B . Representative negative stain class averages of PlexAecto correlate well with ring - like conformation of mouse PlxnA1 . ring - like conformation of mouse PlxnA1 is shown in surface representation rainbow colour ramped from blue ( N terminus ) to red ( C terminus ) . Scale bar , 10 nm .

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B. Representative negative stain class averages of PlexAecto correlate well with the ring-like conformation of mouse PlxnA1. The ring-like conformation of mouse PlxnA1 is shown in surface representation rainbow colour ramped from blue (N terminus) to red (C terminus). Scale bar, 10 nm.


E . In vitro FRET - FLIM measurement of PlexA - FRET Biosensor shows that PlexA ectodomain undergoes a conformational change upon treatment with Sema1b resulting in more open conformation . boxcharts represent average lifetime . Box limits indicate 25th 75th percentiles , centred lines show median , squares represent sample means , whiskers extend 1 . 5 - fold interquartile range from 25th 75th percentiles , p - value was calculated by one - way of variance ( ANOVA ) . * * * P < 0 . 001 .

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E. In vitro FRET-FLIM measurement of the PlexA-FRET Biosensor shows that the PlexA ectodomain undergoes a conformational change upon treatment with Sema1b resulting in more open conformation. The boxcharts represent the average lifetime. Box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, p-value was calculated by one-way analysis of variance (ANOVA). ***P<0.001.


F . Relative change of COS - 7 surface upon treatment with Sema1a - Fc . expressing PlexAFL - mClover or PlexAFL - mCover Sema1b - mRuby2 wild - type or mutants were treated with purified Sema1a - Fc at a final of 5 . 8 µM . were acquired every minute for 30 minutes . surface was calculated using ImageJ before after stimulation . Data are presented as means ± . * P < 0 . 05 , * * * P < 0 . 001 , p - value calculated by one - way of variance ( ANOVA ) .

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F. Relative change of COS-7 cells surface area upon treatment with Sema1a-Fc. Cells expressing PlexAFL-mClover or PlexAFL-mCover and Sema1b-mRuby2 wild-type or mutants were treated with purified Sema1a-Fc at a final concentration of 5.8 µM. Images were acquired every minute for 30 minutes. Cell surface area was calculated using ImageJ before and after stimulation. Data are presented as means ± sem. *P<0.05, ***P<0.001, p-value calculated by one-way analysis of variance (ANOVA).


A . Cultured dorsal root ganglion ( DRG ) neurons from E12 . 5 Sema6A knockout ( KO ) were transfected with EGFP , EGFP - tagged wild - type ( WT ) Sema6A ( Sema6A - WT ) or different EGFP - tagged Sema6A mutants ( Sema6A - mutA , Sema6A - mutB , Sema6A - mutA + B ) at 1 day in vitro ( DIV1 ) treated with purified Sema6A - Fc at a of 75 nM for 1 hour at 37 ° C at DIV4 . Transfection of EGFP was used as a control ( full collapse ) . DRG neurons from Sema6A KO acquired sensitivity to Sema6A growth cone collapse was observed in three independent experiments . Transfection of Sema6A - WT , Sema6A - mutA Sema6A - mutB but not of EGFP or Sema6A - mutantA + B prevented Sema6A - mediated growth cone collapse ( Fisher ' s exact p < 0 . 0001 ) . of growth cone collapse was performed using a growth cone matrix ( lower part panel A ) . Data is presented as of morphologically distinct growth cones , divided ( represented by dotted ) in two categories , from uncollapsed ( 1 - 4 ) to fully collapsed ( 5 - 8 ) . Totally , 20 - 40 growth cones were analyzed for each condition per experiment ( n = 3 experiments ) . Scale bar 20 µm .

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A. Cultured dorsal root ganglion (DRG) neurons from E12.5 Sema6A knockout (KO) embryos were transfected with EGFP, EGFP-tagged wild-type (WT) Sema6A (Sema6A-WT) or different EGFP-tagged Sema6A mutants (Sema6A-mutA, Sema6A-mutB, and Sema6A-mutA+B) at 1 day in vitro (DIV1) and treated with purified Sema6A-Fc at a concentration of 75 nM for 1 hour at 37°C at DIV4. Transfection of EGFP was used as a control (full collapse). DRG neurons from Sema6A KO embryos acquired sensitivity to Sema6A and growth cone collapse was observed in three independent experiments. Transfection of Sema6A-WT, Sema6A-mutA and Sema6A-mutB but not of EGFP or Sema6A-mutantA+B prevented Sema6A-mediated growth cone collapse (Fisher's exact test p<0.0001). Quantification of growth cone collapse was performed using a growth cone morphology matrix (lower part panel A). Data is presented as percentage of morphologically distinct growth cones, divided (represented by the dotted line) in two categories, from uncollapsed (1-4) to fully collapsed (5-8). Totally, 20-40 growth cones were analyzed for each condition per experiment (n=3 experiments). Scale bar 20 µm.


B . Representative of growth cones of mouse DRG neurons transfected with WT or mutant constructs treated with 75 nM Sema6A - Fc visualized with immunofluorescent to detect GFP ( green ) phalloidin ( red ) . Average for different constructs are shown in grey color . Scale bar 20 µm .

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B. Representative images of growth cones of mouse DRG neurons transfected with WT or mutant constructs treated with 75 nM Sema6A-Fc and visualized with immunofluorescent staining to detect GFP (green) and phalloidin (red). Average intensity images for all different constructs are shown in grey color. Scale bar 20 µm.


( A ) Hep3B hepatocytes were treated with nontargeting control siRNA ( siNT ) or siRNA targeting Dyn2 ( siDyn2 ) , followed by re - expression of either vector alone , or GFP - tagged versions of Dyn2 , loaded with 150 µM oleate overnight starved for 48 h in medium containing 0 . 1 % FBS . LDs were visualized by immunofluorescence using Oil Red O . boundaries are outlined those re - expressing GFP , GFP - wtDyn2 , or GFP - - K44A are denoted with asterisks . Bars , 20 µM . ( B ) Representative blot showing efficiency of Dyn2 knockdown in these . ( C D ) Quantitation of average LD number ( in pixels2 ) per from three independent experiments . data are represented as mean ± SE . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 . NS , not significant .

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(A) Hep3B hepatocytes were treated with nontargeting control siRNA (siNT) or siRNA targeting Dyn2 (siDyn2), followed by re-expression of either vector alone, or GFP-tagged versions of Dyn2, loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS. LDs were visualized by immunofluorescence using Oil Red O staining. Cell boundaries are outlined and those cells re-expressing GFP, GFP-wtDyn2, or GFP--K44A are denoted with asterisks. Bars, 20 µM. (B) Representative blot showing the efficiency of the Dyn2 knockdown in these cells. (C and D) Quantitation of the average LD number and area (in pixels2) per cell from three independent experiments. The data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. NS, not significant.


HuH - 7 ( A ) Hep3B ( B ) hepatocytes were loaded with 150 µM oleate overnight starved for 48 h in medium containing 0 . 1 % FBS in presence of Dyn2 inhibitors or DMSO as indicated . Representative of inhibitor - treated control ( stained with Oil Red O ) are shown in A B together with quantitation of average LD per from three independent experiments . Pharmacological inhibitors used were : Dynasore ( inhibits Dyn2 GTPase activity ) , MiTMAB ( targets PH domain interferes with binding ) , Dynole 34 - 2 ( allosteric GTPase inhibitor ) , Dynole 31 - 2 ( negative control for Dynole 34 - 2 ) . Bars , 20 µM .

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HuH-7 (A) and Hep3B (B) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS in the presence of Dyn2 inhibitors or DMSO as indicated. Representative images of inhibitor-treated and control cells (stained with Oil Red O) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments. Pharmacological inhibitors used were: Dynasore (inhibits Dyn2 GTPase activity), MiTMAB (targets PH domain and interferes with membrane binding), Dynole 34-2 (allosteric GTPase inhibitor), and Dynole 31-2 (negative control for Dynole 34-2). Bars, 20 µM.


( C ) Representative from control Dyn2 knockout MEFs after an overnight lipid loading with 400 µM Dyn2 for 17 h . Knockout of Dyn2 was induced by treatment with 2 µM 4 - hydroxy - tamoxifen for 7 d was confirmed by immunostaining of endogenous Dyn2 ( top row ) by immunoblot ( D ) . Bars , 20 µM . ( E F ) Average LD number ( E ) ( F ) per from five independent experiments . data are represented as mean ± SE . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 .

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(C) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM Dyn2 for 17 h. Knockout of Dyn2 was induced by treatment with 2 µM 4-hydroxy-tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 (top row) and by immunoblot (D). Bars, 20 µM. (E and F) Average LD number (E) and area (F) per cell from five independent experiments. All data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01.


( G ) Whole - lysates LD fractions isolated from HuH - 7 hepatocytes under resting or starved ( 2 h HBSS starvation ) conditions .

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(G) Whole-cell lysates and LD fractions isolated from HuH-7 hepatocytes under resting or starved (2 h HBSS starvation) conditions.


( H ) hepatocyte expressing Dyn2 - GFP , showing an absence of colocalization with LD surface ( stained with Oil Red O ) . Bar , 20 µM . Inset shows magnification of boxed region ( bar , 2 µM ) .

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(H) Primary hepatocyte expressing Dyn2-GFP, showing an absence of colocalization with the LD surface (stained with Oil Red O). Bar, 20 µM. Inset shows magnification of boxed region (bar, 2 µM).


Hep3B treated with either a nontargeting control siRNA ( A B , siNT ) or an siRNA targeting human Dyn2 ( C D , siDyn2 ) were fixed co - stained with antibodies specific for LAMP1 ( red ) LC3 ( green ) . After Dyn2 knockdown , a juxtanuclear aggregation enlargement of LAMP1 - positive compartment is observed ( C D , arrows ) . Increased labeling of LC3 is also detectable after knockdown of Dyn2 .

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Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and co-stained with antibodies specific for LAMP1 (red) and LC3 (green). After Dyn2 knockdown, a juxtanuclear aggregation and enlargement of the LAMP1-positive compartment is observed (C and D, arrows). Increased labeling of LC3 is also detectable after knockdown of Dyn2.


( E ) Western blotting of Hep3B lysates after a 3 - d treatment with either control or Dyn2 - targeted siRNA further treatment with or without 50 µM leupeptin . Densitometry - of six independent experiments is shown at bottom of figure .

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(E) Western blotting of Hep3B lysates after a 3-d treatment with either the control or Dyn2-targeted siRNA and further treatment with or without 50 µM leupeptin. Densitometry-based analysis of six independent experiments is shown at the bottom of the figure.


( F ) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore , in presence or absence of 50 µM leupeptin . Quantitation of LC3 - II relative to control are shown below blots . data are represented as mean ± SE ; * , P ≤ 0 . 05 .

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(F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.


Dyn2 knockdown results in formation of enlarged autophagic structures . ( A - D ) Transmission electron ( TEMs ) of oleate - loaded Hep3B hepatocytes treated with nontargeting control ( siNT ) or Dyn2 ( siDyn2 ) siRNA for 72 h . Bars : ( A B ) 2 µm ; ( A ′ B ′ ) 1 µm ; ( C D ) 0 . 5 µm . Insets in A B show of LAMP1 - stained ( bars , 10 µM ) . Control ( A ) contain an abundance of small ( < 1 µm ) electron - dense lysosomes autolysosomes ( arrowheads ) . Under conditions in which Dyn2 expression is suppressed ( B - D ) , far fewer small lysosomes are observed . Instead , are populated by larger autolysosomes ( ≥ 1 µm ) with aberrant morphologies containing putative LDs ( * ) . ( E ) Quantitative measure of autolysosomal from in which Dyn2 was knocked down as compared with control . Values represent fold - increase in number of autophagic structures of a given diameter ( measured in microns ) observed over siNT - treated control . Data were obtained from a single experiment with n = 6 examined by electron microscopy from each condition .

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Dyn2 knockdown results in the formation of enlarged autophagic structures. (A-D) Transmission electron micrographs (TEMs) of oleate-loaded Hep3B hepatocytes treated with nontargeting control (siNT) or Dyn2 (siDyn2) siRNA for 72 h. Bars: (A and B) 2 µm; (A′ and B′) 1 µm; (C and D) 0.5 µm. Insets in A and B show fluorescent micrographs of LAMP1-stained cells (bars, 10 µM). Control cells (A) contain an abundance of small (<1 µm) electron-dense lysosomes and autolysosomes (arrowheads). Under conditions in which Dyn2 expression is suppressed (B-D), far fewer small lysosomes are observed. Instead, the cells are populated by larger autolysosomes (≥1 µm) with aberrant morphologies and containing putative LDs (*). (E) Quantitative measure of autolysosomal size from cells in which Dyn2 was knocked down as compared with control cells. Values represent the fold-increase in the number of autophagic structures of a given diameter (measured in microns) observed over siNT-treated control cells. Data were obtained from a single experiment with n = 6 cells examined by electron microscopy from each condition.


Hep3B treated with either a nontargeting control siRNA ( A B , siNT ) or an siRNA targeting human Dyn2 ( C D , siDyn2 ) were fixed stained with an antibody specific for LAMP1 after a 24 - h starvation period in media containing 0 . 1 % FBS . Control displayed normal lysosomal compartments , with LAMP1 - stained structures dispersed throughout cytosol , some containing short reformation tubules ( B ′ ) . After Dyn2 knockdown , many contained enlarged autolysosomes with a noticeable increase in persistent lengthy reformation tubules emanating from LAMP1 - positive compartments ( C ′ D ′ ) . Bars : ( A - D ) 20 µM ; ( A ′ B ′ ) 5 µM ; ( C ′ D ′ ) 10 µM . ( E F ) Measurements of ( E ) distribution ( F ) of LAMP1 - stained ALR tubules in control ( siNT ) siDyn2 - treated . Data represent mean derived from measurements of tubules in a minimum of 40 for each experimental group over 6 independent experiments . Error bars represent SE ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 .

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Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and stained with an antibody specific for LAMP1 after a 24-h starvation period in media containing 0.1% FBS. Control cells displayed normal lysosomal compartments, with LAMP1-stained structures dispersed throughout the cytosol, some containing short reformation tubules (B′). After Dyn2 knockdown, many cells contained enlarged autolysosomes with a noticeable increase in persistent and lengthy reformation tubules emanating from LAMP1-positive compartments (C′ and D′). Bars: (A-D) 20 µM; (A′ and B′) 5 µM; (C′ and D′) 10 µM. (E and F) Measurements of the length (E) and distribution (F) of LAMP1-stained ALR tubules in control (siNT) and siDyn2-treated cells. Data represent the mean derived from measurements of tubules in a minimum of 40 cells for each experimental group over 6 independent experiments. Error bars represent SE; *, P ≤ 0.05; **, P ≤ 0.01.


( G ) of siDyn2 - treated Hep3B hepatocyte exhibiting enlarged autolysosomal structures with extensive tubulation ( arrows ) . Bar , 2 µm . ( G ′ ) Inset showing enlargement of autolysosomal tubule ( arrows ) . Bar , 1 µm . ( H ) Tubulating autolysosome with engulfed lipid droplet . Bar , 0 . 5 µm .

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(G) TEM of siDyn2-treated Hep3B hepatocyte exhibiting enlarged autolysosomal structures with extensive tubulation (arrows). Bar, 2 µm. (G′) Inset showing enlargement of autolysosomal tubule (arrows). Bar, 1 µm. (H) Tubulating autolysosome with engulfed lipid droplet. Bar, 0.5 µm.


Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation ( ALR ) lysosomal tubule scission . ( A - D ) Still frames from time - lapse movies of Hep3B expressing LAMP1 - mCherry . were starved for 2 h in HBSS subsequently treated for 30 min with either DMSO ( A B ) or 40 µM Dynasore ( C D ) , which induced extensive tubulation of LAMP1 - positive compartments . Bars ( A - D ) : 20 µM ; ( A ′ - B ′ ) 2 µM ; ( C ′ - D ′ ) 10 µM . ( E - G ) To demonstrate reversibility of this tubulation , Dynasore - treated were washed extensively with drug - free media containing 10 % FBS monitored by time - lapse microscopy for 45 min . Frequently , after drug washout , LAMP1 - positive tubules exhibited noticeable varicosities ( E F , arrows ; bars , 10 µM ) along their . These sites are suggestive of areas of scission resumed budding of nascent protolysosomes from reformation tubules ( G ; bars , 10 µM ) . ( H ) Tubules from undergoing drug washout were quantified by tracing their lengths at beginning end of these movies . Still frames from a representative movie show tubule content at t = 10 45 min after drug washout . Bars , 20 µM . ( I ) of five independent movies showed an average decrease in total tubulation of ∼ 50 % after drug washout . Data represent average relative change in total tubule between first last frames of time - lapse movies . Error bars represent SE ; * , P < 0 . 05 .

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Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. (A-D) Still frames from time-lapse movies of Hep3B cells expressing LAMP1-mCherry. Cells were starved for 2 h in HBSS and subsequently treated for 30 min with either DMSO (A and B) or 40 µM Dynasore (C and D), which induced extensive tubulation of LAMP1-positive compartments. Bars (A-D): 20 µM; (A′-B′) 2 µM; (C′-D′) 10 µM. (E-G) To demonstrate the reversibility of this tubulation, Dynasore-treated cells were washed extensively with drug-free media containing 10% FBS and monitored by time-lapse microscopy for 45 min. Frequently, after drug washout, LAMP1-positive tubules exhibited noticeable varicosities (E and F, arrows; bars, 10 µM) along their length. These sites are suggestive of areas of scission and resumed budding of nascent protolysosomes from the reformation tubules (G; bars, 10 µM). (H) Tubules from cells undergoing drug washout were quantified by tracing their lengths at the beginning and end of these movies. Still frames from a representative movie show tubule content at t = 10 and 45 min after drug washout. Bars, 20 µM. (I) Analysis of five independent movies showed an average decrease in total tubulation of ∼50% after drug washout. Data represent the average relative change in total tubule length between the first and last frames of the time-lapse movies. Error bars represent SE; *, P < 0.05.


( A B ) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS treated with 40 µM Dynasore , to induce tubule formation , as in Fig . 5 . were lysed ( WCL ) , post - supernatant ( PNS ) was pelleted by centrifugation to produce a crude lysosomal fraction ( CLF ) high - speed supernatant ( HSS ) . CLF was subsequently loaded onto an 8 - 27 % discontinuous iodixanol ( OptiPrep ) gradient for separation by ultracentrifugation . Nine fractions were collected from top of gradient blotted for Dyn2 LAMP1 resident , LAMP1 . Lysosomal acid phosphatase activity roughly correlates with LAMP1 in each fraction . of Dyn2 are highest in fraction 2 , corresponding with both peak of LAMP1 lysosomal activity . ( A ) data shown are from a single representative experiment out of three repeats . ( B ) Blotting for subcellular components shows that Dyn2 is specific for these same fractions , unlike other organelle markers such as EEA1 ( early endosomes ) COXIV ( mitochondria ) .

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(A and B) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS and treated with 40 µM Dynasore, to induce tubule formation, as in Fig. 5. Cells were lysed (WCL), and the post-nuclear supernatant (PNS) was pelleted by centrifugation to produce a crude lysosomal fraction (CLF) and high-speed supernatant (HSS). The CLF was subsequently loaded onto an 8-27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected from the top of the gradient and blotted for Dyn2 and the LAMP1 resident protein, LAMP1. Lysosomal acid phosphatase activity roughly correlates with LAMP1 protein levels in each fraction. Levels of Dyn2 are highest in fraction 2, corresponding with both the peak levels of LAMP1 and lysosomal activity. (A) The data shown are from a single representative experiment out of three repeats. (B) Blotting for subcellular components shows that Dyn2 is specific for these same fractions, unlike other organelle markers such as EEA1 (early endosomes) and COXIV (mitochondria).


( C D ) Dyn2 localizes to surface of LAMP1 - positive compartments . imaging of Hep3B hepatocytes transfected with Dyn2 - GFP LAMP1 - mCherry under resting ( C C ′ ) or starvation ( D ) conditions . ( C ′ ′ ) Dyn2 - GFP localizing to LAMP1 - labeled lysosome structures under starvation conditions . Arrows indicate regions of colocalization . Dyn2 ( arrows ) is present at site of scission of LAMP1 - positive tubules ( arrowhead ) from large autolysosomal structures . Bars : ( C ) 5 µM ; ( C ′ , C ′ ′ , D ) 3 µM .

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

(C and D) Dyn2 localizes to the surface of LAMP1-positive compartments. Fluorescence imaging of Hep3B hepatocytes transfected with Dyn2-GFP and LAMP1-mCherry under resting (C and C′) or starvation (D) conditions. (C′′) Dyn2-GFP localizing to LAMP1-labeled lysosome structures under starvation conditions. Arrows indicate regions of protein colocalization. Dyn2 (arrows) is present at the site of scission of LAMP1-positive tubules (arrowhead) from large autolysosomal structures. Bars: (C) 5 µM; (C′, C′′, and D) 3 µM.


A ) Box blot of vitrealANG - 1 from newly diagnosed diagnosed with wAMD , DR , proliferative DR , RVO compared to controls ( macular whole ) . B ) Box blot of vitrealANG - 2 from as in A ) . interquartile range of data is indicated by box . A non - parametric Kruskal - Wallis ( * * , P = 0 . 029 ) followed by Dunn ' s method for multiple comparisons was used to show significant differences of groups to control which are indicated by an asterisks . ANG - 1 did not differ significantly , but ANG - 2 were significantly different : control vs AMD ( * , P = 0 . 0451 ) , vs DR ( * , P < 0 . 0001 ) , pDR ( * , P < 0 . 0001 ) RVO ( P < 0 . 0001 ) .

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 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 O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 O O O O O O O O O O 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) Box blot of vitrealANG-1 levels from newly diagnosed patients diagnosed with wAMD, DR, proliferative DR, and RVO compared to controls (macular whole).B) Box blot of vitrealANG-2 levels from patients as in A). The interquartile range of the data is indicated by the box. A non-parametric Kruskal-Wallis analysis (**, P = 0.029) followed by Dunn's method for multiple comparisons was used to show significant differences of the groups to control which are indicated by an asterisks. ANG-1 levels did not differ significantly, but ANG-2 levels were significantly different:control vs AMD (*, P = 0.0451), vs DR (*, P < 0.0001), pDR (*,P < 0.0001) and RVO (P < 0.0001).


D ) 24 - h of ANG - 2 in supernatants of basal site of culture stimulated with 5 ng / ml of VEGF - A , 200 ng / ml of ANG - 1 or combination . Error bars show with one sided ANOVA ( * * * * , P < 0 . 0001 ) Tukey ' s multiple T - for five independent experiments indicating significance for control vs VEGF - A ( * * , P = 0 . 0028 ) ; VEGF - A vs ANG - 1 ( * * * * , P < 0 . 0001 ) ; ANG - 1 vs VEGF - A ANG1 ( * * , P = 0 . 0061 ) ; VEGF - A vs VEGF - A ANG - 1 ( ns , P = 0 . 0625 ) .

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

D) 24-h concentration of ANG-2 in supernatants of the basal site of the culture stimulated with 5 ng/ml of VEGF-A, 200 ng/ml of ANG-1 or the combination. Error bars show SEM with one sided ANOVA (****, P < 0.0001) and Tukey's multiple T-test for five independent experiments indicating significance for control vs VEGF-A (**, P = 0.0028); VEGF-A vs ANG-1 (****, P <0.0001); ANG-1 vs VEGF-A ANG1 (**, P = 0.0061); VEGF-A vs VEGF-A ANG-1 (ns, P = 0.0625).


E ) Humanendothelial plated on filters were assessed for endothelial barrier function over time after addition of 5 ng / ml VEGF - A alone or in combination with 200 ng / ml ANG - 1 or 10 μg / ml anti - ANG - 2 or three items . final time point was used for statistical . Error bars show with one sided ANOVA ( * * * * , P < 0 . 0001 ) Tukey ' s multiple T - for three independent experiments indicating significance of VEGF - A vs untreated ( * * * * , P < 0 . 0001 ) ; vs VEGF - A ANG - 1 anti - ANG - 2 ( * * * , P = 0 . 0004 ) ; vs VEGF - A anti - ANG - 2 ( P = 0 . 038 ) ; vs VEGF - A ANG - 1 , ( P = 0 . 040 ) . Furthermore , VEGF - A anti - ANG - 2 ANG - 1 is significantly different vs VEGF - A ANG - 1 ( * , P = 0 . 0451 ) VEGF - A anti - ANG - 2 ( * , P = 0 . 0474 ) untreated is significantly different from vs VEGF - A ANG - 1 ( * , P = 0 . 0009 ) VEGF - A anti - ANG - 2 ( * , P = 0 . 0008 ) .

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

E) Humanendothelial cells plated on filters were assessed for endothelial barrier function over time after addition of 5 ng/ml VEGF-A alone or in combination with 200 ng/ml ANG-1 or 10 μg/ml anti-ANG-2 or all three test items. The final time point was used for statistical analysis. Error bars show SEM with one sided ANOVA (****, P < 0.0001) and Tukey's multiple T-test for three independent experiments indicating significance of VEGF-A vs untreated (****, P < 0.0001); vs VEGF-A ANG-1 anti-ANG-2 (***, P = 0.0004); vs VEGF-A anti-ANG-2 (P = 0.038); vs VEGF-A ANG-1, (P = 0.040). Furthermore, VEGF-A anti-ANG-2 ANG-1 is significantly different vs VEGF-A ANG-1 (*, P = 0.0451) and VEGF-A anti-ANG-2 (*, P = 0.0474) and untreated is significantly different from vs VEGF-A ANG-1 (*, P = 0.0009) and VEGF-A anti-ANG-2 (*, P = 0.0008).


A , B ) Real - time qPCR of ANG - 2 in retina RPE / choroid complexes of JR5558 C57Bl / 6J ( C57 ) mice . A ) Relative expression of ANG - 2 were significantly increased in RPE / choroid complexes of JR5558mice in comparison to C57 , at 50 62 days old ( P = 0 . 022 at D50 P = 0 . 042 at D62 ) . B ) By contrast , retina of ANG - 2 were not significantly different between C57 JR5558mice , indicating neovascularization is driven locally by ANG - 2 . is shown as error bar with n = 4 per group . * Denotes statistical significance of JR5558mice compared to wild type C57bl6 using unpaired T - for each time point analyzed separately .

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A,B) Real-time qPCR analysis of ANG-2 levels in retina and RPE/choroid complexes of JR5558 and C57Bl/6J (C57) mice. A) Relative expression levels of ANG-2 were significantly increased in the RPE/choroid complexes of JR5558mice in comparison to C57, at 50 and 62 days old (P = 0.022 at D50 and P = 0.042 and at D62). B) By contrast, retina levels of ANG-2 were not significantly different between C57 and JR5558mice, indicating neovascularization is driven locally by ANG-2. SEM is shown as error bar with n = 4 animals per group. *Denotes statistical significance of JR5558mice compared to wild type C57bl6 using unpaired T-test for each time point analyzed separately.


D - I ) Representative examples of angiograms of IgG control ( D ) , anti - VEGF - A ( E ) , anti - ANG - 2 ( F ) ( 5 mg / kg ) three doses of anti - VEGF - A / ANG - 2 ( G , H , I ) , ( at 3 , 5 , 10 mg / kg ) . J , K ) Bar graph of numbers of spontaneously occurring lesion number ( J ) by angiography ( K ) after two weekly doses of antibody ( antibodies at 5 mg / kg IP anti - VEGF - A / ANG - 2 at 10 [ high ] , 5 [ mid ] , 3 [ low ] mg / kg IP ) followed by a week later in early intervention model . L , M ) Bar graph of numbers of spontaneously occurring lesion numbers ( L ) by angiography ( M ) after two weekly doses of antibody ( 3 mg / kg IP ) followed by a week later in late intervention model .

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

D-I) Representative examples of fluorescence angiograms of IgG control (D), anti-VEGF-A (E), anti-ANG-2 (F) (all the 5 mg/kg) and three doses of anti-VEGF-A/ANG-2 (G,H,I), (at 3, 5, and 10 mg/kg).J,K) Bar graph of numbers of spontaneously occurring lesion number (J) and area by fluorescence angiography (K) after two weekly doses of antibody (antibodies at 5 mg/kg IP and anti-VEGF-A/ANG-2 at 10 [high], 5 [mid], and 3 [low] mg/kg IP) followed by analysis a week later in the early intervention model.L,M) Bar graph of numbers of spontaneously occurring lesion numbers (L) and area by fluorescence angiography (M) after two weekly doses of antibody (3 mg/kg IP) followed by analysis a week later in the late intervention model.


A ) Bar graph showing relative change in OCT - measured volume between outer layer RPE layer following intervention with articles . Anti - VEGF - A anti - ANG - 2 were given at 5 mg / kg anti - VEGF - A / ANG - 2 at 1 , 5 , 10 mg / kg ( low , mid , high dose ) . B ) Representative OCT converted to heat maps with scaling grade given on right of figure delineating fluid filled areas of retina . Baseline ( D46 ) 14 days treatments of IgG control , anti - VEGF - A , anti - ANG - 2 , bispecific anti - VEGF - A / ANG - 2 at end of experiment ( D60 ) are shown . For region of interest , corresponding FA image at D60 is also shown as comparator .

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

A) Bar graph showing the relative change in OCT-measured volume between the outer nuclear layer and the RPE layer following intervention with test articles. Anti-VEGF-A and anti-ANG-2 were given at 5 mg/kg and anti-VEGF-A/ANG-2 at 1, 5, and 10 mg/kg (low, mid, and high dose).B) Representative OCT images converted to heat maps with the scaling grade given on the right of the figure delineating fluid filled areas of the retina. Baseline (D46) and 14 days treatments of IgG control, anti-VEGF-A, anti-ANG-2, and bispecific anti-VEGF-A/ANG-2 at the end of the experiment (D60) are shown. For the region of interest, the corresponding FA image at D60 is also shown as comparator.


A ) Bar graph of number of IBA - 1 + in retina of IgG control , anti - VEGF - A , anti - ANG - 2 ( 5 mg / kg IP ) , bispecific anti - VEGF - A / ANG - 2 ( at 10 , 5 , 3 mg / kg ) - treated mice in late interference model at D60 . B ) IgG control , C ) anti - VEGF - A , D ) anti - ANG - 2 , E - G ) anti - VEGF - A / ANG - 2 low , mid high : representative of whole - mount eyecup preparations stained with isolectin - B4 - positive ( green ) to stain vessels anti - IBA1 ( red ) to stain for macrophages show a decrease both in absolute numbers clustering of subretinal IBA - 1 - expressing associated with neovascular complexes in anti - VEGF - A / ANG - 2 treatment groups . Error bars show with n = 6 per group ( Scale bar , 200µm ) . significant differences are shown by * after one sided ANOVA ( * * * , P < 0 . 0008 ) Tukey ' s multiple t - . IgG control is significant against anti - VEGF - A / ANG - 2 low ( * , P < 0 . 0134 ) , - mid ( * * , P < 0 . 0078 ) - high ( * * , P < 0 . 0042 ) ; furthermore anti - ANG - 2 against anti - VEGF - A / ANG - 2 mid ( * , P < 0 . 0353 ) .

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A) Bar graph of number of IBA-1+cells in the retina of IgG control, anti-VEGF-A, anti-ANG-2 (all 5 mg/kg IP), and bispecific anti-VEGF-A/ANG-2 (at 10, 5, and 3 mg/kg)-treated mice in the late interference model at D60.B) IgG control, C) anti-VEGF-A, D) anti-ANG-2, E-G) anti-VEGF-A/ANG-2 low, mid and high: representative micrographs of whole-mount eyecup preparations stained with isolectin-B4-positive (green) to stain vessels and anti-IBA1 (red) to stain for macrophages show a decrease both in absolute numbers and clustering of subretinal IBA-1-expressing cells associated with neovascular complexes in anti-VEGF-A/ANG-2 treatment groups. Error bars show SEM with n = 6 animals per group (Scale bar, 200µm). All significant differences are shown by * after one sided ANOVA (***, P < 0.0008) and Tukey's multiple t-test. IgG control is significant against anti-VEGF-A/ANG-2 low (*, P < 0.0134), -mid (**, P < 0.0078) and -high (**, P < 0.0042); furthermore anti-ANG-2 against anti-VEGF-A/ANG-2 mid (*, P < 0.0353).


A ) Bar graph of TUNEL - positive in retina of mice treated with 5 mg / kg anti - VEGF - A , anti - ANG - 2 , 3 , 5 , 10 mg / kg anti - VEGF - A / ANG - 2 . Error bars show with n = 6 per group * denote significant changes after one sided ANOVA ( * * , P = 0 . 0041 ) Tukey ' s multiple t - . IgG control is significantly different from anti - VEGF - A / ANG - 2 mid ( * * , P = 0 . 0077 ) - high ( * * , P = 0 . 0028 ) . B ) IgG control , C ) anti - VEGF - A , D ) anti - ANG - 2 , E - G ) anti - VEGF - A / ANG - 2 low , mid , high : representative of whole - mount retinae preparations display reduction of TUNEL - positive photoreceptor ( green ) clustered around focal neovascular lesions in anti - VEGF - A / ANG - 2 ( E - G ) , to a lesser extent , anti - VEGF - A - ( C ) anti - ANG - 2 ( D ) - treated groups compared with IgG - treated controls ( B ) , scale bar 200 μM .

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A) Bar graph of TUNEL-positive cells in the retina of mice treated with 5 mg/kg anti-VEGF-A, anti-ANG-2, and 3, 5, and 10 mg/kg anti-VEGF-A/ANG-2. Error bars show SEM with n = 6 animals per group and * denote all significant changes after one sided ANOVA (**, P = 0.0041) and Tukey's multiple t-test. IgG control is significantly different from anti-VEGF-A/ANG-2 mid (**, P = 0.0077) and -high (**, P = 0.0028). B) IgG control, C) anti-VEGF-A, D) anti-ANG-2, E-G) anti-VEGF-A/ANG-2 low, mid, high: representative micrographs of whole-mount retinae preparations display the reduction of TUNEL-positive photoreceptor cells (green) clustered around focal neovascular lesions in anti-VEGF-A/ANG-2 (E-G) and, to a lesser extent, anti-VEGF-A- (C) and anti-ANG-2 (D)-treated groups compared with IgG-treated controls (B), scale bar 200 μM.


H ) ERG was used to assess retinal function in response to large - field flash stimuli under both scotopic photopic conditions . Representative photopic ( - adapted ) scotopic ( - adapted ) flash response series show increased amplitude reduced latency after treatment with bispecific VEGF - A / ANG - 2 compared with IgG control ( both 3 mg / kg ) . ERG shows reduced depression of B - wave responses in JR5558mice treated with anti - VEGF - A / ANG - 2 compared to IgG control . I ) Maximum photopic ERG amplitude was increased using rodent crossreactive bispecific VEGF - A / ANG - 2 compared to single anti - VEGF - A or anti - ANG - 2 treatments ( at 3 mg / kg ) . There was a significant increase in amplitude at stimuli > 2 log ( cd * s / m2 ) between bispecific VEGF - A / ANG - 2 IgG control group under scoptopic phototopic conditions . * Denotes significance after ANOVA Dunnett ' s multiple t - compared to IgG control compared for each stimuli separately , error bars show with n = 8 per group . Under phototopic conditions anti - VEGF - A / ANG - 2 reached significance at 2 . 58 2 . 30 cd * s / m2 with P = 0 . 0498 * P = 0 . 0479 , respectively . Under scotopic conditions stimuli reached significance for anti - VEGF - A / ANG - 2 with P - values of - 2 . 3 cd * s / m2 ( P = 0 . 0498 ) ; - 1 . 93 cd * s / m2 ( P = 0 . 0035 ) ; - 1 . 33 - 1 . 03 cd * s / m2 ( P 0 . 0001 ) ; - 0 . 73 cd * s / m2 ( P = 0 . 0002 ) ; - 0 . 43 cd * s / m2 , ( P = 0 . 0004 ) ; - 0 . 13 cd * s / m2 , ( P = 0 . 0024 ) 0 . 18 cd * s / m2 ( P = 0 . 0031 ) . In addition anti - ANG - 2 treatment reached significance at - 0 . 13 cd * s / m2 ( P = 0 . 011 ) 0 . 18 cd * s / m2 ( P = 0 . 005 ) , respectively .

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H) ERG was used to assess retinal function in response to large-field flash stimuli under both scotopic and photopic conditions. Representative photopic (light-adapted) and scotopic (dark-adapted) flash response series show increased amplitude and reduced latency after treatment with bispecific VEGF-A/ANG-2 compared with IgG control (both 3 mg/kg). ERG shows reduced depression of B-wave responses in JR5558mice treated with anti-VEGF-A/ANG-2 compared to IgG control.I) Maximum photopic ERG amplitude was increased using the rodent crossreactive bispecific VEGF-A/ANG-2 compared to single anti-VEGF-A or anti-ANG-2 treatments (all at 3 mg/kg). There was a significant increase in amplitude at stimuli >2 log (cd*s/m2) between the bispecific VEGF-A/ANG-2 and IgG control group under scoptopic and phototopic conditions. *Denotes significance after ANOVA and Dunnett's multiple t-test compared to IgG control compared for each stimuli separately, error bars show SEM with n = 8 animals per group. Under phototopic conditions anti-VEGF-A/ANG-2 reached significance at 2.58 and 2.30 cd*s/m2 with P = 0.0498 and *P = 0.0479, respectively. Under scotopic conditions all stimuli reached significance for anti-VEGF-A/ANG-2 with P-values of -2.3 cd*s/m2 (P = 0.0498); -1.93 cd*s/m2 (P = 0.0035); -1.33 -1.03 cd*s/m2 (P 0.0001); -0.73 cd*s/m2 (P = 0.0002); -0.43 cd*s/m2, (P = 0.0004); -0.13 cd*s/m2, (P = 0.0024) and 0.18 cd*s/m2 (P = 0.0031). In addition anti-ANG-2 treatment reached significance at -0.13 cd*s/m2 (P = 0.011) and 0.18 cd*s/m2 (P = 0.005), respectively.


B ) Summary table of experimental binding affinities of parental components targeting VEGF - A ( ranibizumab ) ANG - 2 ( LC10 ) as well as of RG7716 measured by isothermal titration calorimetry . RG7716 binds VEGF - A121 - 165 with an affinity comparable to ranibizumab ( 3 . 3 vs . 3 . 1 nM ) . ANG - 2 binding of RG7716 is slightly decreased to 22 nM compared to α - ANG - 2 IgG1 ( LC10 ) with 4 . 1 nM . No binding to ANG - 1 was detectable for RG7716 or α - ANG - 2 IgG1 ( LC10 ) .

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B) Summary table of experimental binding affinities of the parental components targeting VEGF-A (ranibizumab) and ANG-2 (LC10) as well as of RG7716 measured by isothermal titration calorimetry. RG7716 binds VEGF-A121 and -165 with an affinity comparable to ranibizumab (3.3 vs. 3.1 nM). ANG-2 binding of RG7716 is slightly decreased to 22 nM compared to α-ANG-2 IgG1 (LC10) with 4.1 nM. No binding to ANG-1 was detectable for RG7716 or α-ANG-2 IgG1 (LC10).


C ) Inhibition of ANG - 1 induced endothelial cellsurvival was tested in presence of increasing concentrations of LC10 ( anti - ANG - 2IgG , ANG - 2 binding part in RG7716 ) LC08 ( ANG - 1 ANG - 2 binding antibody ) . An average of three independent experiments is shown with .

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C) Inhibition of ANG-1 induced endothelial cellsurvival was tested in the presence of increasing concentrations of LC10 (anti-ANG-2IgG, ANG-2 binding part in RG7716) and LC08 (ANG-1 and ANG-2 binding antibody). An average of three independent experiments is shown with SEM.


D ) ability of RG7716 to bind to ANG - 2 VEGF - A simultaneously independent is demonstrated by a surface plasmon resonance experiment . RG7716 is captured via an anti - Fc antibody on chip surface . green curve depicts experiment with ANG - 2 in first VEGF - A in second binding event . red curve shows binding events in other .

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D) The ability of RG7716 to bind to ANG-2 and VEGF-A simultaneously and independent is demonstrated by a surface plasmon resonance experiment. RG7716 is captured via an anti-Fc antibody on the chip surface. The green curve depicts the experiment with ANG-2 in the first and VEGF-A in the second binding event. The red curve shows the binding events in the other sequence.


Time - dependent binding of RG7716 is shown in pink , anti - VEGF - A / ANG - 2 without modification of Fc - part in red ( wild - type ) , anti - VEGF - A / ANG - 2 with modification of FcRn binding site only in yellow ( FcRn - ) , anti - VEGF - A / ANG - 2 with modification of FcγR binding in grey ( FcγR - ) . A ) Shows SPR binding profile over time to immobilized FcγR - I , B ) to immobilized FcγR - II , C ) to immobilized FcγR - III , D ) to immobilized FcRn . E ) Summary data table of binding data for RG7716 in comparison to anti - VEGF - A / ANG - 2 CrossMAbs without modifications in Fc - part .

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Time-dependent binding of RG7716 is shown in pink, anti-VEGF-A/ANG-2 without modification of the Fc- part in red (wild-type), anti-VEGF-A/ANG-2 with modification of the FcRn binding site only in yellow (FcRn-), and anti-VEGF-A/ANG-2 with modification of the FcγR binding in grey (FcγR-).A) Shows SPR binding profile over time to immobilized FcγR-I, B) to immobilized FcγR-II, C) to immobilized FcγR-III, D) to immobilized FcRn. E) Summary data table of binding data for RG7716 in comparison to anti-VEGF-A/ANG-2 CrossMAbs without modifications in the Fc-part.


A ) Bar graph demonstrating platelet aggregation induced by complexes of heparin , VEGF - A165 , anti - VEGF - A ( IgG1 ) in vitro using washed human platelets . Introduction of modification of Fc - region in RG7716 does not allow platelet aggregation to happen as compared to anti - VEGF - A as wild type IgG1 . three components are needed as heparin / anti - VEGF - A alone does not activate platelet aggregation . Error bars show of three independent experiments * * * * denotes significance using ANOVA ( P < 0 . 0001 ) Dunnett ' s multiple t - comparing to IgG control with P < 0 . 0001 of Heparin / VEGF - A / anti - VEGF - A against IgG control .

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A) Bar graph demonstrating platelet aggregation induced by complexes of heparin, VEGF-A165, and anti-VEGF-A (IgG1) in vitro using washed human platelets. Introduction of modification of the Fc-region in RG7716 does not allow platelet aggregation to happen as compared to anti-VEGF-A as wild type IgG1. All three components are needed as heparin/anti-VEGF-A alone does not activate platelet aggregation. Error bars show SEM of three independent experiments and **** denotes significance using ANOVA (P < 0.0001) and Dunnett's multiple t-test comparing to IgG control with P < 0.0001 of Heparin/VEGF-A/anti-VEGF-A against IgG control.


B ) Kinetic presentation of mean serum , vitreous , aqueous humor concentrations after single intravitreal administration of 0 . 5 mg of RG7716 in cynomolgus monkeys .

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B) Kinetic presentation of mean serum, vitreous, and aqueous humor concentrations after single intravitreal administration of 0.5 mg of RG7716 in cynomolgus monkeys.


C ) Summary table of key experimental PK parameters of RG7716 anti - VEGF - A / ANG - 2 - FcγR - ( with intact FcRn binding ) .

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C) Summary table of key experimental PK parameters of RG7716 and anti-VEGF-A/ANG-2-FcγR- (with intact FcRn binding).


B ) Inhibition of neovascularization measured in severity grades , change of severity from baseline is shown for each treatment ; treatment significantly reduced severity grade compared to IgG control . In addition , efficacy of RG7716 ( 150 kDa molecule at 90 µg ) was significantly better at equal molar of binding sites than anti - VEGF - A ( ranibizumab , 50 kDa molecule at 30 µg ) , anti - ANG - 2 low dose of RG7716 ( 30 µg ) . C - G ) Representative figures of fundusangiograms at day 28 H - L ) of cross sections of a hematoxylin of a spot at end of experiment from eyes treated with IgG control , anti - VEGF - A , anti - ANG - 2 RG7716 at 30 90 μg , respectively .

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B) Inhibition of neovascularization measured in severity grades, change of severity from baseline is shown for each treatment; all treatment significantly reduced the severity grade compared to IgG control. In addition, efficacy of RG7716 (150 kDa molecule at 90 µg) was significantly better at equal molar concentration of binding sites than anti-VEGF-A (ranibizumab, 50 kDa molecule at 30 µg), anti-ANG-2 and the low dose of RG7716 (30 µg).C-G) Representative figures of fluorescence fundusangiograms at day 28 and H-L) of cross sections of a hematoxylin staining of a spot at the end of the experiment from eyes treated with IgG control, anti-VEGF-A, anti-ANG-2 and RG7716 at 30 and 90 μg, respectively.


A ) VEGF - A only slightly increased from D16 to D30 in IgG control . At D16 of IgG control are significantly higher compared to anti - VEGF - A ( + + + + , P < 0 . 0001 ) ; RG7716 , 30 μg ( + + + + , P < 0 . 0001 ) RG7716 , 90 μg ( + + + + , P < 0 . 0001 ) . Anti - ANG - 2 are comparable to IgG control therefore significantly different from anti - VEGF - A ( + + + + , P < 0 . 0001 ) ; RG7716 , 30 μg ( + + + + , P < 0 . 0001 ) RG7716 , 90 μg ( + + + + , P < 0 . 0001 ) . At D30 of VEGF - A are significantly reduced in RG7716 , 90 μg group are compared to IgG control ( * , P = 0 . 0309 ) . Anti - ANG - 2 remains at comparable to IgG control is therefore significantly higher than RG7716 , 30 μg ( * , P = 0 . 0107 ) , RG7716 , 90 μg ( * * , P = 0 . 002 ) anti - VEGF - A ( * , P = 0 . 0162 ) .

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

A) VEGF-A levels only slightly increased from D16 to D30 in the IgG control. At D16 levels of IgG control are significantly higher compared to anti-VEGF-A (++++, P < 0.0001); RG7716, 30 μg (++++, P < 0.0001) and RG7716, 90 μg (++++, P < 0.0001). Anti-ANG-2 levels are comparable to IgG control and therefore significantly different from anti-VEGF-A (++++, P < 0.0001); RG7716, 30 μg (++++, P < 0.0001) and RG7716, 90 μg (++++, P < 0.0001). At D30 the levels of VEGF-A are significantly reduced in the RG7716, 90 μg group are compared to IgG control (*, P = 0.0309). Anti-ANG-2 remains at levels comparable to IgG control and is therefore significantly higher than RG7716, 30 μg (*, P = 0.0107), RG7716, 90 μg (**, P = 0.002) and anti-VEGF-A (*, P = 0.0162).


B ) ANG - 2 increased from D16 to D30 in IgG control . same patterns , with a weaker total increase , were seen for ANG - 2 both RG7716 treatment groups . ANG - 2 were significantly higher at D16 in anti - VEGF - A treated group compared to IgG control ( + + , P = 0 . 002 ) , to anti - ANG - 2 ( + + + , P = 0 . 0004 ) , RG7716 , 30 μg ( + + + + , P < 0 . 0001 ) RG7716 , 90 μg ( + + + + , P < 0 . 0001 ) .

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B) ANG-2 levels increased from D16 to D30 in the IgG control. The same patterns, with a weaker total increase, were seen for the ANG-2 and both RG7716 treatment groups. ANG-2 levels were significantly higher at D16 in the anti-VEGF-A treated group compared to IgG control (++, P = 0.002), to anti-ANG-2 (+++, P = 0.0004), RG7716, 30 μg (++++, P < 0.0001) and RG7716, 90 μg (++++, P < 0.0001).


C ) reductions of treatments compared to IgG control at day 30 did not reach significance . IL - 6 only very slightly increased from D16 to D30 in IgG control ; while for other treatment groups there was a trend to reduced IL - 6 at D30 compared to baseline with high dose of RG7716 reaching significance , IgG control vs RG7716 , 90 μg ( * , P = 0 . 023 ) .

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C) The reductions of treatments compared to IgG control at day 30 did not reach significance. IL-6 levels only very slightly increased from D16 to D30 in the IgG control; while for all other treatment groups there was a trend to reduced IL-6 levels at D30 compared to baseline with the high dose of RG7716 reaching significance, IgG control vs RG7716, 90 μg (*, P = 0.023).


D ) IL - 8 increased from D16 to D30 in IgG control group . Compared to IgG control of IL - 8 were reduced in anti - VEGF - A ( * , P = 0 . 0262 ) , RG7716 , 30 μg ( * , P = 0 . 0188 ) RG7716 , 90 μg ( * , P = 0 . 0107 ) treatments at at D30 .

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D) IL-8 levels increased from D16 to D30 in the IgG control group. Compared to the IgG control the levels of IL-8 were reduced in the anti-VEGF-A (*, P = 0.0262), RG7716, 30 μg (*, P = 0.0188) and RG7716, 90 μg (*, P = 0.0107) treatments at at D30.


E ) Substitution of arginines in hnRNPA2 LC with lysine prevents partitioning of hnRNPF PLD into hnRNPA2 LCRK droplets . Removing charged residues except for arginine from hnRNPA2 LC ( hnRNPA2 LCCD , R ) allows partitioning of hnRNPF PLD into droplets , indicating arginine in hnRNPA2 LC is required necessary for hnRNPF partitioning . hnRNPA2 LCRK does not phase separate much as hnRNPA2 LC at these conditions , see Figure S1J for of phase separation of variants . Conditions : 20 µM , 20 mM MES pH 5 . 5 50 mM NaCl , 150 mM urea . Scale bar : 10 µm .

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E) Substitution of all arginines in hnRNPA2 LC with lysine prevents the partitioning of hnRNPF PLD into hnRNPA2 LCRK droplets. Removing all charged residues except for arginine from hnRNPA2 LC (hnRNPA2 LCCD,R) allows partitioning of hnRNPF PLD into droplets, indicating arginine in hnRNPA2 LC is required and necessary for hnRNPF partitioning. hnRNPA2 LCRK does not phase separate much as hnRNPA2 LC at these conditions, see Figure S1J for quantification of phase separation of variants. Conditions: 20 µM proteins, 20 mM MES pH 5.5 50 mM NaCl, 150 mM urea. Scale bar: 10 µm.


A ) AlexaFluor488 - tagged ( green ) hnRNPA2 LC undergoes LLPS , while AlexaFluor555 - tagged ( red ) TOG D1 does not . However , TOG D1 partitions into hnRNPA2 LC droplets when mixed at a 1 : 1 . Conditions : 20 µM indicated ( ~ 1 % fluorescently tagged ) , 20 mM MES pH 5 . 5 , 50 mM NaCl , 150 mM urea . Scale bar : 10 µm . hnRNPA2 LC control duplicated from Figure 1A as hnRNPF PLD TOG D1 samples were made concurrently . B ) Similar to hnRNPF PLD , AlexaFluor555 - tagged TOG D1 does not undergo LLPS at 300 µM or partition into AlexaFluor488 - tagged FUS LC droplets with both at 300 µM . Conditions : 300 µM ( ~ 1 % fluorescently tagged ) , 20 mM MES pH 5 . 5 150 mM NaCl , 150 mM urea . Scale bar : 10 µm .

O O O O O O 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 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O 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) AlexaFluor488-tagged (green) hnRNPA2 LC undergoes LLPS, while AlexaFluor555-tagged (red) TOG D1 does not. However, TOG D1 partitions into hnRNPA2 LC droplets when mixed at a 1:1 ratio. Conditions: 20 µM indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. hnRNPA2 LC control duplicated from Figure 1A as hnRNPF PLD and TOG D1 samples were made concurrently. B) Similar to hnRNPF PLD, AlexaFluor555-tagged TOG D1 does not undergo LLPS at 300 µM or partition into AlexaFluor488-tagged FUS LC droplets with both proteins at 300 µM. Conditions: 300 µM proteins (~1% fluorescently tagged), 20 mM MES pH 5.5 150 mM NaCl, 150 mM urea. Scale bar: 10 µm.


E ) AlexaFluor555 - tagged hnRNPF PLD or FL AlexaFluor405 - tagged TOG D1 partition simultaneously into AlexaFluor488 - tagged hnRNPA2 LC droplets . Conditions : 20 µM of each indicated ( ~ 1 % fluorescently tagged ) , 20 mM MES pH 5 . 5 , 50 mM NaCl , 150 mM urea . Scale bar : 20 µm .

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

E) AlexaFluor555-tagged hnRNPF PLD or FL and AlexaFluor405-tagged TOG D1 partition simultaneously into AlexaFluor488-tagged hnRNPA2 LC droplets. Conditions: 20 µM of each indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 20 µm.


C ) showing that although TOG D1 hnRNPF PLD partition into hnRNPA2 LC droplets ( rows 1 3 ) , they are unable to partition into tyrosine phosphorylated hnRNPA2 LC droplets ( rows 2 4 ) . Conditions : 20 µM ( ~ 1 % fluorescently labeled ) , 20 mM MES pH 5 . 5 , 50 mM NaCl , 150 mM urea . Scale bar : 10 µm . D ) Phosphomimic variants containing 5 or 12 serine to glutamate substitutions ( hnRNPA2 LC5E hnRNPA2 LC12E , respectively ) both allow partitioning of TOG D1 hnRNPF PLD , even though hnRNPA2 LC12E barely phase separates at these conditions , indicating that increased negative charge is insufficient to prevent partitioning of TOG D1 hnRNPF PLD . See Appendix Figure S7B for of LLPS of hnRNPA2 LC5E hnRNPA2 LC12E . Conditions : 20 µM ( ~ 1 % fluorescently labeled ) , 20 mM MES pH 5 . 5 , 50 mM NaCl , 150 mM urea . Scale bar : 10 µm .

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

C) Fluorescence micrographs showing that although TOG D1 and hnRNPF PLD partition into hnRNPA2 LC droplets (rows 1 and 3), they are unable to partition into tyrosine phosphorylated hnRNPA2 LC droplets (rows 2 and 4). Conditions: 20 µM proteins (~1% fluorescently labeled), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. D) Phosphomimic variants containing 5 or 12 serine to glutamate substitutions (hnRNPA2 LC5E and hnRNPA2 LC12E, respectively) both allow partitioning of TOG D1 and hnRNPF PLD, even though hnRNPA2 LC12E barely phase separates at these conditions, indicating that increased negative charge is insufficient to prevent partitioning of TOG D1 and hnRNPF PLD. See Appendix Figure S7B for quantification of LLPS of hnRNPA2 LC5E and hnRNPA2 LC12E. Conditions: 20 µM proteins (~1% fluorescently labeled), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm.


A ) After cleavage of a C - terminal maltose binding solubility tag , hnRNPA2 FL WT undergoes LLPS disease mutants D290V P298L aggregate . In contrast , phosphorylated hnRNPA2 FL WT P298L do not undergo LLPS or aggregation in time frame tested . Phosphorylated hnRNPA2 FL D290V can form some structures resembling liquid droplets but does not form amorphous in time frame tested . Conditions : 20 µM , 20 mM Tris pH 7 . 4 , 50 mM NaCl . Scale bar : 10

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 B-MEASURED_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

A) After cleavage of a C-terminal maltose binding protein solubility tag, hnRNPA2 FL WT undergoes LLPS and disease mutants D290V and P298L aggregate. In contrast, phosphorylated hnRNPA2 FL WT and P298L do not undergo LLPS or aggregation in the time frame tested. Phosphorylated hnRNPA2 FL D290V can form some structures resembling liquid droplets but does not form amorphous aggregates in the time frame tested. Conditions: 20 µM proteins, 20 mM Tris pH 7.4, 50 mM NaCl. Scale bar: 10 µm


D ) After 22 hours of paraquat induced oxidative stress , both HRPA - 1mScarlet HRPA - 1HsLCWTmScarlet assemble more than when unstressed ( ANOVA p < 0 . 0001 ) . No significant differences were found within no stress ( p = 0 . 8 ) stress groups ( p = 0 . 45 ) by ANOVA , indicating that there is no difference in assembly between HRPA - 1mScarlet HRPA - 1HsLCWTmScarlet . N = 12 per genotype .

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

D) After 22 hours of paraquat induced oxidative stress, both HRPA-1mScarlet and HRPA-1HsLCWTmScarlet assemble more than when unstressed (ANOVA p<0.0001). No significant differences were found within the no stress (p=0.8) stress groups (p=0.45) by ANOVA, indicating that there is no difference in assembly between HRPA-1mScarlet and HRPA-1HsLCWTmScarlet. N = 12 animals per genotype.


F ) There is no significant difference in number of spots in HRPA - 1HsLCWTmScarlet lines with or without Fyn , although there seems to be a trend of reduced spots in HRPA - 1HsLCD290VmScarlet lines in also expressing Fyn * ; this difference is not statistically significant ( ANOVA ) . N = 12 per genotype . G ) After exposure to 22 hours paraquat induced oxidative stress there is no significant difference between HRPA - 1HsLCWTmScarlet lines with or without Fyn , although there seems to be a trend of reduced spots in HRPA - 1HsLCD290VmScarlet lines in also expressing Fyn * ; this difference is not statistically significant ( ANOVA ) . N = 12 per genotype .

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

F) There is no significant difference in number of spots in HRPA-1HsLCWTmScarlet lines with or without Fyn, although there seems to be a trend of reduced spots in HRPA-1HsLCD290VmScarlet lines in animals also expressing Fyn*; this difference is not statistically significant (ANOVA). N = 12 animals per genotype. G) After exposure to 22 hours paraquat induced oxidative stress there is no significant difference between HRPA-1HsLCWTmScarlet lines with or without Fyn, although there seems to be a trend of reduced spots in HRPA-1HsLCD290VmScarlet lines in animals also expressing Fyn*; this difference is not statistically significant (ANOVA). N = 12 animals per genotype.


D ) Loss of C . elegans TDP - 43 ortholog , tdp - 1 , rescues glutamatergic tail / phasmid neurodegeneration caused by hrpa - 1HsLCD290V after 22 hours paraquat induced oxidative stress , but not hrpa - 1 ( ∆ ) . N = 4 - 12 / genotype / trial , significance from two t - ; tdp - 1 ∆ = tdp - 1 ( tgx58 ) . E ) Loss of C . elegans TDP - 43 ortholog , tdp - 1 , rescues glutamatergic tail / phasmid neurodegeneration caused by hrpa - 1HsLCD290V after 22 hours paraquat induced oxidative stress , but not hrpa - 1 ( ∆ ) . N = 4 - 12 / genotype / trial , significance from two t - ; tdp - 1 ∆ = tdp - 1 ( ok803 ) .

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

D) Loss of C. elegans TDP-43 ortholog, tdp-1, rescues glutamatergic tail/phasmid neurodegeneration caused by hrpa-1HsLCD290V after 22 hours paraquat induced oxidative stress, but not hrpa-1(∆). N=4-12 animals/genotype/trial, significance from two tailed t-test; tdp-1∆=tdp-1(tgx58). E) Loss of C. elegans TDP-43 ortholog, tdp-1, rescues glutamatergic tail/phasmid neurodegeneration caused by hrpa-1HsLCD290V after 22 hours paraquat induced oxidative stress, but not hrpa-1(∆). N=4-12 animals/genotype/trial, significance from two tailed t-test; tdp-1∆=tdp-1(ok803).


G ) Expression of Fyn * in sensory glutamatergic neurons ( osm - 10 promoter , unc - 54 3 ' UTR ) reduces glutamatergic tail / phasmid neuron degeneration in hrpa - 1HsLCD290V after 22 hours paraquat induced oxidative stress . N = 9 - 12 / genotype / trial , significance from two t - . H ) Expression of an empty Fyn control ( Fynempty ) in sensory glutamatergic neurons ( osm - 10 promoter , unc - 54 3 ' UTR ) does not alter glutamatergic phasmid neuron degeneration in hrpa - 1HsLCD290V after 22 hours paraquat induced oxidative stress . N = 9 - 12 / genotype / trial .

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

G) Expression of Fyn* in sensory glutamatergic neurons (osm-10 promoter, unc-54 3'UTR) reduces glutamatergic tail/phasmid neuron degeneration in hrpa-1HsLCD290V animals after 22 hours paraquat induced oxidative stress. N=9-12 animals/genotype/trial, significance from two tailed t-test. H) Expression of an empty Fyn control (Fynempty) in sensory glutamatergic neurons (osm-10 promoter, unc-54 3'UTR) does not alter glutamatergic phasmid neuron degeneration in hrpa-1HsLCD290V animals after 22 hours paraquat induced oxidative stress. N=9-12 animals/genotype/trial.


C . were treated with BMP2 for 1 h . D . were stimulated with increasing ( doubling ) doses of BMP4 / 7 ( 0 . 625 - 20 ng / mL ) for 1 h . Data information : In cases , whole extracts were Western blotted using indicated antibodies . Representative experiments are shown . of pSMAD1 / 5 relative to Actin were determined are expressed as a fold change relative to parental HEK293T untreated sample or normalized to 20 ng / mL ligand dose in parental HEK293T Act , Activin A ; Par , parental HEK293T , KO1 , ACVR1 KO clone 1 .

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

C. Cells were treated with BMP2 for 1 h. D. Cells were stimulated with increasing (doubling) doses of BMP4/7 (0.625-20 ng/mL) for 1 h. Data information: In all cases, whole cell extracts were Western blotted using the indicated antibodies. Representative experiments are shown. The levels of pSMAD1/5 relative to Actin were determined and are expressed as a fold change relative to the parental HEK293T untreated sample or normalized to the 20 ng/mL ligand dose in parental HEK293T cells Act, Activin A; Par, parental HEK293T cells, KO1, ACVR1 KO clone 1.


, B . Parental HEK293T , HET HOM1 were treated with BMP2 for indicated times . Quantifications are shown on right . Data information : quantifications are average normalized intensities ± of independent experiments in B , quantifications are expressed as fold changes relative to 1 h of ligand stimulation . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . p - values are from two - way ANOVA with Dunnett ' s post - hoc . In panels , Western blots of whole lysates were probed with antibodies indicated .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Parental HEK293T, HET and HOM1 cells were treated with BMP2 for the indicated times. Quantifications are shown on the right. Data information: quantifications are the average normalized intensities ± SEM of independent experiments in B , the quantifications are expressed as fold changes relative to 1 h of ligand stimulation. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. The p-values are from two-way ANOVA with Dunnett's post-hoc test. In all panels, Western blots of whole cell lysates were probed with the antibodies indicated.


D . Parental HEK293T , S2 / 3 dKO1 HOM1 were treated with Activin A for indicated times . Data information : ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . p - values are from two - way ANOVA with Dunnett ' s post - hoc . In D quantifications are from a representative experiment are intensities of pSMAD1 / 5 normalized to Actin , expressed as fold change relative to untreated for each clone . In panels , Western blots of whole lysates were probed with antibodies indicated .

O O O O O B-CONTROLLED_VAR O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Parental HEK293T, S2/3 dKO1 and HOM1 cells were treated with Activin A for the indicated times. Data information: ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. The p-values are from two-way ANOVA with Dunnett's post-hoc test. In D quantifications are from a representative experiment and are the intensities of pSMAD1/5 normalized to Actin, expressed as fold change relative to untreated cells for each clone. In all panels, Western blots of whole cell lysates were probed with the antibodies indicated.


B . Parental HEK293T , HET HOM1 were treated with either Activin A or BMP4 / 7 ( both at 20 ng / mL ) for 1 h , in presence or absence of either 1 µM LDN - 193189 or 10 µM SB - 431542 . Quantifications are of pSMAD1 / 5 normalized to loading control ( Actin or Tubulin ) , expressed as fold change relative to untreated for each clone . Quantifications are average normalized intensities ± of 3 - 5 independent experiments . Note that Activin A quantifications also includes data generated with high dose Activin A in Fig . 3A as they are independent replicates . Data information : In panels , Western blots of whole lysates were probed with antibodies indicated . hi , high ; Act , Activin A ; SB , SB - 431542 ; LDN , LDN - 193189 . p - values ( for A , B D ) are from a t - with Holm - Sidak correction , for comparison between indicated pairs . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 , * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . p - values in C are from two - way ANOVA with Tukey ' s post hoc correction .

O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Parental HEK293T, HET and HOM1 cells were treated with either Activin A or BMP4/7 (both at 20 ng/mL) for 1 h, in the presence or absence of either 1 µM LDN-193189 or 10 µM SB-431542. Quantifications are the levels of pSMAD1/5 normalized to the loading control (Actin or Tubulin), expressed as fold change relative to untreated for each clone. Quantifications are the average normalized intensities ± SEM of 3-5 independent experiments. Note that the Activin A quantifications also includes data generated with high dose Activin A in Fig. 3A as they are independent replicates. Data information: In all panels, Western blots of whole cell lysates were probed with the antibodies indicated. hi, high; Act, Activin A; SB, SB-431542; LDN, LDN-193189. The p-values (for A, B and D) are from a t-test with Holm-Sidak correction, for comparison between indicated pairs. ns, not significant; *, p< 0.05; **, p<0.01, ***, p<0.001; ****, p<0.0001. The p-values in C are from two-way ANOVA with Tukey's post hoc correction.


A . Parental HEK293T , ACVR2A KO , ACVR2B KO , ACVR2A / B dKO , HOM1 , HOM1 ACVR2A KO , HOM1 ACVR2B KO , HOM1 ACVR2A / B dKO were treated with Activin A or BMP2 for 1 h . Data information : In panels , Western blots of whole lysates were probed with antibodies indicated . Representative experiments are shown . Par , parental HEK293T .

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

A. Parental HEK293T, ACVR2A KO, ACVR2B KO, ACVR2A/B dKO, HOM1, HOM1 ACVR2A KO, HOM1 ACVR2B KO, HOM1 ACVR2A/B dKO cells were treated with Activin A or BMP2 for 1 h. Data information: In all panels, Western blots of whole cell lysates were probed with the antibodies indicated. Representative experiments are shown. Par, parental HEK293T cells.


C . NIH - 3T3 were either untransfected or transfected with either FLAG - SMAD1 or GFP - SMAD3 together with Opto - ACVR1B * . Post transfection , were either kept in or exposed to blue for 1 h , in presence or absence of 50 µM SB - 505124 or 1 µM LDN - 193189 . Data information : In panels , Western blots of whole lysates were probed with antibodies against pSMAD1 / 5 ( that detected both endogenous pFLAG - SMAD1 ) , SMAD1 ( which detects both endogenous FLAG - SMAD1 ) , HA ( which detects Opto - receptors ) , pSMAD3 ( which detects pGFP - SMAD3 ) , SMAD3 ( which detects GFP - SMAD3 ) either Tubulin or Actin as loading controls . Lane numbers are given below each blot for clarity .

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

C. NIH-3T3 cells were either untransfected or transfected with either FLAG-SMAD1 or GFP-SMAD3 together with Opto-ACVR1B*. Post transfection, cells were either kept in the dark or exposed to blue light for 1 h, in the presence or absence of 50 µM SB-505124 or 1 µM LDN-193189. Data information: In all panels, Western blots of whole cell lysates were probed with antibodies against pSMAD1/5 (that detected both endogenous and pFLAG-SMAD1), SMAD1 (which detects both endogenous and FLAG-SMAD1), HA (which detects the Opto-receptors), pSMAD3 (which detects pGFP-SMAD3), SMAD3 (which detects GFP-SMAD3) and either Tubulin or Actin as the loading controls. Lane numbers are given below each blot for clarity.


H . Average brightness of His - Activin A - Atto647N in conditions ( A - G ) . Means ± are plotted . Each point represents one analyzed image , collected from 2 - 3 biological replicates . Data information : In H I p values are from one - way ANOVA with Tukey ' s post hoc correction . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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

H. Average brightness of His-Activin A-Atto647N in conditions (A-G). Means ± SEM are plotted. Each point represents one analyzed image, collected from 2-3 biological replicates. Data information: In H and I the p values are from one-way ANOVA with Tukey's post hoc correction. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


I . Average brightness of His - Activin A - Atto647N in ICR - B169 HSJD - DIPG - 007 alone or with pre - incubation with 1 . 5 µg / mL of rhACVR2A Fc 15 µg / mL of rhACVR2B Fc . Source data is in Fig . EV4B - F . Means ± are plotted . Each point represents one analyzed image , collected from 2 - 4 biological replicates . Data information : In H I p values are from one - way ANOVA with Tukey ' s post hoc correction . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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I. Average brightness of His-Activin A-Atto647N in ICR-B169 and HSJD-DIPG-007 cells alone or with pre-incubation with 1.5 µg/mL of rhACVR2A Fc and 15 µg/mL of rhACVR2B Fc. Source data is in Fig. EV4B-F. Means ± SEM are plotted. Each point represents one analyzed image, collected from 2-4 biological replicates. Data information: In H and I the p values are from one-way ANOVA with Tukey's post hoc correction. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


A , B . HEK293T HOM1 were plated on a planar lipid bilayer containing ( NIP ) 1 - H12 - Hylight647 ( A ) or His - Activin A - Atto647N ( B ) an automated time - lapse was started , where 2 - 4 positions within a well were imaged every 40 s during a 27 - min time period ; 3 time points from one position are shown . TIRF , upper panels , brightfield , lower panels . cluster intensities from positions were quantified at 6 time points indicated ( right panel ) plotted as cluster values . horizontal gray bar denotes mean . + bilayer denotes bilayer areas under , as identified from brightfield , bilayer denotes areas where no were present . A representative experiment from 2 biological replicates quantified is shown . Scale bar 10 μm . In B , region under 2 is shown enlarged in top right - corner .

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A, B. HEK293T HOM1 cells were plated on a planar lipid bilayer containing (NIP)1-H12-Hylight647 (A) or His-Activin A-Atto647N (B) and an automated time-lapse was started, where 2-4 positions within a well were imaged every 40 s during a 27-min time period; 3 time points from one position are shown. TIRF, upper panels, brightfield, lower panels. The cluster intensities from all positions were quantified at 6 time points indicated (right panel) and plotted as individual cluster values. The horizontal gray bar denotes the mean. Cells + bilayer denotes bilayer areas under the cells, as identified from the brightfield images, and bilayer denotes areas where no cells were present. A representative experiment from 2 biological replicates quantified is shown. Scale bar 10 μm. In B, the region under cell 2 is shown enlarged in the top right-hand corner.


F Minimal inhibitory concentrations of DHA treated wild - type , heterozygous ( YME1 / yme1 ) homozygous ( yme1 / yme1 ) mutant strains . Plates were imaged after 72h .

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F Minimal inhibitory concentrations of DHA treated wild-type, heterozygous (YME1/yme1) and homozygous (yme1/yme1) mutant strains. Plates were imaged after 72h.


D Competitive growth of DHA resistant single clones . Wild - type ( mCherry + _ Cre _ Puro ) knockout ( GFP + _ Puro ) sister clones were derived from mutant ( retrovirus - intron RE , darker shading , Tol2 transposon - intron T2 , lighter shading ) resistant colonies , treated with DHA , analyzed with flow cytometry Sanger sequenced for insertion site mapping .

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D Competitive growth assay of DHA resistant single cell clones. Wild-type (mCherry+_Cre_Puro) and knockout (GFP+_Puro) sister clones were derived from mutant (retrovirus - intron RE, darker shading, Tol2 transposon - intron T2, lighter shading) resistant colonies, treated with DHA, analyzed with flow cytometry and Sanger sequenced for insertion site mapping.


A , B survival of dihydroartemisinin treated ESCs in combination with ( A ) Ppox inhibitor acifluorfen or upon ( B ) 5 - ALA administration . Alamar Blue was used to assess viability after 72h of treatment .

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A, B Cell survival of dihydroartemisinin treated ESCs in combination with (A) the Ppox inhibitor acifluorfen or upon (B) 5-ALA administration. Alamar Blue staining was used to assess viability after 72h of treatment.


C , D survival of DHA treated human glioblastoma from cancer in combination with ( C ) Ppox inhibitor acifluorfen ( BTL90 ) or upon ( D ) 5 - ALA administration ( VBT12 ) . Viability was assessed using CellTiter - Glo after 72h . Experiments were performed in triplicate .

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C, D Cell survival of DHA treated primary human glioblastoma cells from cancer patients and in combination with (C) the Ppox inhibitor acifluorfen (BTL90 cells) or upon (D) 5-ALA administration (VBT12 cells). Viability was assessed using CellTiter-Glo after 72h. Experiments were performed in triplicate.


E , F ( E ) ROS ( DHE , PE 582 / 15 nm - MFI ) ( F ) survival of DHA ( 0 . 5μM ) , 5 - ALA ( 0 . 25mM ) or Ppox inhibitor ( 10μM ) treated human neuroblastoma ( SHSY5Y ) . of DHE ( ROS ) numbers ( survival ) were assessed by flow cytometry automated counting . experiments were performed two times , in triplicate each .

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E, F (E) ROS levels (DHE staining, PE 582/15 nm - MFI) and (F) cell survival of DHA (0.5μM), 5-ALA (0.25mM) or Ppox inhibitor (10μM) treated human neuroblastoma cells (SHSY5Y). Fluorescence of DHE (ROS levels) and cell numbers (survival) were assessed by flow cytometry and automated cell counting. The experiments were performed two times, in triplicate each.


C , D ( C ) ( D ) brightfield of DMSO ( control ) , 5 - ALA ( 0 . 0625mM ) , DHA ( 1μM ) , or 5 - ALA DHA ( 0 . 0625mM 1μM ) treated organoids ( CNS - PNET - like , c - MYC overexpression ) . Organoids were imaged on day 1 , day 5 day 7 . One experiment is shown , representative of four independent experiments .

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C, D (C) Fluorescence and (D) brightfield images of DMSO (control), 5-ALA (0.0625mM), DHA (1μM), or 5-ALA and DHA (0.0625mM and 1μM) treated tumor organoids (CNS-PNET-like, c-MYC overexpression). Organoids were imaged on day 1, day 5 and day 7. One experiment is shown, representative of four independent experiments.


G ROS / DHE flow cytometry of dissociated organoids ( PE , 582 / 15 nm ) . Mean intensities ( MFI ) of DHE stained GFP + GFP - control are indicated . n = 2 , values are mean ± SD .

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G ROS/ DHE staining and flow cytometry analysis of dissociated tumor organoids (PE, 582/15 nm). Mean fluorescence intensities (MFI) of DHE stained GFP+ tumor cells and GFP- control cells are indicated. n=2, values are mean ± SD.


H of γH2AX + in cerebral organoids . 6 cryo - sections of 3 organoids each were stained for γH2AX , scanned 25 regions of interest ( ROI 2 . 500μm2 ) were analyzed per condition . Raw data were normalized to organoid ( per ROI ) are shown as box plots ( 25th - 75th percentiles , median ) .

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H Quantification of γH2AX+ cells in cerebral tumor organoids. 6 cryo-sections of 3 organoids each were stained for γH2AX, scanned and 25 regions of interest (ROI 2.500μm2) were analyzed per condition. Raw data were normalized to organoid area (per ROI) and are shown as box plots (25th-75th percentiles, median).