = 49 WT and 71 ATG16L1HM cells).


g , Chart of selected genes for which mRNA is enriched in either ATG16L1 - deficient Paneth ( baseline is WT Paneth ) or ATG16L1 - deficient thymocytes ( baseline is WT thymocytes ) . Only haemoglobin is in common in these two sets of analyses . NS , not significant . Scale bars : a , b , 10 µm ; c , d , 500 nm .

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g, Chart of selected genes for which mRNA is enriched in either ATG16L1-deficient Paneth cells (baseline is WT Paneth cells) or ATG16L1-deficient thymocytes (baseline is WT thymocytes). Only haemoglobin is in common in these two sets of analyses. NS, not significant. Scale bars: a, b, 10 µm; c, d, 500 nm.


a , b , Haematoxylin - - eosin - stained sections of uninvolved areas from ileo - colic resections from with Crohn ' s disease homozygous for safe ( a ) or risk ( b ) allele of ATG16L1 ( blue dashed denotes crypt unit ) .

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a, b, Haematoxylin-and-eosin-stained sections of uninvolved areas from ileo-colic resections from patients with Crohn's disease homozygous for the safe (a) or risk (b) allele of ATG16L1 (blue dashed line denotes crypt unit).


c - f , Immunofluorescence of Paneth from control ( c , e ) with risk allele ( d , f ) stained for lysozyme ( red ; c , d ) double - labelled additionally for leptin ( green ; e , f ; yellow dashed denotes crypt unit ) .

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

c-f, Immunofluorescence images of Paneth cells from control patients (c, e) and patients with the risk allele (d, f) stained for lysozyme (red; c, d) and double-labelled additionally for leptin (green; e, f; yellow dashed line denotes the crypt unit).


g , Aberrant lysozyme expression was quantified using same criteria as that used for mouse sections in Fig . 3 ( n = 6 , 829 for at risk 8 , 182 for control , n = 5 for each genotype ) . Leptin - positive D3 were quantified in samples ( n = 4 per genotype ) homozygous for risk allele ( 76 out of 322 D3 were positive from a total of 580 crypts examined ) homozygous for safe allele ( 11 out of 93 D3 were positive from a total 749 crypts examined ) . Scale bars : a - f , 10 µm . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 . P values were calculated using two - Student ' s t - . Error bars , s . e . m .

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 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

g, Aberrant lysozyme expression was quantified using the same criteria as that used for mouse sections in Fig. 3 (n = 6,829 cells for at risk patients and 8,182 for control, n = 5 patients for each genotype). Leptin-positive D3 cells were quantified in patient samples (n = 4 patients per genotype) homozygous for the risk allele (76 out of 322 D3 cells were positive from a total of 580 crypts examined) and homozygous for the safe allele (11 out of 93 D3 cells were positive from a total 749 crypts examined). Scale bars: a-f, 10 µm. *P 0.05, **P 0.01, ***P 0.001. P values were calculated using two-tailed Student's t-test. Error bars, s.e.m.


MIP of cerebral vessels of 3dpf Tg ( kdrl : HRAS - mCherry ) s916 ( grey LUT ; inverted ) . Higher magnification panel showing two kugeln ( arrowheads ) arising from middle mesencephalic central artery ( MMCtA ) .

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

MIP of cerebral vessels of 3dpf Tg(kdrl:HRAS-mCherry)s916 embryo (grey LUT; inverted). Higher magnification panel showing two kugeln (arrowheads) arising from the middle mesencephalic central artery (MMCtA).


Diameter of kugeln at 3dpf ( mean ± s . e . m . 10 . 13 ± 0 . 49 ; n = 93 kugeln from 32 3dpf ; 3 experimental repeats ) .

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

Diameter of kugeln at 3dpf (mean ± s.e.m. 10.13 ± 0.49; n=93 kugeln from 32 3dpf embryos; 3 experimental repeats).


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

Number of kugeln per embryo in 3, 4 and 5dpf animals was not statistically significantly different (mean ± s.e.m. 3dpf 5.47 ± 1.09, 4dpf 7.47 ± 1.46, 5dpf 7.09 ± 1.55; p = 0.8571; 3dpf: 175 kugeln from 32 embryos; 4dpf: 239 kugeln from 32 embryos, 5dpf: 227 kugeln from 32 embryos; 4 experimental repeats; Kruskal-Wallis test).


Kugeln ( arrowheads ) could be observed in 28dpf .

Kugeln (arrowheads) could be observed in 28dpf animals.


MIP of cerebral vessels of 4dpf Tg ( fli1aep : CAAX - eGFP ) ( grey LUT ; inverted ) . Higher magnification panel shows a kugel ( arrowhead ) protruding from posterior mesencephalic central artery ( PMCtA ) .

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

MIP of cerebral vessels of 4dpf Tg(fli1aep:CAAX-eGFP) embryo (grey LUT; inverted). Higher magnification panel shows a kugel (arrowhead) protruding from the posterior mesencephalic central artery (PMCtA).


Single Z - plane micrograph of cerebral vessels of a 3dpf Tg ( gata1 : dsRed ) , injected with a Tol2 - fli1a : myr - Cherry construct showing two kugeln ( arrowheads ) in higher magnification panel protruding from MMCtA .

O O O O O O O O O O O O O O O O O O O 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 Z-plane micrograph of cerebral vessels of a 3dpf Tg(gata1:dsRed), injected with a Tol2-fli1a:myr-Cherry construct showing two kugeln (arrowheads) in the higher magnification panel protruding from the MMCtA.


Locations proportion of kugeln on cerebral vessels ( colour coded by vessel ; total n = 107 kugeln from 34 4dpf ; 3 experimental repeats ) . AMCtA - anterior mesencephalic central artery , BCA - basal communicating artery , CaDI - caudal division of internal carotid artery , CMV - communicating vessel , CtA - central arteries , MMCtA - middle mesencephalic central artery , MtA - metencephalic artery , PCS - posterior communicating segment , PMBC - posterior midbrain channel , PMCtA - posterior mesencephalic central artery , PrA - prosencephalic artery .

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

Locations and proportion of kugeln on cerebral vessels (colour coded by vessel; total n=107 kugeln from 34 4dpf embryos; 3 experimental repeats). AMCtA - anterior mesencephalic central artery, BCA - basal communicating artery, CaDI - caudal division of internal carotid artery, CMV - communicating vessel, CtA - central arteries, MMCtA - middle mesencephalic central artery, MtA - metencephalic artery, PCS - posterior communicating segment, PMBC - posterior midbrain channel, PMCtA - posterior mesencephalic central artery, PrA - prosencephalic artery.


MIPs of a time - series sheet acquisition shows three different kugeln ( kugel 1 - black arrowhead ; kugel 2 - grey arrowhead ; kugel 3 - unfilled arrowhead ) protruding retracting from parent vessels ( 2min intervals ; inverted LUT ) .

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

MIPs of a time-series light sheet acquisition shows three different kugeln (kugel 1 - black arrowhead; kugel 2 - grey arrowhead; kugel 3 - unfilled arrowhead) protruding and retracting from parent vessels (2min intervals; inverted LUT).


Kugeln persisted for variable durations before regression into parent vessel ( 43 kugeln from 9 4dpf embyros ; 4 experimental repeats ) .

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

Kugeln persisted for variable durations before regression into the parent vessel (43 kugeln from 9 4dpf embyros; 4 experimental repeats).


MIPs taken 20min apart showing examples of different kugel behaviour including shape changes ( asterisk ) , expansion ( circle ) , retraction ( triangle ) , or little change ( pentagon ) .

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

MIPs taken 20min apart showing examples of different kugel behaviour including shape changes (asterisk), expansion (circle), retraction (triangle), or little change (pentagon).


MIPs taken 1h apart show that kugeln may be observed at one time - point ( red arrowhead , 55 : 20 : 00 ) but not 1h later in same ( 56 : 20 : 00 ) to develop on other vessels 1h later ( 57 : 20 : 00 ) which again have regressed by 58 : 20 : 00 .

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

MIPs taken 1h apart show that kugeln may be observed at one time-point (red arrowhead, 55:20:00) but not 1h later in the same animal (56:20:00) and to develop on other vessels 1h later (57:20:00) which again have regressed by 58:20:00.


Kymographs generated by - scanning across diameter of a typical kugel shows that kugel diameter oscillated with a periodicity of minutes , while no such oscillations were observed in adjacent similarly sized cerebral vessels ( grey LUT ; inverted ) .

Kymographs generated by line-scanning across the diameter of a typical kugel shows that kugel diameter oscillated with a periodicity of minutes, while no such oscillations were observed in adjacent similarly sized cerebral vessels (all images grey LUT; inverted).


Double transgenic visualizing endothelial ( red ) endothelial nuclei ( green ) . Endothelial nuclei ( arrowhead ) were observed close to but never within kugel ( unfilled arrowhead ) .

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

Double transgenic visualizing endothelial membrane (red) and endothelial nuclei (green). Endothelial nuclei (arrowhead) were observed close to but never within the kugel (unfilled arrowhead).


Double transgenic showing endothelial ( red ) endothelial F - actin ( green ) . F - actin was found to localize at neck of

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

Double transgenic showing endothelial membrane (red) and endothelial F-actin (green). F-actin was found to localize at the neck of the kugeln


Double transgenic showing endothelial ( red ) endothelial cytoplasm ( green ) . reporter was visible in parent vessel but not in kugel ( unfilled arrowhead ) .

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

Double transgenic showing endothelial membrane (red) and endothelial cytoplasm (green). The cytoplasmic reporter was visible in the parent vessel but not in the kugel (unfilled arrowhead).


Triple transgenic showing endothelial ( red ) , neurons ( green ) erythrocytes ( red ) . Surrounding neurons were excluded from volume of kugel ( unfilled arrowhead ) erythrocytes were not observed inside kugeln ( arrowhead ) .

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

Triple transgenic showing endothelial membrane (red), neurons (green) and erythrocytes (red). Surrounding neurons were excluded from the volume of the kugel (unfilled arrowhead) and erythrocytes were not observed inside kugeln (arrowhead).


Treatment with inhibitor of actin polymerization Latrunculin B statistically significantly increased number of kugeln per ( 100nM 1 hour ; * * p = 0 . 0041 ; control n = 21 8 . 05 ± 1 . 76 ( mean ± s . e . m . ) ; Latrunculin n = 21 17 . 19 ± 2 . 93 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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

Treatment with the inhibitor of actin polymerization Latrunculin B statistically significantly increased number of kugeln per embryo (100nM 1 hour; **p = 0.0041; control n=21 embryos 8.05 ± 1.76 (mean ± s.e.m.); Latrunculin n=21 embryos 17.19 ± 2.93 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Latrunculin B treatment statistically significantly reduced kugel diameter ( * p = 0 . 0164 ; control n = 169 kugeln from 21 7 . 56 ± 2 . 25 ( mean ± s . e . m . ) ; Latrunculin n = 361 kugeln from 21 5 . 91 ± 1 . 78 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Student ' s t - ) .

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 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

Latrunculin B treatment statistically significantly reduced kugel diameter (*p = 0.0164; control n=169 kugeln from 21 embryos 7.56 ± 2.25 (mean ± s.e.m.); Latrunculin n=361 kugeln from 21 embryos 5.91 ± 1.78 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Student's t-test).


Treatment with Myosin II inhibitor Blebbistatin statistically significantly reduced number of kugeln per ( 25 µM 1h ; * * * * p < 0 . 0001 ; control n = 22 3 . 77 ± 0 . 56 ( mean ± s . e . m . ) , Blebbistatin n = 24 1 . 08 ± 0 . 22 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

O O B-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 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

Treatment with the Myosin II inhibitor Blebbistatin statistically significantly reduced number of kugeln per embryo (25 µM 1h; ****p <0.0001; control n=22 embryos 3.77 ± 0.56 (mean ± s.e.m.), Blebbistatin n=24 embryos 1.08 ± 0.22 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Blebbistatin treatment had no effect on kugel diameter ( p = 0 . 3731 ; control n = 83 kugeln from 22 6 . 27 ± 0 . 72 ( mean ± s . e . m . ) , Blebbistatin n = 26 kugeln from 24 7 . 02 ± 0 . 89 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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

Blebbistatin treatment had no effect on kugel diameter (p = 0.3731; control n=83 kugeln from 22 embryos 6.27 ± 0.72 (mean ± s.e.m.), Blebbistatin n=26 kugeln from 24 embryos 7.02 ± 0.89 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


MIP of cerebral vessels of a 4dpf before exsanguination ( grey LUT ; inverted ) . MIP of same after exsanguination , which did not alter kugel .

MIP of the cerebral vessels of a 4dpf embryo before exsanguination (grey LUT; inverted). MIP of the same embryo after exsanguination, which did not alter kugel size.


Time - lapse imaging of an with transiently halted cardiac contraction ( using Tricaine ) . Despite absent blood flow kugeln still changed shape ( grey arrowhead ) , retained shape ( arrowhead ) or protruded retracted ( black arrowhead ; time post cessation of flow is indicated on ; 3dpf ; grey inverted LUT ) . Time - lapse of an with transiently halted cardiac contraction as in ( C ) showed that kugel diameter still oscillates ( time post cessation of flow ) .

Time-lapse imaging of an embryo with transiently halted cardiac contraction (using Tricaine). Despite absent blood flow kugeln still changed shape (grey arrowhead), retained shape (white arrowhead) or protruded and retracted (black arrowhead; time post cessation of flow is indicated on micrographs; 3dpf; grey inverted LUT). Time-lapse of an embryo with transiently halted cardiac contraction as in (C) showed that kugel diameter still oscillates (time post cessation of flow).


Dextran microangiography filled perfused vessels with dextran ( arrowhead ) , while dextran was not observed to enter kugeln ( unfilled arrowhead ) .

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

Dextran microangiography filled perfused vessels with dextran (arrowhead), while dextran was not observed to enter kugeln (unfilled arrowhead).


Inhibition of cardiac contraction by tnnt2a morpholino ( MO ) knockdown statistically significantly reduced kugel number per ( * * * * p < 0 . 0001 ; control n = 20 5 . 10 ± 1 . 47 ( mean ± s . e . m . ) , tnnt2a MO = 18 0 . 06 ± 0 . 06 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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 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

Inhibition of cardiac contraction by tnnt2a morpholino (MO) knockdown statistically significantly reduced kugel number per embryo (****p<0.0001; control n=20 embryos 5.10 ± 1.47 (mean ± s.e.m.), tnnt2a MO=18 embryos 0.06 ± 0.06 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Kugeln were studied in double - transgenic Tg ( fli1a : LifeAct - mClover ) sh467 , Tg ( kdrl : HRAS - mCherry ) s916 . BLECs were mClover positive mCherry - negative ( BLECs - black arrowheads ; kugeln - arrowheads ) .

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

Kugeln were studied in the double-transgenic Tg(fli1a:LifeAct-mClover)sh467, Tg(kdrl:HRAS-mCherry)s916. BLECs were mClover positive and mCherry-negative (BLECs - black arrowheads; kugeln - white arrowheads).


No direct physical interaction between kugeln ( BLECs - black arrowheads ; kugeln - arrowheads ) BLECs was observed ( n = 21 4dpf ; 3 experimental repeats ) . Depth - coded MIPs showed that BLECs kugeln are present on different anatomical planes ( depths ; purple ventral ( v ) , dorsal ( d ) ; BLECs - black arrowheads ; kugeln - arrowheads ) .

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

No direct physical interaction between kugeln (BLECs - black arrowheads; kugeln - white arrowheads) and BLECs was observed (n=21 4dpf embryos; 3 experimental repeats). Depth-coded MIPs showed that BLECs and kugeln are present on different anatomical planes (depths; purple ventral (v), white dorsal (d); BLECs - black arrowheads; kugeln - white arrowheads).


Ccbe1 morpholino ( MO ) injection led to a loss of lymphatics ( arrowhead ) .

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

Ccbe1 morpholino (MO) injection led to a loss of lymphatics (white arrowhead).


Kugel number was not statistically significantly altered by ccbe1 morpholino ( MO ) knockdown ( p = 0 . 3496 ; control MO n = 22 0 . 95 ± 0 . 28 ( mean ± s . e . m . ) , ccbe1 MO n = 23 0 . 57 ± 0 . 14 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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 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

Kugel number was not statistically significantly altered by ccbe1 morpholino (MO) knockdown (p = 0.3496; control MO n=22 embryos 0.95 ± 0.28 (mean ± s.e.m.), ccbe1 MO n=23 embryos 0.57 ± 0.14 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Kugel diameter was not statistically significantly altered by ccbe1 MO knockdown ( p = 0 . 8783 ; control MO n = 21 kugeln from 22 7 . 75 ± 1 . 29 ( mean ± s . e . m . ) , ccbe1 MO n = 13 kugeln from 23 8 . 93 ± 2 . 35 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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 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

Kugel diameter was not statistically significantly altered by ccbe1 MO knockdown (p = 0.8783; control MO n=21 kugeln from 22 embryos 7.75 ± 1.29 (mean ± s.e.m.), ccbe1 MO n=13 kugeln from 23 embryos 8.93 ± 2.35 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Injection of IgG - conjugated Alexa 647 into tectum showed no uptake ( blue ; unfilled arrowhead ) by kugeln ( arrowhead ; 140 kugeln from 17 4dpf ; 2 experimental repeats ) .

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

Injection of IgG-conjugated Alexa 647 into the tectum showed no uptake (blue; unfilled arrowhead) by kugeln (white arrowhead; 140 kugeln from 17 4dpf embryos; 2 experimental repeats).


possible interaction of macrophages with kugeln was studied in transgenic Tg ( kdrl : HRAS - mCherry ) s916 , Tg ( fms : GAL4 . VP16 ) i186 , Tg ( UAS - E1b : nfsB . mCherry ) il149 , visualizing macrophages vasculature simultaneously . Coloured depth - coding indicates different anatomical depths of kugeln macrophages ( ventral purple , dorsal ) .

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

The possible interaction of macrophages with kugeln was studied in the transgenic Tg(kdrl:HRAS-mCherry)s916, Tg(fms:GAL4.VP16)i186, Tg(UAS-E1b:nfsB.mCherry)il149, visualizing macrophages and the vasculature simultaneously. Coloured depth-coding indicates the different anatomical depths of kugeln and macrophages (ventral purple, dorsal white).


Maximum projections of z - stacks acquired every 10 minutes during a time - lapse showed no interaction of macrophages ( unfilled arrowhead ) with kugeln ( filled arrowhead ; n = 21 kugeln in 8 3dpf ) . Macrophages are , indicating they are on a different z - plane to vessels seen .

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

Maximum intensity projections of z-stacks acquired every 10 minutes during a time-lapse showed no interaction of macrophages (unfilled arrowhead) with kugeln (filled arrowhead; n=21 kugeln in 8 3dpf embryos). Macrophages are all white, indicating they are on a different z-plane to the vessels seen.


VEGF inhibition by 2h treatment with AV951 statistically significantly increased kugel number ( * * * * p < 0 . 0001 ; DMSO control n = 30 8 . 53 ± 2 . 62 ( mean ± s . e . m . ) ; AV951 n = 31 21 . 10 ± 2 . 71 ( mean ± s . e . m . ) ; 4dpf ; 4 experimental repeats ; Mann - Whitney U ) .

B-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 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

VEGF inhibition by 2h treatment with AV951 statistically significantly increased kugel number (****p<0.0001; DMSO control n=30 embryos 8.53 ± 2.62 (mean ± s.e.m.); AV951 n=31 embryos 21.10 ± 2.71 (mean ± s.e.m.); 4dpf; 4 experimental repeats; Mann-Whitney U test).


Mean kugel diameter was not statistically significantly different after AV951 treatment ( p = 0 . 7890 ; DMSO control n = 243 kugeln from 30 9 . 94 ± 0 . 76 ( mean ± s . e . m . ) ; AV951 n = 652 kugeln from 31 9 . 73 ± 0 . 32 ( mean ± s . e . m . ) ; 4dpf ; 4 experimental repeats ; Student ' s t - ) .

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 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

Mean kugel diameter was not statistically significantly different after AV951 treatment (p = 0.7890; DMSO control n=243 kugeln from 30 embryos 9.94 ± 0.76 (mean ± s.e.m.); AV951 n=652 kugeln from 31 embryos 9.73 ± 0.32 (mean ± s.e.m.); 4dpf; 4 experimental repeats; Student's t-test).


DAF - FM , a vital dye for nitric oxide ( NO ) , showed that 57 . 56 % of kugeln were positive for nitric oxide reactivity ( 2 . 5µM incubation from 96 - 102hpf ; n = 22 4dpf ; 118 of 205 kugeln filled ; 3 experimental repeats ) . show representative " filled " ( DAF - FM positive ) " unfilled " ( DAF - FM negative ) kugeln .

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

DAF-FM staining, a vital dye for nitric oxide (NO), showed that 57.56% of kugeln were positive for nitric oxide reactivity (2.5µM incubation from 96-102hpf; n=22 4dpf embryos; 118 of 205 kugeln filled; 3 experimental repeats). Images show representative "filled" (DAF-FM positive) and "unfilled" (DAF-FM negative) kugeln.


Time - lapse acquisition with DAF - FM revealed that kugeln contained NO early in their biogenesis ( grey LUT ; inverted ) .

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

Time-lapse acquisition with DAF-FM revealed that kugeln contained NO early in their biogenesis (grey LUT; inverted).


Application of LysoTracker , a vital dye that stains lysosomes or acidic compartments , showed that 17 . 08 % of kugeln contained acidic contents ( 8 . 33µM ; 96 - 101hpf ; n = 22 4dpf ; 62 of 363 kugeln filled ; 3 experimental repeats ) . show representative " filled " ( Lysotracker positive ) " unfilled " ( Lysotracker negative ) kugeln .

Application of LysoTracker, a vital dye that stains lysosomes or acidic compartments, showed that 17.08% of kugeln contained acidic contents (8.33µM; 96-101hpf; n=22 4dpf embryos; 62 of 363 kugeln filled; 3 experimental repeats). Images show representative "filled" (Lysotracker positive) and "unfilled" (Lysotracker negative) kugeln.


nitric oxide synthase ( NOS ) inhibitor L - NAME had no statistically significant effect on kugel number per ( 0 . 5mM L - NAME 18h ; p = 0 . 4870 ; control n = 22 14 . 27 ± 3 . 35 ( mean ± s . e . m . ) , L - NAME n = 24 7 . 46 ± 1 . 61 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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 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 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

The nitric oxide synthase (NOS) inhibitor L-NAME had no statistically significant effect on kugel number per embryo (0.5mM L-NAME 18h; p = 0.4870; control n=22 embryos 14.27 ± 3.35 (mean ± s.e.m.), L-NAME n=24 embryos 7.46 ± 1.61 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Diameter of kugeln was not affected by L - NAME ( p = 0 . 4161 ; control n = 315 kugeln from 22 8 . 77 ± 0 . 49 ( mean ± s . e . m . ) , L - NAME n = 179 kugeln from 24 8 . 66 ± 0 . 71 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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 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

Diameter of kugeln was not affected by L-NAME (p = 0.4161; control n=315 kugeln from 22 embryos 8.77 ± 0.49 (mean ± s.e.m.), L-NAME n=179 kugeln from 24 8.66 ± 0.71 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Kugel number was significantly decreased by inhibition of Notch signalling by 12h treatment with 50µM DAPT ( * * * * p < 0 . 0001 ; control n = 24 9 . 38 ± 1 . 77 ( mean ± s . e . m . ) , DAPT n = 24 0 . 92 ± 0 . 28 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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

Kugel number was significantly decreased by inhibition of Notch signalling by 12h treatment with 50µM DAPT (****p<0.0001; control n=24 embryos 9.38 ± 1.77 (mean ± s.e.m.), DAPT n=24 embryos 0.92 ± 0.28 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Mean kugel diameter was not statistically significantly altered by DAPT treatment ( p = 0 . 0832 ; control n = 225 kugeln from 24 8 . 64 ± 0 . 54 ( mean ± s . e . m . ) ; DAPT n = 22 kugeln from 24 6 . 88 ± 0 . 86 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Student ' s t - ) .

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 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

Mean kugel diameter was not statistically significantly altered by DAPT treatment (p = 0.0832; control n=225 kugeln from 24 embryos 8.64 ± 0.54 (mean ± s.e.m.); DAPT n=22 kugeln from 24 embryos 6.88 ± 0.86 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Student's t-test).


A transgenic dll4 reporter showed no indication of different dll4 expression at sites of kugeln ( arrowheads pointing to kugeln ; n = 122 kugeln from 23 3dpf ; 2 experimental repeats ) .

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

A transgenic dll4 reporter line showed no indication of different dll4 expression at the sites of kugeln (white arrowheads pointing to kugeln; n=122 kugeln from 23 3dpf embryos; 2 experimental repeats).


Studying Notch signalling in Notch reporter Tg ( TP1glob : venusPest ) s940 , showed high Notch in mid - brain , but , again , no difference in expression at sites of kugeln ( n = 45 kugeln from 19 4dpf ; 2 experimental repeats ) .

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

Studying Notch signalling in the Notch reporter line Tg(TP1glob:venusPest)s940, showed high Notch levels in the mid-brain, but, again, no difference in expression at the sites of kugeln (n=45 kugeln from 19 4dpf embryos; 2 experimental repeats).


Kugel number was statistically significantly increased by inhibition of Wnt signalling by 4h treatment with 10µm XAV - 939 ( * * * p = 0 . 0003 ; control n = 22 1 . 32 ± 0 . 24 ( mean ± s . e . m . ) ; XAV - 939 n = 21 4 . 29 ± 0 . 71 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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

Kugel number was statistically significantly increased by inhibition of Wnt signalling by 4h treatment with 10µm XAV-939 (***p = 0.0003; control n=22 embryos 1.32 ± 0.24 (mean ± s.e.m.); XAV-939 n=21 embryos 4.29 ± 0.71 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Kugel diameter was not statistically significantly altered by XAV - 939 ( p = 0 . 4098 ; control n = 29 kugeln from 22 8 . 78 ± 0 . 76 ( mean ± s . e . m . ) ; XAV - 939 n = 90 kugeln from 21 7 . 99 ± 0 . 56 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Student ' s t - )

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 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

Kugel diameter was not statistically significantly altered by XAV-939 (p = 0.4098; control n=29 kugeln from 22 embryos 8.78 ± 0.76 (mean ± s.e.m.); XAV-939 n=90 kugeln from 21 embryos 7.99 ± 0.56 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Student's t-test)


Kugel number was statistically significantly increased by activation of Wnt signalling by 4h treatment with 10µm GSK3 inhibition XV ( p 0 . 0359 ; control n = 22 1 . 04 ± 0 . 34 ( mean ± s . e . m . ) ; GSK3 inhibitor n = 21 4 . 24 ± 1 . 28 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U ) .

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 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

Kugel number was statistically significantly increased by activation of Wnt signalling by 4h treatment with 10µm GSK3 inhibition XV (p 0.0359; control n=22 embryos 1.04 ± 0.34 (mean ± s.e.m.); GSK3 inhibitor n=21 embryos 4.24 ± 1.28 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


diameter of kugel was not statistically significantly altered by GSK3 inhibition ( p 0 . 5555 ; control n = 23 kugeln from 22 7 . 26 ± 0 . 89 ( mean ± s . e . m . ) ; GSK3 inhibitor n = 89 kugeln from 21 8 . 09 ± 0 .

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

The diameter of kugel was not statistically significantly altered by GSK3 inhibition (p 0.5555; control n=23 kugeln from 22 embryos 7.26 ± 0.89 (mean ± s.e.m.); GSK3 inhibitor n=89 kugeln from 21 embryos 8.09 ± 0.97


( C ) Co - immunoprecipitation between VP30 indicated PPxPxY containing host factors . Empty expression plasmid ( Vector ) or FLAG - tagged host expression plasmids were co - transfected with an HA - VP30 expression plasmid in HEK293T . An anti - FLAG immunoprecipitation ( IP : Anti - FLAG ) was performed . Western blots of IP whole lysates ( WCL ) are shown . Anti - β - tubulin was used as a loading control for WCLs .

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

(C) Co-immunoprecipitation between VP30 and the indicated PPxPxY containing host factors. Empty expression plasmid (Vector) or FLAG-tagged host protein expression plasmids were co-transfected with an HA-VP30 expression plasmid in HEK293T cells. An anti-FLAG immunoprecipitation (IP: Anti-FLAG) was performed. Western blots of IP and whole cell lysates (WCL) are shown. Anti-β-tubulin was used as a loading control for the WCLs.


( D ) were transfected with empty vector or HA - VP30 expression plasmid IPs were performed with anti - HA antibody ( IP : Anti - HA ) . Western blotting with anti - HA tag or antibodies to indicated host was performed on IP on whole lysates ( WCL ) .

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

(D) Cells were transfected with empty vector or HA-VP30 expression plasmid and IPs were performed with anti-HA antibody (IP: Anti-HA). Western blotting with anti-HA tag or antibodies to the indicated host proteins was performed on IP and on whole cell lysates (WCL).


( B ) Co - immunoprecipitation between HA - VP30 either GFP or GFP - fused to peptides derived from host was performed . Western blots of co - immunoprecipitation whole - lysate ( WCL ) are shown .

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

(B) Co-immunoprecipitation between HA-VP30 and either GFP or GFP-fused to peptides derived from the host proteins was performed. Western blots of the co-immunoprecipitation and whole-cell lysate (WCL) are shown.


( C ) Equilibrium dissociation curves of FITC - RBBP6 from eVP30130 - 272 in presence of increasing concentrations ( 0 . 13 - 500 μM ) of PPxPxY containing peptides . polarization was determined with constant concentrations of FITC - RBBP6 eVP30130 - 272 , at 0 . 50 μM 3 . 8 μM respectively . Experiments were performed in two independent replicates . Error bars represent standard deviation .

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 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

(C) Equilibrium dissociation curves of FITC-RBBP6 from eVP30130-272 in the presence of increasing concentrations (0.13-500 μM) of the PPxPxY containing peptides. Fluorescence polarization was determined with constant concentrations of FITC-RBBP6 and eVP30130-272, at 0.50 μM and 3.8 μM respectively. Experiments were performed in two independent replicates. Error bars represent standard deviation.


( A ) HDX kinetics data for VP30 alone for indicated host peptides incubated with VP30 are shown . VP30 peptide acid residue positions sequences are indicated at top of each curve . error bars indicate mean ± S . D . ( N = 3 ) .

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

(A) HDX kinetics data for VP30 alone and for the indicated host peptides incubated with VP30 are shown. VP30 peptide amino acid residue positions and sequences are indicated at the top of each curve. The error bars indicate mean ± S.D. (N=3).


( A ) Co - immunoprecipitation experiment demonstrating that NP interferes with VP30 - PPxPxY binding . VP30 was co - expressed with indicated HA - tagged PPxPxY either empty vector ( - ) or increasing amounts of FLAG - tagged NP expression plasmid . IPs were performed with anti - HA antibodies followed by Western blots of IPs or WCL by using anti - HA , anti - FLAG anti - VP30 antibodies .

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

(A) Co-immunoprecipitation experiment demonstrating that NP interferes with VP30-PPxPxY binding. VP30 was co-expressed with the indicated HA-tagged PPxPxY proteins and either empty vector (-) or increasing amounts of FLAG-tagged NP expression plasmid. IPs were performed with anti-HA antibodies followed by Western blots of IPs or WCL by using anti-HA, anti-FLAG and anti-VP30 antibodies.


( C ) Co - immunoprecipitations between VP30 indicated host upon titration of RBBP6 . VP30 was co - expressed with FLAG - tagged PPxPxY containing host either a 5 - fold or 10 - fold excess of RBBP6 plasmid . IP was performed using anti - FLAG beads followed by Western blotting using indicated antibodies .

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

(C) Co-immunoprecipitations between VP30 and the indicated host proteins upon titration of RBBP6. VP30 was co-expressed with FLAG-tagged PPxPxY containing host proteins and either a 5-fold or 10-fold excess of RBBP6 plasmid. IP was performed using anti-FLAG beads followed by Western blotting using the indicated antibodies.


( B ) Equilibrium dissociation curves of FITC - RBBP6 from eVP30130 - 272 as it is outcompeted by increasing concentrations ( 0 . 13 - 500 μM ) of RBBP6 alanine mutants . polarization was determined with constant concentrations of FITC - RBBP6 eVP30130 - 272 at 0 . 50 µM 3 . 8 µM respectively . Experiments were performed in two independent replicates . Error bars represent standard deviation .

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 B-CONTROLLED_VAR 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

(B) Equilibrium dissociation curves of FITC-RBBP6 from eVP30130-272 as it is outcompeted by increasing concentrations (0.13-500 μM) of RBBP6 alanine mutants. Fluorescence polarization was determined with constant concentrations of FITC-RBBP6 and eVP30130-272 at 0.50 µM and 3.8 µM respectively. Experiments were performed in two independent replicates. Error bars represent the standard deviation.


( D ) Minigenome ( MG ) activity upon titration of plasmids encoding GFP fused to wildtype or single point mutant - RBBP6 peptides . GFP alone or GFP - RBBP6 peptide plasmids were transfected at 12 . 5 25 ng amounts along with plasmids of MG system . data represent mean ± S . D . from one representative experiment in which each transfection condition was performed in triplicate ( n = 3 ) . experiment was reproduced in two additional , independent experiments ( see Appendix Figure S2 ) . Statistical significance was calculated using ANOVA with Tukey ' s multiple comparisons . * * * * p < 0 . 00005 ; * * * p < 0 .

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

(D) Minigenome (MG) activity upon titration of plasmids encoding GFP fused to wildtype or single point mutant-RBBP6 peptides. GFP alone or GFP-RBBP6 peptide plasmids were transfected at 12.5 and 25 ng amounts along with the plasmids of the MG system. The data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments (see Appendix Figure S2). Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005


( C ) MG activity upon knockdown of host genes . HEK293T were transfected with scrambled siRNA or siRNA targeting RBBP6 , hnRNP L , hnRNPUL1 or PEG10 . Twenty - four hours post - transfection , were transfected with MG plasmids including VP30 at 12 . 5 25ng doses . Data information : , data represent mean ± S . D . from one representative experiment in which each transfection condition was performed in triplicate ( n = 3 ) . experiment was reproduced in two additional , independent experiments Statistical significance was calculated using ANOVA with Tukey ' s multiple comparisons . * * * * p < 0 . 00005 ; * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 .

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 I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O 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) MG activity upon knockdown of host genes. HEK293T cells were transfected with scrambled siRNA or siRNA targeting RBBP6, hnRNP L, hnRNPUL1 or PEG10. Twenty-four hours post-transfection, cells were transfected with MG assay plasmids including VP30 at 12.5 and 25ng doses. Data information: , the data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005, **p<0.005, *p<0.05.


( A ) HeLa were transfected with scrambled siRNA ( SCR si ) or siRNAs targeting hnRNP L , hnRNPUL1 or PEG10 , 48 h post - transfection , were challenged with EBOV - EGFP at an MOI of 0 . 5 . 24 h post - infection , were fixed , stained for nuclei with Hoechst dye imaged . Scale bar is 200 µm . graph ( right ) depicts relative number of infected as determined by calculating of number of infected to nuclei shown relative to SCR siRNA . data shows a representative result of two independent experiments with mean ± S . D . calculated from three independently treated wells . Statistical significance was calculated relative to values obtained in SCR siRNA - treated using ANOVA with Dunnett ' s multiple comparisons . * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 .

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 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

(A) HeLa cells were transfected with scrambled siRNA (SCR si) or siRNAs targeting hnRNP L, hnRNPUL1 or PEG10, 48 h post-transfection, cells were challenged with EBOV-EGFP at an MOI of 0.5. 24 h post-infection, cells were fixed, stained for nuclei with Hoechst dye and imaged. Scale bar is 200 µm.The graph (right) depicts relative number of infected cells as determined by calculating the ratio of number of infected cells to nuclei and shown relative to SCR siRNA. The data shows a representative result of two independent experiments with the mean ± S.D. calculated from three independently treated wells. Statistical significance was calculated relative to the values obtained in SCR siRNA-treated cells using ANOVA with Dunnett's multiple comparisons test. ***p<0.0001, ***p<0.001, **p<0.01


( B ) of transcription replication in MG upon over - expression of indicated host factors . MG assays were performed with 50 500 ng of host expression plasmid . was isolated analyzed by qRT - PCR to measure of viral mRNA / cRNA vRNA . sample with vector control was set to 100 % mean ± S . D . values from two independent experiments are shown ( each experiment was performed in duplicate , n = 2 ) . * * p < 0 . 005 , * p < 0 . 05 . Statistical significance was calculated relative to vector control using ANOVA with Tukey ' s multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Levels of transcription and replication in the MG assay upon over-expression of the indicated host factors. MG assays were performed with 50 and 500 ng of host protein expression plasmid. RNA was isolated and analyzed by qRT-PCR to measure the levels of viral mRNA/cRNA and vRNA. The sample with vector control was set to 100% and mean ± S.D. values from two independent experiments are shown (each experiment was performed in duplicate, n=2). **p<0.005, *p<0.05. Statistical significance was calculated relative to vector control using ANOVA with Tukey's multiple comparisons test.


( C ) Immunoblots to assess VP30 pVP30 in lysates from MG upon knockdown of RBBP6 , hnRNP L or hnRNPUL1 .

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

(C) Immunoblots to assess VP30 and pVP30 levels in lysates from MG assay upon knockdown of RBBP6, hnRNP L or hnRNPUL1.


( D ) Immunoblots to assess of VP30 , pVP30 NP in EBOV infected lysates upon knockdown of NPC1 or RBBP6 . HeLa were mock transfected or transfected with siRNA targeting NPC1 or RBBP6 . 48 h post - transfection were infected with EBOV at MOI = 0 . 1 . At 24 h post - infection , were lysed in TRIzol . was extracted from TRIzol reagent analyzed by Western blotting to determine of VP30 , pSer29 VP30 NP .

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

(D) Immunoblots to assess the levels of VP30, pVP30 and NP in EBOV infected lysates upon knockdown of NPC1 or RBBP6. HeLa cells were mock transfected or transfected with siRNA targeting NPC1 or RBBP6. 48 h post-transfection cells were infected with EBOV at MOI=0.1. At 24 h post-infection, cells were lysed in TRIzol. Protein was extracted from the TRIzol reagent and analyzed by Western blotting to determine levels of VP30, pSer29 VP30 and NP.


( E ) Western blots detecting pVP30 , VP30 NP in EBOV infected HeLa upon hnRNP L , hnRNPUL1 or PEG10 knockdown .

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

(E) Western blots detecting pVP30, VP30 and NP levels in EBOV infected HeLa cells upon hnRNP L, hnRNPUL1 or PEG10 knockdown.


liposome permeabilization assays were performed with C - terminally His - tagged BakΔTM BclxLΔTM attached to Ni2 + - lipid doped MOM - like liposomes . ( a - d ) Liposome permeabilization assays detecting release of a dye from liposomes . ( a ) Pore formation of 50 nM BakΔTM ( red ) is activated by 20 nM cBid ( blue ) . 20 nM cBid alone ( orange ) shows no liposome permeabilization . Addition of 25 nM BclxLΔTM ( green ) inhibits pore - formation . ( b ) Increasing amounts of cBid are titrated to 50 nM BakΔTM in absence ( blue ) presence ( green ) of 25 nM BclxLΔTM . ( c , d ) BakΔTM becomes autoactive at increasing concentrations . concentrations are color coded as indicated in ( d ) . ( c ) averaged kinetics for increasing BakΔTM concentrations . Bar diagrams in ( b ) ( d ) represent data from kinetic experiments averaged between 9000 - 10000 s . standard deviation was calculated from three measurements . (

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

All liposome permeabilization assays were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (a-d) Liposome permeabilization assays detecting the release of a fluorescence dye from liposomes. (a) Pore formation of 50 nM BakΔTM (red) is activated by 20 nM cBid (blue). 20 nM cBid alone (orange) shows no liposome permeabilization. Addition of 25 nM BclxLΔTM (green) inhibits pore-formation. (b) Increasing amounts of cBid are titrated to 50 nM BakΔTM in the absence (blue) and presence (green) of 25 nM BclxLΔTM. (c,d) BakΔTM becomes autoactive at increasing concentrations. The concentrations are color coded as indicated in (d). (c) The averaged kinetics for increasing BakΔTM concentrations. Bar diagrams in (b) and (d) represent data from the kinetic experiments averaged between 9000 -10000 s. The standard deviation was calculated from three measurements. (


CD measurements were performed with C - terminally His - tagged BakΔTM BclxLΔTM attached to Ni2 + - lipid doped MOM - like liposomes . ( e ) Far - UV - CD spectra of BakΔTM + / - liposomes activators cBid or Puma - BH3 peptide as indicated .

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All CD measurements were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (e) Far-UV-CD spectra of BakΔTM +/- liposomes and the activators cBid or Puma-BH3 peptide as indicated.


CD measurements were performed with C - terminally His - tagged BakΔTM BclxLΔTM attached to Ni2 + - lipid doped MOM - like liposomes . ( f ) CD - detected thermal melting experiments of BakΔTM ( black ) with liposomes ( red ) Puma - BH3 ( blue ) .

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All CD measurements were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes. (f) CD-detected thermal melting experiments of BakΔTM (black) with liposomes (red) and Puma-BH3 (blue).


( c , d ) Liposome permeabilization comparing variants in ( a , green ) ( b , blue ) to wild - type BakΔTM ( black ) . ( c ) Comparison of 50 nM of wild - type BakΔTM ( black ) to two variants after activation by 25 nM cBid . ( d ) Bar diagram comparing behavior of BakΔTM variants alone in presence of 25 nM cBid 25 nM BclxLΔTM . data derived from liposome permeabilization kinetics shown in panel ( c ) is averaged for time interval of 9000 - 10000 s standard deviations were derived from three technical replicates . (

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(c, d) Liposome permeabilization assay comparing the variants in (a, green) and (b, blue) to wild-type BakΔTM (black). (c) Comparison of 50 nM of wild-type BakΔTM (black) to the two variants after activation by 25 nM cBid. (d) Bar diagram comparing the behavior of the BakΔTM variants alone and in the presence of 25 nM cBid and 25 nM BclxLΔTM. The data derived from the liposome permeabilization kinetics shown in panel (c) is averaged for the time interval of 9000-10000 s and the standard deviations were derived from three technical replicates. (


A . HeLa , H1299 A549 were infected with lentiviruses expressing control shRNA or lncRNA - MIF shRNA . Forty - eight hours after infection , lysates were subjected to Western blot with anti - c - Myc antibody .

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A. HeLa, H1299 and A549 cells were infected with lentiviruses expressing control shRNA or lncRNA-MIF shRNA. Forty-eight hours after infection, cell lysates were subjected to Western blot analysis with anti-c-Myc antibody.


C . Total from indicated lines was subjected to Northern blot to determine molecular of lncRNA - MIF . Actin was also included as a positive control .

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C. Total RNA from the indicated cell lines was subjected to Northern blot analysis to determine the molecular size of lncRNA-MIF. Actin was also included as a positive control.


D . HeLa were infected with lentiviruses expressing control , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , lysates were analyzed by Western blotting with anti - c - Myc anti - GAPDH antibodies .

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D. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with anti-c-Myc and anti-GAPDH antibodies.


E . HeLa were infected with lentiviruses expressing control , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , total was extracted from these subjected to real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

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E. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, total RNA was extracted from these cells and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


F . HeLa expressing either control or lncRNA - MIF were treated with or without MG132 ( 20 μM ) for 6 h . lysates were analyzed by Western blot with indicated antibodies .

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F. HeLa cells expressing either control RNA or lncRNA-MIF were treated with or without MG132 (20 μM) for 6 h. Cell lysates were analyzed by Western blot with the indicated antibodies.


G . HeLa expressing either control shRNA or lncRNA - MIF shRNA were treated with MG132 for 6 h . lysates were analyzed by Western blotting with indicated antibodies .

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G. HeLa cells expressing either control shRNA or lncRNA-MIF shRNA were treated with MG132 for 6 h. Cell lysates were analyzed by Western blotting with the indicated antibodies.


H . HeLa were infected with lentiviruses expressing control , lncRNA - MIF or lncRNA - MIF - AS as indicated . Forty - eight hours after infection , were treated with cycloheximide ( CHX , 50 μg / ml ) for indicated periods of time . lysates were then analyzed by Western blotting to examine c - Myc half - life . band were analyzed by ImageJ . I . Band of c - Myc in Fig 1H was analyzed by ImageJ .

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

H. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, cells were treated with cycloheximide (CHX, 50 μg/ml) for the indicated periods of time. Cell lysates were then analyzed by Western blotting to examine the c-Myc protein half-life. Protein band intensity were analyzed by ImageJ.I. Band intensity of c-Myc in Fig 1H was analyzed by ImageJ.


J . HeLa were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 as indicated . Forty - eight hours after infection , were treated with CHX for indicated periods of time . lysates were then analyzed by Western blotting to examine half - life of c - Myc . K . Band of c - Myc in Fig 1J was analyzed by ImageJ .

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J. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3 as indicated. Forty-eight hours after infection, cells were treated with CHX for the indicated periods of time. Cell lysates were then analyzed by Western blotting to examine the half-life of c-Myc protein.K. Band intensity of c-Myc in Fig 1J was analyzed by ImageJ.


A . P493 - 6 ( human B lymphoma ) carrying a c - Myc tet - off system were treated with Doxycycline ( 1 μg / ml ) for indicated periods of time . Total was subjected to real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - t - ) . lysates were also analyzed by Western blotting with indicated antibodies .

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A. P493-6 cells (human B cell lymphoma cell line) carrying a c-Myc tet-off system were treated with Doxycycline (1 μg/ml) for the indicated periods of time. Total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test). Cell lysates were also analyzed by Western blotting with the indicated antibodies.


B . HeLa , HCT116 MCF10A were infected with lentiviruses expressing control shRNA or c - Myc shRNA . Forty - eight hours after infection , total lysates were analyzed by real - time RT - PCR Western blotting , respectively . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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B. HeLa, HCT116 and MCF10A cells were infected with lentiviruses expressing control shRNA or c-Myc shRNA. Forty-eight hours after infection, total RNA and cell lysates were analyzed by real-time RT-PCR and Western blotting, respectively. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).


C . HeLa , HCT116 MCF10A were transfected with control vector or Flag - c - Myc . Twenty - four hours after transfection , total was extracted from these subjected to real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - t - ) . lysates were also analyzed by Western blotting .

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C. HeLa, HCT116 and MCF10A cells were transfected with control vector or Flag-c-Myc. Twenty-four hours after transfection, total RNA was extracted from these cells and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test). Cell lysates were also analyzed by Western blotting.


E . HeLa were co - transfected with either Flag - c - Myc or control vector plus indicated reporter constructs Renilla luciferase plasmid . Twenty - four hours after transfection , reporter activity was measured plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - t - ) .

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E. HeLa cells were co-transfected with either Flag-c-Myc or control vector plus the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


F . HeLa expressing control shRNA or c - Myc shRNA were co - transfected with indicated reporter constructs Renilla luciferase plasmid . Twenty - four hours after transfection , reporter activity was measured plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - t - ) .

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F. HeLa cells expressing control shRNA or c-Myc shRNA were co-transfected with the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


G . P493 - 6 were co - transfected with indicated reporter constructs Renilla luciferase plasmid . Twenty - four hours after trasfection , were treated with doxycycline for 0 hour 12 hours respectively . Then reporter activity was measured plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - t - ) .

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G. P493-6 cells were co-transfected with the indicated reporter constructs and Renilla luciferase plasmid. Twenty-four hours after trasfection, cells were treated with doxycycline for 0 hour and 12 hours respectively. Then reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


H . Lysates from HeLa were subjected to ChIP . ChIP products were amplified by qPCR reaction with indicated pairs of primers . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

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H. Lysates from HeLa cells were subjected to ChIP assay. ChIP products were amplified by qPCR reaction with the indicated pairs of primers. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


A . Lysates from HeLa were incubated with in vitro synthesized biotin - labeled sense or antisense probes against lncRNA - MIF for biotinpull - down , followed by Western blot with indicated antibodies . Lysates from HeLa were incubated with in vitro synthesized biotin - labeled lncRNA - MIF or biotin - labeled antisense for biotinpull - down , followed by Western blot with indicated antibodies .

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A. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled sense or antisense DNA probes against lncRNA-MIF for biotinpull-down assay, followed by Western blot analysis with the indicated antibodies. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled lncRNA-MIF or biotin-labeled antisense RNA for biotinpull-down assay, followed by Western blot analysis with the indicated antibodies.


B . HeLa were infected with lentiviruses expressing control , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , lysates were analyzed by Western blotting with indicated antibodies .

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B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with the indicated antibodies.


C . HeLa were infected with lentiviruses expressing control , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , total was subjected to real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).


E . Lysates from HeLa were incubated with in vitro synthesized biotin - labeled sense or antisense probes against lncRNA - MIF for biotinpull - down , followed by real - time RT - PCR to examine miR - 586 . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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E. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled sense or antisense DNA probes against lncRNA-MIF for biotinpull-down assay, followed by real-time RT-PCR analysis to examine miR-586 levels. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).


F . Lysates from HeLa were incubated with in vitro synthesized biotin - labeled lncRNA - MIF antisense for biotinpull - down , followed by real - time RT - PCR to examine miR - 586 miR - 34a . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - t - ) .

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F. Lysates from HeLa cells were incubated with in vitro synthesized biotin-labeled lncRNA-MIF and antisense RNA for biotinpull-down assay, followed by real-time RT-PCR analysis to examine miR-586 and miR-34a levels. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


H . miR - 586 or control mimics were transfected into HeLa together with indicated pSICHECK2 - luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

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H. miR-586 or control mimics were transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


I . miR - 586 or control inhibitors were transfected into HeLa together with indicated pSICHECK2 - luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

I. miR-586 or control inhibitors were transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


J . HeLa were subjected to fractionation into ( C ) ( N ) extracts . Total extracted from each fraction was analyzed for indicated expression by real - time RT - PCR . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) . Actin PARP were used as markers for fractions , respectively .

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J. HeLa cells were subjected to fractionation into cytoplasmic (C) and nuclear (N) extracts. Total RNA extracted from each fraction was analyzed for the indicated RNA expression by real-time RT-PCR. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test). Actin and PARP were used as markers for cytoplasmic and nuclear fractions, respectively.


K . HeLa were subjected to cytoplasm or nucleus fractionation before each fraction was incubated with in vitro synthesized biotin - labeled sense or antisense probes of lncRNA - MIF for biotinpull - down , followed by real - time RT - PCR to examine miR - 586 . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , * * P < 0 . 01 , two - t - ) .

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K. HeLa cells were subjected to cytoplasm or nucleus fractionation before each fraction was incubated with in vitro synthesized biotin-labeled sense or antisense DNA probes of lncRNA-MIF for biotinpull-down assay, followed by real-time RT-PCR analysis to examine miR-586 levels. Data shown are mean ± SD (n = 3; *P < 0.05, **P < 0.01, two-tailed t-test).


B . miR - 586 mimics or negative control mimics ( NC mimics ) were transfected into HeLa together with indicated pSICHECK2 - luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

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 O O O O O O O

B. miR-586 mimics or negative control mimics (NC mimics) were transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


C . miR - 586 inhibitor or negative control inhibitor ( NC inhibitor ) was transfected into HeLa together with indicated pSICHECK2 - luciferase reporter construct . Twenty - four hours after transfection , reporter activity was measured plotted after normalizing with respect to Renilla luciferase activity . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - t - ) .

C. miR-586 inhibitor or negative control inhibitor (NC inhibitor)was transfected into HeLa cells together with the indicated pSICHECK2-based luciferase reporter construct. Twenty-four hours after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


D . HeLa were transfected with miR - 586 mimics or inhibitors as indicated . Forty - eight hours after transfection , lysates were analyzed by Western blotting .

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D. HeLa cells were transfected with miR-586 mimics or inhibitors as indicated. Forty-eight hours after transfection, cell lysates were analyzed by Western blotting.