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 B-ORGANISM O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O B-TISSUE O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O B-TISSUE O O B-ORGANISM O O O B-ORGANISM O O O O 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-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O

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 B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O 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-TISSUE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O B-EXP_ASSAY O O 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 B-EXP_ASSAY O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O 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 ) .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

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 .

O O O O O B-EXP_ASSAY O O O B-TISSUE O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O 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) 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 ) .

O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O

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 .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O

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 .

O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

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 .

O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

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 .

O O O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

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 .

O O O O O O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM O

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 ) .

O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O

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 .

O O O O 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-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-TISSUE O O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O

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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 ) .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

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 ) .

O O O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O

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 .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

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 .

O O O O B-SMALL_MOLECULE O B-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O

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 B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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 B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY B-GENEPROD O O 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-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O 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-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD 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 B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-DISEASE I-DISEASE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-DISEASE I-DISEASE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD 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-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O 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-GENEPROD O O B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE 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-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O B-CELL_LINE B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD 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 B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY 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 B-CELL_LINE O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 B-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O 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-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CELL_LINE O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-CELL_LINE 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 B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD 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 B-GENEPROD O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I. 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 .

O O O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

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 .

O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O 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-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O

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 .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O

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 .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-ORGANISM B-DISEASE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

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 .

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O B-ORGANISM B-DISEASE O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

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).


A ( left panel ) brightfield ( right panel ) of 5 - ALA DHA treated human cerebral organoids ( glioblastoma - like ) . Organoids were monitored on day 1 , day 5 , day 8 day 11 . One experiment is shown , representative of two independent experiments . Scale bar 500μm . B , C Image of GFP - positive areas of treated organoids . Four organoids per group were analyzed on day ( B ) d5 ( C ) d11 , normalized to day 1 . Data is shown as box plots ( 25th - 75th percentiles , median ) n = 4 .

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A Fluorescence (left panel) and brightfield (right panel) images of 5-ALA and DHA treated human cerebral tumor organoids (glioblastoma-like). Organoids were monitored on day 1, day 5, day 8 and day 11. One experiment is shown, representative of two independent experiments. Scale bar 500μm. B, C Image analysis and quantification of GFP-positive tumor areas of the treated organoids. Four organoids per group were analyzed on day (B) d5 and (C) d11, normalized to day 1. Data is shown as box plots (25th-75th percentiles, median) n=4. D


A Representative brightfield of 5 - ALA DHA treated - derived glioblastoma spheroids ( VBT92 ) . were taken on day 3 of culture left untreated or exposed to indicated treatments . Scale bar 500μm .

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A Representative brightfield images of 5-ALA and DHA treated patient-derived glioblastoma spheroids (VBT92). Images were taken on day 3 of culture left untreated or exposed to the indicated treatments. Scale bar 500μm.


B Re - plating of spheroids after 3 days of 5 - ALA DHA treatment ( duplicates ) . Representative brightfield are shown for glioblastoma VBT92 .

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B Re-plating of spheroids after 3 days of 5-ALA and DHA treatment (duplicates). Representative brightfield images are shown for glioblastoma VBT92.


C of re - adhesion capacity of re - plated VBT92 spheroids as assessed by crystal violet absorbance measurement . Data is shown as mean ± SD . Experiments were performed in quadruplicates .

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C Quantification of the re-adhesion capacity of re-plated VBT92 spheroids as assessed by crystal violet staining and absorbance measurement. Data is shown as mean ± SD. Experiments were performed in quadruplicates.


D Lipid peroxidation in VBT92 treated with 5 - ALA DHA . Lipid peroxidation was measured using BODIPY ™ 581 / 591 C11 green ( from red ) shift was determined using flow cytometry . Experiments were performed in triplicate ; values are shown as mean ± SD .

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D Lipid peroxidation in VBT92 cells treated with 5-ALA and DHA. Lipid peroxidation was measured using BODIPY™ 581/591 C11 staining and green (from red) fluorescence shift was determined using flow cytometry. Experiments were performed in triplicate; values are shown as mean ± SD.


B In vivo efficacy comparison of solvents ( control ) , 5 - ALA , artesunate ( ARS ) combined treatment in an orthotopic glioblastoma model . 11 - 14 days after stereotactic intracerebral injection of 2 x 105 VBT529 , mice received following treatments : Control ( solvents ) , 5 - ALA ( 80 mg / kg ) , ARS ( 40 mg / kg ) , or 5 - ALA plus ARS 5 times per week via intraperitoneal injection ( n ≥ 5 ) .

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B In vivo efficacy comparison of solvents (control), 5-ALA, artesunate (ARS) and combined treatment in an orthotopic glioblastoma model. 11-14 days after stereotactic intracerebral injection of 2 x 105 VBT529 cells, mice received the following treatments: Control (solvents), 5-ALA (80 mg/kg), ARS (40 mg/kg), or 5-ALA plus ARS 5 times per week via intraperitoneal injection (n ≥ 5).


C , D representative of VBT529 flank luminescence signal . 6 - 7 days after injection of 1 . 5 x 105 VBT529 into flank , mice received following treatments : Control ( solvents ) or 5 - ALA plus ARS 4 - 5 times per week for 5 via intraperitoneal injection ( n = 6 ) . After 4 days of treatment , concentrations of 5 - ALA ( 100 mg / kg ) ARS ( 50 mg / kg ) were increased to 120 mg / kg 60 mg / kg , respectively . 20 % dosage increase of both drugs was used as - , behaviour overall health of mice were not affected by both , lower as well as higher dosages . E , F representative of VBT529 brain luminescence signals . 6 - 7 days after stereotactic intracerebral injection of 1 . 5 x 105 VBT529 , mice received treatments as described in ( C D ) ( n = 6 ) .

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C, D Quantification and representative images of the VBT529 flank tumor luminescence signal. 6-7 days after injection of 1.5 x 105 VBT529 cells into the flank, mice received the following treatments: Control (solvents) or 5-ALA plus ARS 4-5 times per week for 5 weeks via intraperitoneal injection (n = 6). After 4 days of treatment, concentrations of 5-ALA (100 mg/kg) and ARS (50 mg/kg) were increased to 120 mg/kg and 60 mg/kg, respectively. The 20% dosage increase of both drugs was used as the body-weight, behaviour and overall health of the mice were not affected by both, the lower as well as higher dosages. E, F Quantification and representative images of VBT529 brain tumor luminescence signals. 6-7 days after stereotactic intracerebral injection of 1.5 x 105 VBT529 cells, mice received the treatments as described in (C and D) (n = 6).


G Kaplan - Meier survival curves of mice injected with VBT529 glioblastoma into flank brain ( same mice as in C - F ; n = 6 ) . Mice were treated with solvents ( control ) or 5 - ALA plus ARS ( same mice as in C - F ) .

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G Kaplan-Meier survival curves of mice injected with VBT529 glioblastoma cells into the flank and brain (same mice as in C-F; n = 6). Mice were treated with solvents (control) or 5-ALA plus ARS (same mice as in C-F).


A Mass spectrometric of LSD1 methylation . Immunoprecipitation ( IP ) was performed using anti - Flag antibody in HEK - 293T overexpressing Flag - tagged LSD1 , followed by liquid chromatography coupled tandem mass spectrometry ( LC - MS / MS ) . fragmentation of LSD1 peptide IADQFLGAMYTLPR identified a dimethylated residue at R838 .

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A Mass spectrometric analysis of LSD1 methylation. Immunoprecipitation (IP) was performed using anti-Flag antibody in HEK-293T cells overexpressing Flag-tagged LSD1, followed by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) analysis. The fragmentation of the LSD1 peptide IADQFLGAMYTLPR identified a dimethylated residue at R838.


B Interactions between LSD1 CARM1 were detected by co - immunoprecipitation ( Co - IP ) with anti - Flag in HEK - 293T transfected with Flag - LSD1 or Flag - CARM1 , respectively .

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B Interactions between LSD1 and CARM1 were detected by co-immunoprecipitation (Co-IP) assay with anti-Flag in HEK-293T cells transfected with Flag-LSD1 or Flag-CARM1, respectively.


C Interaction of endogenous LSD1 with CARM1 was analyzed by Co - IP using anti - CARM1 antibody in MDA - MB - 231 .

C Interaction of endogenous LSD1 with CARM1 was analyzed by Co-IP assay using anti-CARM1 antibody in MDA-MB-231 cells.


Purified GST - tagged CARM1 was pulled down with Flag - LSD1 purified from HEK - 293T . amounts of GST GST - tagged CARM1 were visualized by Coomassie Blue ( asterisk : GST - CARM1 ) .

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Purified GST-tagged CARM1 was pulled down with the Flag-LSD1 purified from HEK-293T cells. The amounts of GST and GST-tagged CARM1 were visualized by Coomassie Blue staining (asterisk: GST-CARM1).


E In vitro methylation assays . Purified fusion of GST - tagged LSD1 WT or four methylated arginine mutants ( R - A ) , or four methylated arginine mutants in combination ( 4RA ) , were incubated with GST - CARM1 in presence of 3H - SAM . Methylated were detected via autoradiography total amounts of were visualized by Coomassie Blue ( arrows : position of LSD1 ; arrowheads : position of CARM1 ) .

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E In vitro methylation assays. Purified fusion protein of GST-tagged LSD1 WT or the four methylated arginine mutants (R-A), or four methylated arginine mutants in combination (4RA), were incubated with GST-CARM1 in the presence of 3H-SAM. Methylated proteins were detected via autoradiography and total amounts of proteins were visualized by Coomassie Blue staining (arrows: the position of LSD1; arrowheads: the position of CARM1).


F Increasing amounts of GST - CARM1 incubated with GST - LSD1 in presence of SAM at 30 ° C for 1 h . methylation of LSD1 was analyzed by immunoblotting with anti - LSD1 R838me2a antibody .

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F Increasing amounts of GST-CARM1 incubated with GST-LSD1 in presence of SAM at 30°C for 1 h. And methylation of LSD1 was analyzed by immunoblotting with anti-LSD1 R838me2a antibody.


G GST - CARM1 incubated with GST - LSD1 WT or GST - LSD1 R838A / R838K in presence of SAM at 30 ° C for 1 h , followed by immunoblotting with anti - LSD1 R838me2a to detect LSD1 methylation .

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G GST-CARM1 incubated with GST-LSD1 WT or GST-LSD1 R838A/R838K in presence of SAM at 30°C for 1 h, followed by immunoblotting with anti-LSD1 R838me2a to detect LSD1 methylation.


A Methylation of LSD1 was assessed in MCF10A , MCF7 MDA - MB - 231 by immunoblotting using anti - LSD1 R838me2a .

A Methylation of LSD1 was assessed in MCF10A, MCF7 and MDA-MB-231 cells by immunoblotting using anti-LSD1 R838me2a.


expression of LSD1 in MDA - MB - 231 transfected with CARM1 shRNAs or control vector , were assessed by western blotting ( B ) ( Error bars represent mean ± SD , n = 3 experimental replicates , ns = not significant , Student ' s t - ) .

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Protein expression levels of LSD1 in MDA-MB-231 cells transfected with CARM1 shRNAs or control vector, were assessed by western blotting (B) (Error bars represent the mean ± SD, n = 3 experimental replicates, ns = not significant, Student's t-test).


relative mRNA expression of LSD1 in MDA - MB - 231 transfected with CARM1 shRNAs or control vector , were assessed by qPCR ( C ) ( Error bars represent mean ± SD , n = 3 experimental replicates , ns = not significant , Student ' s t - ) .

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relative mRNA expression levels of LSD1 in MDA-MB-231 cells transfected with CARM1 shRNAs or control vector, were assessed by qPCR analysis (C) (Error bars represent the mean ± SD, n = 3 experimental replicates, ns = not significant, Student's t-test).


D MDA - MB - 231 were treated with indicated amounts of MS049 for 48 h , LSD1 R838me2a LSD1 were assessed by western blotting using indicated antibodies .

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D MDA-MB-231 cells were treated with the indicated amounts of MS049 for 48 h, LSD1 R838me2a and LSD1 levels were assessed by western blotting using indicated antibodies.


E MDA - MB - 231 were treated with increasing amounts of MS049 for 48 h in presence of MG132 ( 10 μM ) or DMSO , before LSD1 level was analyzed by immunoblotting .

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E MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h in presence of MG132 (10 μM) or DMSO, before LSD1 level was analyzed by immunoblotting.


F MDA - MB - 231 were transfected with control or CARM1 shRNAs then treated with MG132 or DMSO for 10 h , lysates were analyzed by western blotting .

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F MDA-MB-231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 or DMSO for 10 h, and the cell lysates were analyzed by western blotting.


G MDA - MB - 231 were transfected with CARM1 shRNA # 2 or control vector , then treated with CHX ( 100 μg / ml ) for indicated time ; LSD1 level was examined by western blotting .

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G MDA-MB-231 cells were transfected with CARM1 shRNA#2 or control vector, and then treated with CHX (100 μg/ml) for the indicated time; LSD1 protein level was examined by western blotting.


H MDA - MB - 231 were transfected with control or CARM1 shRNAs then treated with MG132 for 10 h . lysates were immunoprecipitated by anti - LSD1 antibody then analysed by immunoblotting .

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H MDA-MB-231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated by anti-LSD1 antibody and then analysed by immunoblotting.


I MDA - MB - 231 were treated with increasing amounts of MS049 for 48 h MG132 for 10 h . ubiquitination level of LSD1 was assessed by immunoblotting after IP with anti - LSD1 antibody .

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I MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h and MG132 for 10 h. The ubiquitination level of LSD1 was assessed by immunoblotting after IP with anti-LSD1 antibody.


J HEK - 293T that transfected with LSD1 WT or LSD1 R838A were treated with CHX for indicated time . LSD1 were examined by western blotting .

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J HEK-293T that transfected with LSD1 WT or LSD1 R838A were treated with CHX for the indicated time. LSD1 were examined by western blotting analysis.


K HEK - 293T were transfected with indicated plasmids then treated with MG132 for 10 h . lysates were immunoprecipitated using anti - Flag antibody then subjected to immunoblotting .

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K HEK-293T cells were transfected with the indicated plasmids and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated using anti-Flag antibody and then subjected to immunoblotting.


L HEK - 293T expressing LSD1 WT or LSD1 R838A were treated with or without MS049 for 48 h MG132 for 10 h . Co - IP was performed using anti - Flag then subjected to western blotting .

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L HEK-293T cells expressing LSD1 WT or LSD1 R838A were treated with or without MS049 for 48 h and MG132 for 10 h. Co-IP assay was performed using anti-Flag and then subjected to western blotting.


A Immunoprecipitation was performed using anti - Flag antibody in MM - 231 - shLSD1 - WT / R838A . interaction of LSD1 DUBs were detected by immunoblotting with indicated antibodies .

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A Immunoprecipitation assay was performed using anti-Flag antibody in MM-231-shLSD1-WT/R838A cells. The interaction of LSD1 and DUBs were detected by immunoblotting with indicated antibodies.


B MDA - MB - 231 were treated with indicated amounts of P5091 for 48 h , LSD1 were assessed by immunoblotting .

B MDA-MB-231 cells were treated with the indicated amounts of P5091 for 48 h, LSD1 protein levels were assessed by immunoblotting.


C MDA - MB - 231 were treated with P5091 ( 10 μM ) for 48 h MG132 for 10 h . LSD1 ubiquitination level was measured by immunoblotting after immunoprecipitated with anti - LSD1 antibody .

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C MDA-MB-231 cells were treated with P5091 (10 μM) for 48 h and MG132 for 10 h. The LSD1 ubiquitination level was measured by immunoblotting after immunoprecipitated with anti-LSD1 antibody.


D HEK - 293T were transfected with USP7 WT or USP7 C223S , then treated with MG132 for 10 h . lysates were immunoprecipitated using anti - LSD1 antibody followed by immunoblotting .

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D HEK-293T cells were transfected with USP7 WT or USP7 C223S, then treated with MG132 for 10 h. Cell lysates were immunoprecipitated using anti-LSD1 antibody followed by immunoblotting analysis.


E MDA - MB - 231 overexpressing Flag - tagged LSD1 WT / R838A after knockdown of endogenous LSD1 then treated with or without P5091 for 48 h MG132 for 10 h . lysates were immunoprecipitated by anti - Flag antibody then analysed by immunoblotting .

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E MDA-MB-231 cells overexpressing Flag-tagged LSD1 WT/R838A after knockdown of endogenous LSD1 and then treated with or without P5091 for 48 h and MG132 for 10 h. Cell lysates were immunoprecipitated by anti-Flag antibody and then analysed by immunoblotting.


F MDA - MB - 231 were transfected with control or CARM1 shRNAs before treated with MG132 for 10 h . lysates were immunoprecipitated with anti - LSD1 then subjected to immunoblotting .

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F MDA-MB-231 cells were transfected with control or CARM1 shRNAs before treated with MG132 for 10 h. And cell lysates were immunoprecipitated with anti-LSD1 then subjected to immunoblotting.


G MDA - MB - 231 were treated with or without MS049 for 48 h MG132 for 10 h , lysates were immunoprecipitated using anti - LSD1 antibody then analysed by immunoblotting .

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G MDA-MB-231 cells were treated with or without MS049 for 48 h and MG132 for 10 h, Cell lysates were immunoprecipitated using anti-LSD1 antibody and then analysed by immunoblotting.


H HEK - 293T were transfected with USP7 WT or USP7 C223S , then treated with or without MS049 for 48 h MG132 for 10 h . lysates were immunoprecipitated with anti - LSD1 antibody followed by immunoblotting .

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H HEK-293T cells were transfected with USP7 WT or USP7 C223S, then treated with or without MS049 for 48 h and MG132 for 10 h. Cell lysates were immunoprecipitated with anti-LSD1 antibody followed by immunoblotting analysis.


Migration assays ( 24 h ) of MM - 231 - shLSD1 - WT / R838A / R838K / R838F ( A ) with or without MS049 treatment . Representative of migrated are shown . Data represent number of derived from mean of five fields ( Error bars indicate mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - ) .

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Migration assays (24 h) of MM-231-shLSD1-WT/R838A/R838K/R838F cells (A) with or without MS049 treatment. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars indicate the mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).


invasion assays ( 48 h ) of MM - 231 - shLSD1 - WT / R838A / R838K / R838F ( B ) with or without MS049 treatment . Representative of invaded are shown . Data represent number of derived from mean of five fields ( Error bars represent mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - ) .

invasion assays (48 h) of MM-231-shLSD1-WT/R838A/R838K/R838F cells (B) with or without MS049 treatment. Representative micrographs of invaded cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars represent mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).


Migration assays ( 24 h ) of MCF7 - LSD1 - WT / R838A / R838K / R838F ( C ) with or without MS049 treatment . Representative of migrated are shown . Data represent number of derived from mean of five fields ( Error bars indicate mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - ) .

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Migration assays (24 h) of MCF7-LSD1-WT/R838A/R838K/R838F cells (C) with or without MS049 treatment. Representative micrographs of migrated cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars indicate the mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).


D invasion assays ( 48 h ) of MCF7 - LSD1 - WT / R838A / R838K / R838F ( D ) with or without MS049 treatment . Representative of invaded are shown . Data represent number of derived from mean of five fields ( Error bars represent mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant , Student ' s t - ) .

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D invasion assays (48 h) of MCF7-LSD1-WT/R838A/R838K/R838F cells (D) with or without MS049 treatment. Representative micrographs of invaded cells are shown. Data represent the number of cells derived from mean cell counts of five fields (Error bars represent mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, Student's t-test).


E MM - 231 - shLSD1 - WT / R838A / R838K / R838F ( 1 × 106 ) were injected into tail veins of 5 - week - old nude mice , 7 later , mice were sacrificed with euthanasia lungs were fixed in paraformaldehyde solutions . number of visible surface metastatic lesions in lungs was counted ( mean of 7 mice ) ( Data are mean ± SD , n = 7 mice for each group , * P < 0 . 05 , * * P < 0 . 01 , ns = not significant , Student ' s t - ) .

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E MM-231-shLSD1-WT/R838A/R838K/R838F cells (1×106) were injected into the tail veins of 5-week-old female nude mice, and 7 weeks later, mice were sacrificed with euthanasia and lungs were fixed in paraformaldehyde solutions. The number of visible surface metastatic lesions in lungs was counted (mean of 7 mice) (Data are mean ± SD, n = 7 mice for each group, *P < 0.05, **P < 0.01, ns = not significant, Student's t-test).


F Lung metastatic nodules confirmed by haematoxylin eosin . Scale bars , 100 μm .

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F Lung metastatic nodules confirmed by haematoxylin and eosin staining. Scale bars, 100 μm.


MDA - MB - 231 were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 antibody immunoprecipitated was analyzed by qPCR using specific primers of E - cadherin ( A ) promoters ( Data are mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , Student ' s t - ) .

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MDA-MB-231 cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of E-cadherin (A) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, Student's t-test).


B MDA - MB - 231 were treated with MS049 or P5091 . ChIP assays were performed using anti - LSD1 antibody immunoprecipitated was analyzed by qPCR using specific primers of vimentin ( B ) promoters ( Data are mean ± SD , n = 3 experimental replicates , * P < 0 . 05 , * * P < 0 . 01 , Student ' s t - ) .

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B MDA-MB-231 cells were treated with MS049 or P5091. ChIP assays were performed using anti-LSD1 antibody and the immunoprecipitated DNA was analyzed by qPCR using specific primers of vimentin (B) promoters (Data are mean ± SD, n = 3 experimental replicates, *P < 0.05, **P < 0.01, Student's t-test).