( E ) Correlation of poison isoform inclusion and GE levels for temperature - dependent poison skipped exon events . Shown are the log2 fold change ( FC ) in GE versus the ∆ PSI of the poison isoform . Decay targets were identified by using data from CHX - treated plants . R² and P ( deviation from zero slope ) are indicated . N =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) Correlation of rhythmic Srsf10 expression ( orange , left y - axis ) and exon 3 inclusion ( black , right y - axis ) in mouse liver samples from the indicated ZTs ( n = 4 , mean + / - SEM ) . Quantifications were obtained by RNA - seq analysis of datasets from ( Atger et al . , 2015 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation of rhythmic Srsf10 expression (orange, left y-axis) and exon 3 inclusion (black, right y-axis) in mouse liver samples from the indicated ZTs (n=4, mean +/- SEM). Quantifications were obtained by RNA-seq analysis of datasets from (Atger et al., 2015).


( C ) Representative Western blot of SRSF10 protein levels from mouse liver nuclear extracts ( NX ) from different ZTs . The different SRSF10 variants are highlighted on the right . SRSF10 - fl and SRSF10 - 2 are the result of differential last exon usage . hnRNP L was used as a loading control . The asterisks could represent hyperphosphorylated SRSF10 - fl through higher CLK activity during the day . ( D ) Quantification of SRSF10 - fl + SRSF10 - 2 relative to ZT0 and hnRNP L ( mean of at least 3 mice ± SEM ) . Student ' s unpaired t test - derived p values * * * p < 0 . 001 .

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

(C) Representative Western blot of SRSF10 protein levels from mouse liver nuclear extracts (NX) from different ZTs. The different SRSF10 variants are highlighted on the right. SRSF10-fl and SRSF10-2 are the result of differential last exon usage. hnRNP L was used as a loading control. The asterisks could represent hyperphosphorylated SRSF10-fl through higher CLK activity during the day. (D) Quantification of SRSF10-fl + SRSF10-2 relative to ZT0 and hnRNP L (mean of at least 3 mice ± SEM). Student's unpaired t test-derived p values ***p<0.001.


( C ) Representative Western blot of SRSF10 protein levels from mouse liver nuclear extracts ( NX ) from different ZTs . The different SRSF10 variants are highlighted on the right . SRSF10 - fl and SRSF10 - 2 are the result of differential last exon usage . hnRNP L was used as a loading control . The asterisks could represent hyperphosphorylated SRSF10 - fl through higher CLK activity during the day .

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

(C) Representative Western blot of SRSF10 protein levels from mouse liver nuclear extracts (NX) from different ZTs. The different SRSF10 variants are highlighted on the right. SRSF10-fl and SRSF10-2 are the result of differential last exon usage. hnRNP L was used as a loading control. The asterisks could represent hyperphosphorylated SRSF10-fl through higher CLK activity during the day.


( A and B ) Root length ( averages ± SD , n ≥ 15 ) ( A ) and cell death staining of cotyledons and primary root tips ( B ) of Arabidopsis seedlings that were exposed to 100 nM Pep2 for 7 days , 2 days after germination . BAK1 ( D416N ) is a kinase - dead variant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) Root length (averages ± SD, n ≥ 15) (A) and cell death staining of cotyledons and primary root tips (B) of Arabidopsis seedlings that were exposed to 100 nM Pep2 for 7 days, 2 days after germination. BAK1 (D416N) is a kinase-dead variant.


( C ) Overview of 3 - week - old plants .

(C) Overview of 3-week-old plants.


( D ) Pep2 induces PEPR1 - FLAG association with SERK1 - , SERK2 - , BKK1 - , and BAK1 - HA in N . benthamiana . Experiments were repeated at least three times , with the same conclusions . IP and IB denote imunoprecipitation and immunoblotting with the indicated antibodies , respectively . NT indicates non - transformed plants . ( * , p < 0 . 05 in two - tailed tests compared to the differences ( ± Pep2 ) from the corresponding values of WT plants . )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pep2 induces PEPR1-FLAG association with SERK1-, SERK2-, BKK1-, and BAK1-HA in N. benthamiana. Experiments were repeated at least three times, with the same conclusions. IP and IB denote imunoprecipitation and immunoblotting with the indicated antibodies, respectively. NT indicates non-transformed plants. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of WT plants.)


( A ) Immunoblot analysis for BIK1 - HA in Arabidopsis protoplasts exposed to Pep2 or flg22 for 10 min . Arrowheads indicate phosphorylated BIK1 - HA .

O O O O 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) Immunoblot analysis for BIK1-HA in Arabidopsis protoplasts exposed to Pep2 or flg22 for 10 min. Arrowheads indicate phosphorylated BIK1-HA.


( B ) Immunoblot analysis for Pep2 - triggered MAPK activation . Ponceau S - stained loading controls are shown ( bottom ) .

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) Immunoblot analysis for Pep2-triggered MAPK activation. Ponceau S-stained loading controls are shown (bottom).


( C ) Root length ( averages ± SD , n ≥ 15 ) of Arabidopsis seedlings that were exposed to 100 nM Pep2 . ( * , p < 0 . 05 in two - tailed tests compared to the differences ( ± Pep2 ) from the corresponding values of bak1 - 3 plants . Two independent experiments were combined for statistical analysis . )

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

(C) Root length (averages ± SD, n ≥ 15) of Arabidopsis seedlings that were exposed to 100 nM Pep2. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of bak1-3 plants. Two independent experiments were combined for statistical analysis.)


( A ) Scatter plots for log2 fold changes of gene expression in WT plants versus bak1 - 3 plants treated with Pep2 for 2 and 10 h . The regression of these scatter plots is indicated by the red lines .

O O O O O O O O O O O O O O O O O O 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) Scatter plots for log2 fold changes of gene expression in WT plants versus bak1-3 plants treated with Pep2 for 2 and 10 h. The regression of these scatter plots is indicated by the red lines.


( B ) Hierarchical analysis of genes exhibiting a > 2 - fold change in expression level in bak1 - 3 plants after Pep2 application for 10 h in a whole - genome microarray analysis . Using Genevestigator v3 , these genes were separately cross - referenced to public databases to show their expression responses to salicylate ( SA ) , methyl - jasmonate ( JA ) , or ethylene ( ET ) .

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

(B) Hierarchical analysis of genes exhibiting a >2-fold change in expression level in bak1-3 plants after Pep2 application for 10 h in a whole-genome microarray analysis. Using Genevestigator v3, these genes were separately cross-referenced to public databases to show their expression responses to salicylate (SA), methyl-jasmonate (JA), or ethylene (ET).


( C and D ) qRT - PCR analysis of defense - related genes in 10 - day - old seedlings exposed to 0 . 5 µM Pep2 for 10 h . Data are averages ( ± SD ) of three biological replicates . ( * , p < 0 . 05 in two - tailed tests compared to the corresponding bak1 - 4 and bak1 - 3 values in C and D , respectively . Relative cycle threshold ( Ct ) values of two independent experiments were combined for statistical analysis . )

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

(C and D) qRT-PCR analysis of defense-related genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. Data are averages (±SD) of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding bak1-4 and bak1-3 values in C and D, respectively. Relative cycle threshold (Ct) values of two independent experiments were combined for statistical analysis.)


( A ) qRT - PCR analysis of PROPEP genes in 10 - day - old seedlings exposed to 0 . 5 µM Pep2 for 10 h . * , p < 0 . 05 in two - tailed tests compared to the corresponding WT values . Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qRT-PCR analysis of PROPEP genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. *, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis.


( B ) Anti - GFP ( upper ) and anti - PROPEP3 ( middle ) immunoblot analysis of PROPEP3 - Venus in seedlings submerged in liquid medium and treated with ( + ) or without ( - ) 0 . 5 M Pep2 for the indicated times . The positions of the molecular weight markers are shown on the left . Arrowheads and asterisks indicate PROPEP3 - Venus and cross - reactive bands , 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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) Anti-GFP (upper) and anti-PROPEP3 (middle) immunoblot analysis of PROPEP3-Venus in seedlings submerged in liquid medium and treated with (+) or without (-) 0.5 M Pep2 for the indicated times. The positions of the molecular weight markers are shown on the left. Arrowheads and asterisks indicate PROPEP3-Venus and cross-reactive bands, respectively.


( C , D and E ) Immunoblot analysis of PEPR1 - FLAG in 10 - day - old seedlings following Pep1 or flg22 application . ( C and D ) Plants were exposed to 1 M Pep1 or flg22 for the indicated times . ( E ) Plants were pre - exposed to MG132 ( 50 µM ) , Wortmannin ( Wm ) ( 30 µM ) and Tyrphostin A23 ( TyrA23 ) ( 100 µM ) for 1 h before being exposed to Pep1 for 10 h . The numbers below the immunoblots represent relative intensities of the PROPEP - Venus ( B ) and PEPR1 - FLAG ( C - E ) bands normalized to the backgrounds in the Ponceau S - stained loading controls ( bottom ) , with the band / background values in WT plant extracts 24 h ( upper ) and 0 h ( middle ) after Pep2 application , respectively , set as 1 . 0 .

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

(C, D and E) Immunoblot analysis of PEPR1-FLAG in 10-day-old seedlings following Pep1 or flg22 application. (C and D) Plants were exposed to 1 M Pep1 or flg22 for the indicated times. (E) Plants were pre-exposed to MG132 (50 µM), Wortmannin (Wm) (30 µM) and Tyrphostin A23 (TyrA23) (100 µM) for 1 h before being exposed to Pep1 for 10 h. The numbers below the immunoblots represent relative intensities of the PROPEP-Venus (B) and PEPR1-FLAG (C-E) bands normalized to the backgrounds in the Ponceau S-stained loading controls (bottom), with the band/background values in WT plant extracts 24 h (upper) and 0 h (middle) after Pep2 application, respectively, set as 1.0.


( A ) qRT - PCR analysis of defense - related genes in 10 - day - old seedlings exposed to 0 . 5 µM Pep2 for 10 h . ( * , p < 0 . 05 in two - tailed tests compared to the corresponding WT values . Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis . )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qRT-PCR analysis of defense-related genes in 10-day-old seedlings exposed to 0.5 µM Pep2 for 10 h. (*, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of two independent experiments with three biological replicates each were combined for statistical analysis.)


( B ) Root length ( averages ± SD , n ≥ 15 ) of Arabidopsis seedlings exposed to 100 nM Pep2 . ( * , p < 0 . 05 in two - tailed tests compared to the differences ( ± Pep2 ) from the corresponding values of WT plants . Two independent experiments were combined for statistical analysis . )

(B) Root length (averages ± SD, n ≥ 15) of Arabidopsis seedlings exposed to 100 nM Pep2. (*, p < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of WT plants. Two independent experiments were combined for statistical analysis.)


( C ) Fresh weight ( averages ± SD , n ≥ 25 ) of 2 - week - old plants grown on soil . ( * , p < 0 . 05 in two - tailed tests compared to the corresponding values of WT plants . ■ , p < 0 . 05 in two - tailed tests . Two independent experiments were combined for statistical analysis . )

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

(C) Fresh weight (averages ± SD, n ≥ 25) of 2-week-old plants grown on soil. (*, p < 0.05 in two-tailed tests compared to the corresponding values of WT plants. ■, p < 0.05 in two-tailed tests. Two independent experiments were combined for statistical analysis.)


( D ) Immunoblot analysis for BAK1 in 10 - day - old seedlings with or without 0 . 5 M Pep1 for 24 h . The numbers below the upper panel represent relative intensities of the BAK1 band normalized to the backgrounds in the Ponceau S - stained loading controls , with the value in non - elicited WT plants set as 1 . 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis for BAK1 in 10-day-old seedlings with or without 0.5 M Pep1 for 24 h. The numbers below the upper panel represent relative intensities of the BAK1 band normalized to the backgrounds in the Ponceau S-stained loading controls, with the value in non-elicited WT plants set as 1.0.


( A ) qRT - PCR analysis in 4 - week - old plant leaves challenged with the indicated P . syringae pv . tomato ( Pst ) DC3000 strains for the indicated times .

O O O O O O O O O O O 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) qRT-PCR analysis in 4-week-old plant leaves challenged with the indicated P. syringae pv. tomato (Pst) DC3000 strains for the indicated times.


( B ) Anti - GFP immunoblot analyses in seedlings challenged with Pst DC3000 ( DC3000 ) and Pst DC3000 ∆ hrcC ( ∆ hrcC ) for the indicated times .

O O O O O O O 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) Anti-GFP immunoblot analyses in seedlings challenged with Pst DC3000 (DC3000) and Pst DC3000 ∆hrcC (∆hrcC) for the indicated times.


. ( C ) Anti - PROPEP3 immunoblot analysis of PROPEP3 - Venus ( upper ) in plant tissues ( expressed in plants ) and in the extracellular fraction ( released into medium ) , 24 h and 48 h after the inoculation with ( + ) or without ( - ) Pst DC3000 . Lower panels indicate Ponceau S - stained loading controls . The numbers below the upper panel represent relative intensities of the PROPEP3 - Venus band normalized to the backgrounds in the Ponceau S - stained loading controls . NT denotes non - transgenic WT plants .

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

. (C) Anti-PROPEP3 immunoblot analysis of PROPEP3-Venus (upper) in plant tissues (expressed in plants) and in the extracellular fraction (released into medium), 24 h and 48 h after the inoculation with (+) or without (-) Pst DC3000. Lower panels indicate Ponceau S-stained loading controls. The numbers below the upper panel represent relative intensities of the PROPEP3-Venus band normalized to the backgrounds in the Ponceau S-stained loading controls. NT denotes non-transgenic WT plants.


( D ) Trypan blue cell death staining of Arabidopsis seedlings challenged with Pst DC3000 or Pst DC3000 AvrRpm1 ( AvrRpm1 ) for 48 h .

O O 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) Trypan blue cell death staining of Arabidopsis seedlings challenged with Pst DC3000 or Pst DC3000 AvrRpm1 (AvrRpm1) for 48 h.


( E ) Anti - PROPEP3 immunoblot analyses in the seedlings exposed to the indicated Pst DC3000 strains or 1 M Pep1 for 48 h .

(E) Anti-PROPEP3 immunoblot analyses in the seedlings exposed to the indicated Pst DC3000 strains or 1 M Pep1 for 48 h.


( F ) Anti - GFP immunoblot analysis for the extracellular fraction of the seedlings exposed to Pst DC3000 AvrRpm1 for 48 h . ( B , C , E and F ) Arrowheads and arrows indicate an apparently full - length and processed form of PROPEP3 - Venus , respectively . Asterisks indicate cross - reactive bands . The positions of the molecular weight markers are shown on the left of the immunoblots .

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

(F) Anti-GFP immunoblot analysis for the extracellular fraction of the seedlings exposed to Pst DC3000 AvrRpm1 for 48 h. (B, C, E and F) Arrowheads and arrows indicate an apparently full-length and processed form of PROPEP3-Venus, respectively. Asterisks indicate cross-reactive bands. The positions of the molecular weight markers are shown on the left of the immunoblots.


( A ) Growth of H . arabidopsidis ( Hpa ) Noco2 in 2 - week - old plants . Spores were counted at 6 days post inoculation ( dpi ) . Results are averages ± SE of three biological replicates . ( * , p < 0 . 05 in two - tailed tests compared to the corresponding values of Col plants . ■ , p < 0 . 05 in two - tailed tests between the indicated values . Three independent experiments were combined for statistical analysis . )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Growth of H. arabidopsidis (Hpa) Noco2 in 2-week-old plants. Spores were counted at 6 days post inoculation (dpi). Results are averages ± SE of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding values of Col plants. ■, p < 0.05 in two-tailed tests between the indicated values. Three independent experiments were combined for statistical analysis.)


( B ) Growth of syringe - infiltrated Pst DC3000 in rosette leaves of 4 - week - old plants pretreated with water ( Mock ) or 1 M flg22 for 24 h . Bacterial titers were determined at 3 dpi . Results are averages ± SE of three independent replicates . ( * , p < 0 . 05 , * * , p < 0 . 01 in two - tailed tests compared to the corresponding values of WT plants . ▲ , p < 0 . 01 in two - tailed tests compared to the differences ( ± flg22 ) from the corresponding values of WT plants . Three independent experiments were combined for statistical analysis . )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Growth of syringe-infiltrated Pst DC3000 in rosette leaves of 4-week-old plants pretreated with water (Mock) or 1 M flg22 for 24 h. Bacterial titers were determined at 3 dpi. Results are averages ± SE of three independent replicates. (*, p < 0.05, **, p < 0.01 in two-tailed tests compared to the corresponding values of WT plants. ▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Three independent experiments were combined for statistical analysis.)


( C ) Total SA levels were determined in the rosette leaves of 4 - week - old plants with or without 1 µM flg22 application for 24 h . Data are averages ( ± SD ) of material from three independent plants . The experiments were repeated twice , with similar results . ( ▲ , p < 0 . 01 in two - tailed tests compared to the differences ( ± flg22 ) from the corresponding values of WT plants . Two independent experiments were combined for statistical analysis . )

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

(C) Total SA levels were determined in the rosette leaves of 4-week-old plants with or without 1 µM flg22 application for 24 h. Data are averages (±SD) of material from three independent plants. The experiments were repeated twice, with similar results. (▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Two independent experiments were combined for statistical analysis.)


( D ) qRT - PCR analysis of the PR1 gene in 4 - week - old plants exposed to 1 µM flg22 . Data are averages ( ± SD ) of three biological replicates . ( * , p < 0 . 05 in two - tailed tests compared to the corresponding WT values . Relative Ct values of four independent experiments were combined for statistical analysis . )

(D) qRT-PCR analysis of the PR1 gene in 4-week-old plants exposed to 1 µM flg22. Data are averages (±SD) of three biological replicates. (*, p < 0.05 in two-tailed tests compared to the corresponding WT values. Relative Ct values of four independent experiments were combined for statistical analysis.)


( E ) Growth of syringe - infiltrated Pst DC3000 in rosette leaves of 4 - week - old plants pretreated with water ( Mock ) or 1 M flg22 for 24 h . Bacterial titers were determined at 3 dpi . Results are averages ± SE of three independent replicates . ( * * , p < 0 . 01 , * * * , p < 1 . 0e - 10 in two - tailed tests compared to the corresponding values of WT plants . ▲ , p < 0 . 01 in two - tailed tests compared to the differences ( ± flg22 ) from the corresponding values of WT plants . Three independent experiments were combined for statistical analysis . )

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

(E) Growth of syringe-infiltrated Pst DC3000 in rosette leaves of 4-week-old plants pretreated with water (Mock) or 1 M flg22 for 24 h. Bacterial titers were determined at 3 dpi. Results are averages ± SE of three independent replicates. (**, p < 0.01, ***, p < 1.0e-10 in two-tailed tests compared to the corresponding values of WT plants. ▲, p < 0.01 in two-tailed tests compared to the differences (± flg22) from the corresponding values of WT plants. Three independent experiments were combined for statistical analysis.)


( A ) Lesion sizes ( left ) were determined 5 days after C . higginsianum ( Ch ) inoculation . Invasion rates ( right ) of Ch on Arabidopsis leaves were determined 3 days after spore inoculation . An average of 3 replicates is shown ( ± SD ) ( left ) and ( ± SE ) ( right ) . ( ■ , p < 0 . 05 in two - tailed tests between the indicated samples . * , p < 0 . 05 in two - tailed tests compared to the corresponding WT values . Two independent experiments were combined for statistical analysis . )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lesion sizes (left) were determined 5 days after C. higginsianum (Ch) inoculation. Invasion rates (right) of Ch on Arabidopsis leaves were determined 3 days after spore inoculation. An average of 3 replicates is shown (±SD) (left) and (±SE) (right). (■, p < 0.05 in two-tailed tests between the indicated samples. *, p < 0.05 in two-tailed tests compared to the corresponding WT values. Two independent experiments were combined for statistical analysis.)


B ) Immunoblot ( left ) and qRT - PCR ( right ) analyses following Ch challenge for the indicated times ( dpi ) . The numbers below the immunoblots represent relative intensities of the indicated bands normalized to the backgrounds , with the values at 0 dpi set as 1 . 0 . Experiments were performed three times with the same conclusions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot (left) and qRT-PCR (right) analyses following Ch challenge for the indicated times (dpi). The numbers below the immunoblots represent relative intensities of the indicated bands normalized to the backgrounds, with the values at 0 dpi set as 1.0. Experiments were performed three times with the same conclusions.


E . Microscale thermophoresis binding experiment for PlexA1 - 4 - mVenus and Sema1becto ( red ) or Sema1becto - mutA ( blue ) or Sema1becto - mutB ( green , ) or Sema1becto - mutA + B ( orange ) . The Sema1becto wild - type binding results are as reported previously ( Rozbesky et al . , 2019 ) . All data were collected at the same time , error bars represent s . d . of three technical replicates .

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

E. Microscale thermophoresis binding experiment for PlexA1-4-mVenus and Sema1becto (red) or Sema1becto-mutA (blue) or Sema1becto-mutB (green,) or Sema1becto-mutA+B (orange). The Sema1becto wild-type binding results are as reported previously (Rozbesky et al., 2019). All data were collected at the same time, error bars represent s.d. of three technical replicates.


A . FRET - FLIM in live COS - 7 cells shows cell surface PlexA - Sema1b cis interaction . Sema1b mutants reveal that both head - to - head and side - on orientations of the PlexA - Sema1b complex are involved in the cis interaction . The cis interaction between Sema1a and PlexA is not observed . The boxcharts represent the average lifetime . Box limits indicate the 25th and 75th percentiles , centred lines show the median , squares represent sample means , whiskers extend 1 . 5 - fold the interquartile range from the 25th and 75th percentiles , p - value was calculated by one - way analysis of variance ( ANOVA ) . * P < 0 . 05 , * * * P < 0 .

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A. FRET-FLIM in live COS-7 cells shows cell surface PlexA-Sema1b cis interaction. Sema1b mutants reveal that both head-to-head and side-on orientations of the PlexA-Sema1b complex are involved in the cis interaction. The cis interaction between Sema1a and PlexA is not observed. The boxcharts represent the average lifetime. Box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, p-value was calculated by one-way analysis of variance (ANOVA). *P<0.05, ***P<0.001


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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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

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


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

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

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


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

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


E ) Humanendothelial 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 ) .

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

E) 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 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 .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 + 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 ) .

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

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

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

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

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


D ) 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 .

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

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 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 .

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


A ) Bar graph demonstrating platelet aggregation induced by complexes of heparin , VEGF - A165 , 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 .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O 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 , and aqueous humor concentrations after single intravitreal administration of 0 . 5 mg of RG7716 in cynomolgus monkeys .

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


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

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


B ) Inhibition of neovascularization measured in severity grades , change of severity from baseline is shown for each treatment ; 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ) .

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

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


C ) 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 ) .

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


D ) IL - 8 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 .

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

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


A ) AlexaFluor488 - tagged ( green ) hnRNPA2 LC undergoes LLPS , while AlexaFluor555 - tagged ( red ) TOG D1 does not . However , TOG D1 partitions into hnRNPA2 LC droplets when mixed at a 1 : 1 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 .

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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 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 .

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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 ) 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 .

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

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


A ) After cleavage of a C - terminal maltose binding 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

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

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


D ) After 22 hours of paraquat induced oxidative stress , both HRPA - 1mScarlet 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 .

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

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.