Figure 9Spinal cord lysates were generated from fresh frozen tissue of 20 - month old mice either expressing GA149 - CFP under the Thy1 promoter ( Tgx GA149 ) or wild type controls . Lysates were immunoblotted for SV2 ( A ) Total protein levels were determined by imaging the Mini - PROTEAN TGX stain - free gel prior to transfer . Quantification of band intensities normalized to total protein loading revealed a significant reduction in SV2 protein levels in the GA - expressing cohort compared with wild type animals , Data presented as mean ± SEM . Unpaired t - test , * p < 0 . 05 . A total of 4 animals per group were examined . Spinal cord lysates were generated from fresh frozen tissue of 20 - month old mice either expressing GA149 - CFP under the Thy1 promoter ( Tgx GA149 ) or wild type controls . Lysates were immunoblotted for synaptophysin ( B ) Total protein levels were determined by imaging the Mini - PROTEAN TGX stain - free gel prior to transfer . synaptophysin levels remained unaltered . Data presented as mean ± SEM . Unpaired t - test , * p < 0 . 05 . A total of 4 animals per group were examined . Spinal cord lysates were generated from fresh frozen tissue of 20 - month old mice either expressing GA149 - CFP under the Thy1 promoter ( Tgx GA149 ) or wild type controls . Lysates were immunoblotted for PSD - 95 ( C ) . Total protein levels were determined by imaging the Mini - PROTEAN TGX stain - free gel prior to transfer . PSD - 95 levels remained unaltered . Data presented as mean ± SEM . Unpaired t - test , * p < 0 . 05 . A total of 4 animals per group were examined .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9Spinal cord lysates were generated from fresh frozen tissue of 20-month old mice either expressing GA149-CFP under the Thy1 promoter (Tgx GA149) or wild type controls. Lysates were immunoblotted for SV2 (A) Total protein levels were determined by imaging the Mini-PROTEAN TGX stain-free gel prior to transfer. Quantification of band intensities normalized to total protein loading revealed a significant reduction in SV2 protein levels in the GA-expressing cohort compared with wild type animals, Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 4 animals per group were examined.Spinal cord lysates were generated from fresh frozen tissue of 20-month old mice either expressing GA149-CFP under the Thy1 promoter (Tgx GA149) or wild type controls. Lysates were immunoblotted for synaptophysin (B) Total protein levels were determined by imaging the Mini-PROTEAN TGX stain-free gel prior to transfer. synaptophysin levels remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 4 animals per group were examined.Spinal cord lysates were generated from fresh frozen tissue of 20-month old mice either expressing GA149-CFP under the Thy1 promoter (Tgx GA149) or wild type controls. Lysates were immunoblotted for PSD-95 (C). Total protein levels were determined by imaging the Mini-PROTEAN TGX stain-free gel prior to transfer. PSD-95 levels remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 4 animals per group were examined.


Figure 10Muscle sections ( 30μm ) were cut from snap frozen tissue of 20 - month old GA149 mice ( Tgx GA149 ) or wild type controls . Immunohistochemical staining was performed to assess synaptic protein levels in proximity to NMJs . Sections were immunostained for SV2 or Synaptophysin , and counterstained with neurofilament . A ) Left Panels : Representative images show SV2 levels ( green ) in proximity to α - bungarotoxin positive NMJs ( red ) in wild type animals ( top ) versus GA149 animals ( bottom ) . Right Panels : Representative images show SV2 levels ( green ) overlapping with neurofilament ( blue ) at synaptic terminals approaching muscle in wild type animals ( top ) versus GA149 animals ( bottom ) . B - C ) Quantification of SV2 ( B ) or Synaptophysin ( C ) colocalized with neurofilament and normalized to neurofilament area . This analysis revealed a significant reduction in SV2 levels in GA149 animals compared with wild types , while synaptophysin remained unaltered . Data presented as mean ± SEM . Unpaired t - test , * p < 0 . 05 . A total of 10 non - overlapping regions of muscle from each of 5 animals per group were evaluated .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 10Muscle sections (30μm) were cut from snap frozen tissue of 20-month old GA149 mice (Tgx GA149) or wild type controls. Immunohistochemical staining was performed to assess synaptic protein levels in proximity to NMJs. Sections were immunostained for SV2 or Synaptophysin, and counterstained with neurofilament. A) Left Panels: Representative images show SV2 levels (green) in proximity to α-bungarotoxin positive NMJs (red) in wild type animals (top) versus GA149 animals (bottom). Right Panels: Representative images show SV2 levels (green) overlapping with neurofilament (blue) at synaptic terminals approaching muscle in wild type animals (top) versus GA149 animals (bottom). B-C) Quantification of SV2 (B) or Synaptophysin (C) colocalized with neurofilament and normalized to neurofilament area. This analysis revealed a significant reduction in SV2 levels in GA149 animals compared with wild types, while synaptophysin remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 10 non-overlapping regions of muscle from each of 5 animals per group were evaluated.


Figure 1A ) 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) 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).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).


Figure 2A , 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A,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.


Figure 3A ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) 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.


Figure 4A ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) 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).


Figure 5A ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) 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.


Figure 6B ) 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B) 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.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.


Figure 7Time - 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Time-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.


Figure 8A ) 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 . C ) Summary table of key experimental PK parameters of RG7716 and anti - VEGF - A / ANG - 2 - FcγR - ( with intact FcRn binding ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A) 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.C) Summary table of key experimental PK parameters of RG7716 and anti-VEGF-A/ANG-2-FcγR- (with intact FcRn binding).


Figure 9B ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9B) 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.


Figure 10A ) 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 10A) 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).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.


Figure 1B . Bimolecular fluorescence complementation between subunits of the Dsl complex and the COPI coat . The BiFC pair β ' - COPVN • Dsl3pVC yielded fluorescent foci that showed dramatically reduced fluorescence intensity in the COPI binding - deficient dsl1 - 5xWA mutant . Schematic yeast cell representations depict the typical BiFC foci localization patterns . Scale bar 10 μm . C . Quantification of the β ' - COPVN • Dsl3pVC BiFC signals in wild type cells and in COPI binding - deficient dsl1 - 5xWA and dsl1 - Δlasso mutants ( as shown exemplarily in ( B ) ) . Mean values + SEM of at least three independent experiments ( n = 3 - 8 ) are displayed . Statistical analysis was carried out by comparing the number of cells with BiFC signals over integrated thresholds to those without signals ( * = p < 0 , 05 ) . dsl1 mutant strains show a significant decrease in brightness , spot number and spot size in comparison with their corresponding DSL1 wild type strains ( two - sample t - test ) . The dsl1 - 5xWA mutation also led to comparable signal reduction in cells carrying the VNI152L variant in the β ' - COPVN I152L • Dsl3pVC combination . D . Subcellular localization of different BiFC - tagged protein pairs . The Dsl1pVN • Dsl3pVC combination involving subunits of the ER - resident Dsl1 complex exhibited typical ER localization . Sec16pVN • Sec16pVC signals in a diploid heterozygous strain displayed an pattern typical for ER exit sites , while diploid cells carrying the α - COPVN • α - COPVC BiFC pair showed a Golgi - like fluorescence pattern . Scale bar 10 μm . E . The localization of COPI • Dsl BiFC foci compared to the autophagy marker Ape1p . Double fluorescence micrographs of the BiFC pair β ' - COPVN • Dsl3pVC and the autophagy marker mRFPApe1p are superimposed with the DIC image . BiFC - YFP signals are pseudocolored green . Fluorescent signals appeared distinct from each other . Also no co - localization was observed when we analyzed COPI • COPII ( Sec16p ) BiFC spots in cells expressing mRFP tagged APE1 . Scale bar 10 µm . F . Growth effects of Venus fragment tags in dsl1 mutant cells . Serial tenfold dilutions of liquid cell cultures were spotted on agar plates and incubated at 30 ° C for two days . The images in the second row show that β ' COPVN producing cells expressing the dsl1 - 5xWA mutation could not grow . The complementation of β ' COPVN by its cognate interaction partners Dsl1pVC and Dsl3pVC , but not by non - cognate BiFC partners ( Sec24pVC , Sec16pVC or VCRer1p ) , suppressed these growth defects . Spot assays of exemplary BiFC partners are depicted . Asterisk : Dsl1pVC carrying the dsl1 - 5xWA defect was expressed from a plasmid . See Fig EV1 , EV2 and Appendix Fig S1 for full data display . G . BiFC signal quantifications of the strains presented in ( F ) . β ' - COPVN yielded BiFC fluorescence signals with all tested complementation partners . This rules out the possibility that the lack of suppression observed in F simply reflects the inability of the BiFC partners to form a complex , and shows that the successful complementation of the split - YFP fragments does not per se suppress the synthetic lethal effect of the VN tag in dsl1 - 5xWA cells . Mean values + SEM of at least three independent experiments ( n = 3 - 8 ) are displayed . H . BiFC signal intensities greatly depend on which BiFC combination is analyzed . Fluorescence intensities of COPI • Dsl BiFC pairs ( left part of H ) were much higher than those of COPII • Dsl BiFC pairs ( right part of H ) . I . Localization of Dsl1pCFP in β ' - COPVN • Dsl3pVCcells . An overall ER localization of Dsl1pCFP was retained in β ' - COPVN • Dsl3pVCcells , while some of the signal was found in the foci . BiFC - YFP signals are pseudocolored red . Scale bar 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Bimolecular fluorescence complementation between subunits of the Dsl complex and the COPI coat. The BiFC pair β'-COPVN•Dsl3pVC yielded fluorescent foci that showed dramatically reduced fluorescence intensity in the COPI binding-deficient dsl1-5xWA mutant. Schematic yeast cell representations depict the typical BiFC foci localization patterns. Scale bar 10 μm.C. Quantification of the β'-COPVN•Dsl3pVC BiFC signals in wild type cells and in COPI binding-deficient dsl1-5xWA and dsl1-Δlasso mutants (as shown exemplarily in (B)). Mean values + SEM of at least three independent experiments (n=3-8) are displayed. Statistical analysis was carried out by comparing the number of cells with BiFC signals over integrated thresholds to those without signals (*=p<0,05). dsl1 mutant strains show a significant decrease in brightness, spot number and spot size in comparison with their corresponding DSL1 wild type strains (two-sample t-test). The dsl1-5xWA mutation also led to comparable signal reduction in cells carrying the VNI152L variant in the β'-COPVN I152L•Dsl3pVC combination.D. Subcellular localization of different BiFC-tagged protein pairs. The Dsl1pVN•Dsl3pVC combination involving subunits of the ER-resident Dsl1 complex exhibited typical ER localization. Sec16pVN•Sec16pVC signals in a diploid heterozygous strain displayed an pattern typical for ER exit sites, while diploid cells carrying the α-COPVN•α-COPVC BiFC pair showed a Golgi-like fluorescence pattern. Scale bar 10 μm.E. The localization of COPI•Dsl BiFC foci compared to the autophagy marker Ape1p. Double fluorescence micrographs of the BiFC pair β'-COPVN•Dsl3pVC and the autophagy marker mRFPApe1p are superimposed with the DIC image. BiFC-YFP signals are pseudocolored green. Fluorescent signals appeared distinct from each other. Also no co-localization was observed when we analyzed COPI•COPII(Sec16p) BiFC spots in cells expressing mRFP tagged APE1. Scale bar 10 µm.F. Growth effects of Venus fragment tags in dsl1 mutant cells. Serial tenfold dilutions of liquid cell cultures were spotted on agar plates and incubated at 30 °C for two days. The images in the second row show that β'COPVN producing cells expressing the dsl1-5xWA mutation could not grow. The complementation of β'COPVN by its cognate interaction partners Dsl1pVC and Dsl3pVC, but not by non-cognate BiFC partners (Sec24pVC, Sec16pVC or VCRer1p), suppressed these growth defects. Spot assays of exemplary BiFC partners are depicted. Asterisk: Dsl1pVC carrying the dsl1-5xWA defect was expressed from a plasmid. See Fig EV1, EV2 and Appendix Fig S1 for full data display.G. BiFC signal quantifications of the strains presented in (F). β'-COPVN yielded BiFC fluorescence signals with all tested complementation partners. This rules out the possibility that the lack of suppression observed in F simply reflects the inability of the BiFC partners to form a complex, and shows that the successful complementation of the split-YFP fragments does not per se suppress the synthetic lethal effect of the VN tag in dsl1-5xWA cells. Mean values + SEM of at least three independent experiments (n=3-8) are displayed.H. BiFC signal intensities greatly depend on which BiFC combination is analyzed. Fluorescence intensities of COPI•Dsl BiFC pairs (left part of H) were much higher than those of COPII•Dsl BiFC pairs (right part of H).I. Localization of Dsl1pCFP in β'-COPVN•Dsl3pVCcells. An overall ER localization of Dsl1pCFP was retained in β'-COPVN•Dsl3pVCcells, while some of the signal was found in the foci. BiFC-YFP signals are pseudocolored red. Scale bar 10 µm.


Figure 2A . Dsl1pVN • ε - COPVC BiFC signal localization pattern throughout the budding cycle . Time - lapse images of agarose - embedded cells were recorded at RT with continuous PM Glc + ura medium supply . Arrowheads mark positions of BiFC foci . Fluorescence signals exhibited a characteristic and reproducible localization pattern , which favored sites of high material turnover during budding . Scale bar 10 µm . B . Dsl1pVN • ε - COPVC BiFC foci showed different subcellular distribution depending on the growth status of cells . Cells were cultured in PM + ura medium with or without 2 % glucose at 30 ° C for 90 min , and imaged . BiFC signals dispersed under glucose starvation , resulting in an ER exit site - like fluorescence pattern . Scale bar 10 µm . C . The effect of carbon source depletion on COPI • Dsl BiFC signal polarization . Cells carrying Dsl1pVN • ε - COPVC were starved in glucose - free PM + ura medium for 90 min . Then the medium was replaced by PM Glc + ura with 2 % glucose , while imaging the cells continuously at 1 min intervals . Two exemplary cells are shown ( upper and lower panel ) . Starvation - induced dispersed BiFC signals reversibly returned to polarized distribution within minutes after addition of glucose - containing medium . Arrowheads mark the point of signal repolarization , arrows indicate resumed bud outgrowth . Scale bar 5 µm . D . βʼ - COPVN • Dsl3pVC BiFC signals during mating . Fluorescence micrographs of the BiFC pair β ' - COPVN • Dsl3pVC ( green ) are superimposed with the DIC image ( grey ) . Two haploid strains carrying βʼ - COPVN • Dsl3pVC were allowed to mate in YEPD liquid culture for 2 h at 30 ° C . Then time - lapse recordings were made in agarose embedding at RT with continuous PM Glc + ura medium supply . Arrowheads mark positions of BiFC foci . Fluorescence signals exhibited a characteristic and reproducible localization at sites of high material turnover during zygote formation . Scale bar 10 µm . E . Comparison of the BiFC localization pattern in COPI • Dsl and COPI • COPI strains . Time - lapse images of agarose - embedded cells carrying the COPI • Dsl BiFC pair βʼ - COPVN • Dsl3pVC or the COPI • COPI BiFC pair βʼ - COPVN • α - COPVC were recorded at RT with continuous PM Glc + ura medium supply at 5 min intervals . Single cell pseudokymographs were generated by concatenating the central section of the cell ( sliced along the pole axis , frames at double the width of the bud neck ) of a time - lapse dataset . The COPI • Dsl strain showed one single dominant spot at the bud tip and later the bud neck . In contrast , the COPI • COPI strain additionally exhibited multiple spots with no apparent polarization tendency , which were localized mainly in the mother cell . Schematic yeast cell representations depict the typical BiFC foci localization pattern of the strains . Scale bar 10 µm . F . Fluorescence recovery after photobleaching of BiFC signals . Time - lapse images of agarose - embedded cells carrying the β ' - COPVN • Dsl3pVC BiFC pair were recorded at RT with PM Glc + ura medium supply at 2 min intervals . Fluorescence of ROIs marked in dashed lines ( where the full cell , full bud , or only the bud foci were bleached ) were depleted by three repetitions of fluorescence excitation at 177 µs / pixel and 100 % laser intensity . Arrowheads : reappearing fluorescence signal ; scale bar 2 µm . G . Densitometric measurements of fluorescence intensities in photobleached cells . Representative data sets from five independent experiments are displayed , featuring cells that had been bleached entirely ( black lines ) , at the bud only ( dark blue lines ) , or at the fluorescent foci at the bud tip only ( light blue line ) . Values are displayed in percentage of initial fluorescence , and were corrected for photobleaching effects by normalization against fluorescence of unbleached cells . Fluorescence recovery was typically not continued to full recovery stage because of the photobleaching artefacts occurring during long - term imaging of the samples .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Dsl1pVN•ε-COPVC BiFC signal localization pattern throughout the budding cycle. Time-lapse images of agarose-embedded cells were recorded at RT with continuous PM Glc+ura medium supply. Arrowheads mark positions of BiFC foci. Fluorescence signals exhibited a characteristic and reproducible localization pattern, which favored sites of high material turnover during budding. Scale bar 10 µm.B. Dsl1pVN•ε-COPVC BiFC foci showed different subcellular distribution depending on the growth status of cells. Cells were cultured in PM+ura medium with or without 2 % glucose at 30 °C for 90 min, and imaged. BiFC signals dispersed under glucose starvation, resulting in an ER exit site-like fluorescence pattern. Scale bar 10 µm.C. The effect of carbon source depletion on COPI•Dsl BiFC signal polarization. Cells carrying Dsl1pVN•ε-COPVC were starved in glucose-free PM+ura medium for 90 min. Then the medium was replaced by PM Glc+ura with 2 % glucose, while imaging the cells continuously at 1 min intervals. Two exemplary cells are shown (upper and lower panel). Starvation-induced dispersed BiFC signals reversibly returned to polarized distribution within minutes after addition of glucose-containing medium. Arrowheads mark the point of signal repolarization, arrows indicate resumed bud outgrowth. Scale bar 5 µm.D. βʼ-COPVN•Dsl3pVC BiFC signals during mating. Fluorescence micrographs of the BiFC pair β'-COPVN•Dsl3pVC (green) are superimposed with the DIC image (grey). Two haploid strains carrying βʼ-COPVN•Dsl3pVC were allowed to mate in YEPD liquid culture for 2 h at 30 °C. Then time-lapse recordings were made in agarose embedding at RT with continuous PM Glc+ura medium supply. Arrowheads mark positions of BiFC foci. Fluorescence signals exhibited a characteristic and reproducible localization at sites of high material turnover during zygote formation. Scale bar 10 µm.E. Comparison of the BiFC localization pattern in COPI•Dsl and COPI•COPI strains. Time-lapse images of agarose-embedded cells carrying the COPI•Dsl BiFC pair βʼ-COPVN•Dsl3pVC or the COPI•COPI BiFC pair βʼ-COPVN•α-COPVC were recorded at RT with continuous PM Glc+ura medium supply at 5 min intervals. Single cell pseudokymographs were generated by concatenating the central section of the cell (sliced along the pole axis, frames at double the width of the bud neck) of a time-lapse dataset. The COPI•Dsl strain showed one single dominant spot at the bud tip and later the bud neck. In contrast, the COPI•COPI strain additionally exhibited multiple spots with no apparent polarization tendency, which were localized mainly in the mother cell. Schematic yeast cell representations depict the typical BiFC foci localization pattern of the strains. Scale bar 10 µm.F. Fluorescence recovery after photobleaching of BiFC signals. Time-lapse images of agarose-embedded cells carrying the β'-COPVN•Dsl3pVC BiFC pair were recorded at RT with PM Glc+ura medium supply at 2 min intervals. Fluorescence of ROIs marked in dashed lines (where the full cell, full bud, or only the bud foci were bleached) were depleted by three repetitions of fluorescence excitation at 177 µs/pixel and 100 % laser intensity. Arrowheads: reappearing fluorescence signal; scale bar 2 µm.G. Densitometric measurements of fluorescence intensities in photobleached cells. Representative data sets from five independent experiments are displayed, featuring cells that had been bleached entirely (black lines), at the bud only (dark blue lines), or at the fluorescent foci at the bud tip only (light blue line). Values are displayed in percentage of initial fluorescence, and were corrected for photobleaching effects by normalization against fluorescence of unbleached cells. Fluorescence recovery was typically not continued to full recovery stage because of the photobleaching artefacts occurring during long-term imaging of the samples.


Figure 3Time - lapse micrographs of agarose - embedded cells were taken at RT with fresh PM Glc + ura medium supply throughout one budding cycle . Representative images of characteristically reoccurring fluorescence localization stages are displayed . In all combinations , fluorescence foci localize to areas of membrane outgrowth in the bud . Often , another prominent fluorescent spot is found in the mother cell on the side of the bud neck . Scale bar 5 µm . Kymographs of all time - lapse datasets shown in this Figure are presented in Fig EV3A . A - D . Cells expressing complementary BiFC - tagged proteins between subunits of the Dsl complex and COPI were analyzed . Panel ( D ) shows a BiFC combination that includes a mutated VC - tag . This mutation in the VC part is equivalent to the A206K mutation in GFP , which prevents the formation of YPF dimers ( Zacharias et al . , 2002 ) . Time - lapse micrographs of agarose - embedded cells were taken at RT with fresh PM Glc + ura medium supply throughout one budding cycle . Representative images of characteristically reoccurring fluorescence localization stages are displayed . In all combinations , fluorescence foci localize to areas of membrane outgrowth in the bud . Often , another prominent fluorescent spot is found in the mother cell on the side of the bud neck . Scale bar 5 µm . Kymographs of all time - lapse datasets shown in this Figure are presented in Fig EV3A . E , F . The VC - tagged COPI vesicle cargo protein Rer1p was examined in combination with the VN - tagged COPI subunit β ' - COP or the VN - tagged Dsl1 protein . Time - lapse micrographs of agarose - embedded cells were taken at RT with fresh PM Glc + ura medium supply throughout one budding cycle . Representative images of characteristically reoccurring fluorescence localization stages are displayed . In all combinations , fluorescence foci localize to areas of membrane outgrowth in the bud . Often , another prominent fluorescent spot is found in the mother cell on the side of the bud neck . Scale bar 5 µm . Kymographs of all time - lapse datasets shown in this Figure are presented in Fig EV3A . G . BiFC spots in COPI • COPII BiFC combination ( β ' - COPVN • Sec16pVC ) were also polarized . Time - lapse micrographs of agarose - embedded cells were taken at RT with fresh PM Glc + ura medium supply throughout one budding cycle . Representative images of characteristically reoccurring fluorescence localization stages are displayed . In all combinations , fluorescence foci localize to areas of membrane outgrowth in the bud . Often , another prominent fluorescent spot is found in the mother cell on the side of the bud neck . Scale bar 5 µm . Kymographs of all time - lapse datasets shown in this Figure are presented in Fig EV3A . H . COPI • COPI ( α - COPVN • α - COPVC ) BiFC signals showed one spot that localized to the bud tip and , at later budding stages , to the bud neck ( compare with A - G ) . Additionally , multiple spots with no apparent polarization tendency were observed , which are reminiscent of a Golgi pattern . Time - lapse micrographs of agarose - embedded cells were taken at RT with fresh PM Glc + ura medium supply throughout one budding cycle . Representative images of characteristically reoccurring fluorescence localization stages are displayed . In all combinations , fluorescence foci localize to areas of membrane outgrowth in the bud . Often , another prominent fluorescent spot is found in the mother cell on the side of the bud neck . Scale bar 5 µm . Kymographs of all time - lapse datasets shown in this Figure are presented in Fig EV3A . I . β ' - COPmGFPfluorescence foci in Dsl1p - depleted GAL - DSL1 cells after incubation in glucose - containing medium ( YEPD ) for 4 h at 30 ° C .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Time-lapse micrographs of agarose-embedded cells were taken at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Representative images of characteristically reoccurring fluorescence localization stages are displayed. In all combinations, fluorescence foci localize to areas of membrane outgrowth in the bud. Often, another prominent fluorescent spot is found in the mother cell on the side of the bud neck. Scale bar 5 µm. Kymographs of all time-lapse datasets shown in this Figure are presented in Fig EV3A.A-D. Cells expressing complementary BiFC-tagged proteins between subunits of the Dsl complex and COPI were analyzed. Panel (D) shows a BiFC combination that includes a mutated VC-tag. This mutation in the VC part is equivalent to the A206K mutation in GFP, which prevents the formation of YPF dimers (Zacharias et al., 2002).Time-lapse micrographs of agarose-embedded cells were taken at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Representative images of characteristically reoccurring fluorescence localization stages are displayed. In all combinations, fluorescence foci localize to areas of membrane outgrowth in the bud. Often, another prominent fluorescent spot is found in the mother cell on the side of the bud neck. Scale bar 5 µm. Kymographs of all time-lapse datasets shown in this Figure are presented in Fig EV3A.E, F. The VC-tagged COPI vesicle cargo protein Rer1p was examined in combination with the VN-tagged COPI subunit β'-COP or the VN-tagged Dsl1 protein.Time-lapse micrographs of agarose-embedded cells were taken at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Representative images of characteristically reoccurring fluorescence localization stages are displayed. In all combinations, fluorescence foci localize to areas of membrane outgrowth in the bud. Often, another prominent fluorescent spot is found in the mother cell on the side of the bud neck. Scale bar 5 µm.Kymographs of all time-lapse datasets shown in this Figure are presented in Fig EV3A.G. BiFC spots in COPI•COPII BiFC combination (β'-COPVN•Sec16pVC) were also polarized.Time-lapse micrographs of agarose-embedded cells were taken at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Representative images of characteristically reoccurring fluorescence localization stages are displayed. In all combinations, fluorescence foci localize to areas of membrane outgrowth in the bud. Often, another prominent fluorescent spot is found in the mother cell on the side of the bud neck. Scale bar 5 µm. Kymographs of all time-lapse datasets shown in this Figure are presented in Fig EV3A.H. COPI•COPI (α-COPVN•α-COPVC) BiFC signals showed one spot that localized to the bud tip and, at later budding stages, to the bud neck (compare with A-G). Additionally, multiple spots with no apparent polarization tendency were observed, which are reminiscent of a Golgi pattern.Time-lapse micrographs of agarose-embedded cells were taken at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Representative images of characteristically reoccurring fluorescence localization stages are displayed. In all combinations, fluorescence foci localize to areas of membrane outgrowth in the bud. Often, another prominent fluorescent spot is found in the mother cell on the side of the bud neck. Scale bar 5 µm. Kymographs of all time-lapse datasets shown in this Figure are presented in Fig EV3A.I. β'-COPmGFPfluorescence foci in Dsl1p-depleted GAL-DSL1 cells after incubation in glucose-containing medium (YEPD) for 4 h at 30 °C.


Figure 4Time - lapse images of agarose - embedded cells were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed cells immunostained for Sed5p . All cells produced the β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . A . RFPHDEL , ER markerTime - lapse images of agarose - embedded cells were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed cells immunostained for Sed5p . All cells produced the β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . B . Sec13pRFP , ER exit sites markerTime - lapse images of agarose - embedded cells were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed cells immunostained for Sed5p . All cells produced the β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . C . RFPSed5p , cis - Golgi markers ( genomic ) Time - lapse images of agarose - embedded cells were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed cells immunostained for Sed5p . All cells produced the β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . D . CFPGos1p , medial Golgi markerTime - lapse images of agarose - embedded cells were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed cells immunostained for Sed5p . All cells produced the β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . E . Sec7pCFP , trans - Golgi markerTime - lapse images of agarose - embedded cells were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed cells immunostained for Sed5p . All cells produced the β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . F . Individual fixed cells carrying the β ' - COPVN • Dsl3pVC BiFC pair along with trans - Golgi marker CFPSec7p were used for this panel . The β ' - COPVN • Dsl3pVC BiFC signals are pseudocolored white . Immunostaining was used to visualize the cis - Golgi marker Sed5p ( red ) . The trans - Golgi marker CFPSec7p is pseudocolored green . Time - lapse images of agarose - embedded cells were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed cells immunostained for Sed5p . All cells produced the β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . G . CFPRer1 , COPI cargoTime - lapse images of agarose - embedded cells were taken at RT with supply of fresh PM Glc + ura medium throughout one budding cycle . Only ( F ) shows fixed cells immunostained for Sed5p . All cells produced the β ' - COPVN • Dsl3pVC BiFC pair . Most strains contained plasmids encoding different organelle markers as indicated below . BiFC signals are pseudocolored green , organelle markers are pseudocolored red . Scale bar 5 µm . H . CFPSec4p , marker for secretory

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.A. RFPHDEL, ER markerTime-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.B. Sec13pRFP, ER exit sites markerTime-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.C. RFPSed5p, cis-Golgi markers (genomic)Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.D. CFPGos1p, medial Golgi markerTime-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.E. Sec7pCFP, trans-Golgi markerTime-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.F. Individual fixed cells carrying the β'-COPVN•Dsl3pVC BiFC pair along with trans-Golgi marker CFPSec7p were used for this panel. The β'-COPVN•Dsl3pVC BiFC signals are pseudocolored white. Immunostaining was used to visualize the cis-Golgi marker Sed5p (red). The trans-Golgi marker CFPSec7p is pseudocolored green.Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.G. CFPRer1, COPI cargoTime-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β'-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.H. CFPSec4p, marker for secretory vesicles


Figure 5A . Fluorescence micrographs of two different COPI • COPII BiFC pairs are shown : Sec16pVN • α - COPVC or Sec16pVN • ε - COPVC . DSL1 cells show dispersed punctate patterns resembling ER exit site patterns . In the dsl1 - 5xWA mutant , cells exhibit additional bright spots . Scale bar 10 µm . B . Quantification of fluorescence intensity differences of the strains shown in ( A ) . COPI • COPII BiFC fluorescence is significantly increased in cells carrying the dsl1 - 5xWA mutant as compared to DSL1 wild type . Mean values + SEM of at least nine independent experiments ( n = 918 ) are displayed ( two - sample unpaired twotailed ttest ) . C . Time - lapse micrographs of cells expressing the β ' - COPVN • Dsl3pVC BiFC pair in a dsl1 - 5xWA background . Cells were imaged embedded in agarose at RT with fresh PM Glc + ura medium supply throughout one budding cycle . Fluorescence signals retain the characteristic polarization pattern as seen DSL1 wild type cells ( see Fig 3 ) . Scale bar 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Fluorescence micrographs of two different COPI•COPII BiFC pairs are shown: Sec16pVN•α-COPVC or Sec16pVN•ε-COPVC. DSL1 cells show dispersed punctate patterns resembling ER exit site patterns. In the dsl1-5xWA mutant, cells exhibit additional bright spots. Scale bar 10 µm.B. Quantification of fluorescence intensity differences of the strains shown in (A). COPI•COPII BiFC fluorescence is significantly increased in cells carrying the dsl1-5xWA mutant as compared to DSL1 wild type. Mean values + SEM of at least nine independent experiments (n=918) are displayed (two-sample unpaired twotailed ttest).C. Time-lapse micrographs of cells expressing the β'-COPVN•Dsl3pVC BiFC pair in a dsl1-5xWA background. Cells were imaged embedded in agarose at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Fluorescence signals retain the characteristic polarization pattern as seen DSL1 wild type cells (see Fig 3). Scale bar 10 μm.


Figure 6A - B . Involvement of filamentous cytoskeletal structures in BiFC foci polarization . Time - lapse micrographs of agarose - embedded cells expressing the Dsl1pVN • ε - COPVC BiFC combination at RT with fresh PM Glc + ura medium supply . ( A ) Upon disruption of actin filaments with 1 , 25 μM latrunculin A , COPI • Dsl BiFC signals dispersed . Arrowheads : polarized signal , asterisks : unpolarized localization . Fluorescent images are displayed with inverted brightness values . Scale bar 10 μm . A - B . Involvement of filamentous cytoskeletal structures in BiFC foci polarization . Time - lapse micrographs of agarose - embedded cells expressing the Dsl1pVN • ε - COPVC BiFC combination at RT with fresh PM Glc + ura medium supply . ( B ) Microtubuli disruption through 24 μg / ml nocodazole did not affect polarization of the COPI • Dsl BiFC signals . Arrowheads : polarized signal , asterisks : unpolarized localization . Fluorescent images are displayed with inverted brightness values . Scale bar 10 μm . C - E . Involvement of myosin motors in BiFC foci polarization . Time - lapse micrographs of agarose - embedded cells carrying the COPI • Dsl BiFC pairs Dsl1pVN • ε - COPVC or β ' COPVN • Dsl3pVC were taken . Either wild type cells ( E ) or mutants carrying the myosin V mutations myo2 - 66 ( C ) or myo4 ∆ ( D ) were analyzed . Cells were grown at RT with fresh PM Glc + ura medium supply . In the myo2 - 66 cells , the BiFC signal polarization was hardly detectable even at permissive temperature , while it was unaffected in myo4 ∆ compared to the control cells . F . Fluorescence micrographs of β ' - COPVN • Dsl3pVCcells carrying the myo2 - 66 defect . COPI • Dsl BiFC , as well as ER or Golgi markers were analyzed . While the BiFC spots were dispersed in these mutants , they retained their association with ER and Golgi . Scale bar 5 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A-B. Involvement of filamentous cytoskeletal structures in BiFC foci polarization. Time-lapse micrographs of agarose-embedded cells expressing the Dsl1pVN•ε-COPVC BiFC combination at RT with fresh PM Glc+ura medium supply. (A) Upon disruption of actin filaments with 1,25 μM latrunculin A, COPI•Dsl BiFC signals dispersed. Arrowheads: polarized signal, asterisks: unpolarized localization. Fluorescent images are displayed with inverted brightness values. Scale bar 10 μm.A-B. Involvement of filamentous cytoskeletal structures in BiFC foci polarization. Time-lapse micrographs of agarose-embedded cells expressing the Dsl1pVN•ε-COPVC BiFC combination at RT with fresh PM Glc+ura medium supply. (B) Microtubuli disruption through 24 μg/ml nocodazole did not affect polarization of the COPI•Dsl BiFC signals. Arrowheads: polarized signal, asterisks: unpolarized localization. Fluorescent images are displayed with inverted brightness values. Scale bar 10 μm.C-E. Involvement of myosin motors in BiFC foci polarization. Time-lapse micrographs of agarose-embedded cells carrying the COPI•Dsl BiFC pairs Dsl1pVN•ε-COPVC or β'COPVN•Dsl3pVC were taken. Either wild type cells (E) or mutants carrying the myosin V mutations myo2-66 (C) or myo4∆ (D) were analyzed. Cells were grown at RT with fresh PM Glc+ura medium supply. In the myo2-66 cells, the BiFC signal polarization was hardly detectable even at permissive temperature, while it was unaffected in myo4∆ compared to the control cells.F. Fluorescence micrographs of β'-COPVN•Dsl3pVCcells carrying the myo2-66 defect. COPI•Dsl BiFC, as well as ER or Golgi markers were analyzed. While the BiFC spots were dispersed in these mutants, they retained their association with ER and Golgi. Scale bar 5 μm.


Figure 7A - B . Immuno - electron micrographs showing the buds and the neck region of two β ' - COPVN • Dsl3pVCcells . Both parts contain an overview image ( left ) and two zoomed details ( right ) . Slides were decorated with antibody against COPI and visualized with protein A - conjugated 10 nm gold particles . Gold particles mark electron - dense areas in apposition to membranes and / or vesicular structures , which are localized at the side of the bud and the bud neck of the mother cell . Scale bar 100 nm . C . Fluorescence micrograph of a budding cell carrying β ' - COPVN • Dsl3pVC ( green ) and RFPHDEL ( red ) . Arrowheads mark BiFC signals which may correspond to the localizations found in ( A ) and ( B ) . Scale bar 2 µm . D . COPI - positive , electron - dense structures in close apposition to perinuclear ER . 10 nm gold particles mark COPI . Scale bar 100 nm . E . Accumulation of COPI - positive vesicles . 10 nm gold particles mark COPI . PM : plasma membrane , nuc : nucleus . Scale bar 100 nm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A-B. Immuno-electron micrographs showing the buds and the neck region of two β'-COPVN•Dsl3pVCcells. Both parts contain an overview image (left) and two zoomed details (right). Slides were decorated with antibody against COPI and visualized with protein A-conjugated 10 nm gold particles. Gold particles mark electron-dense areas in apposition to membranes and/or vesicular structures, which are localized at the side of the bud and the bud neck of the mother cell. Scale bar 100 nm.C. Fluorescence micrograph of a budding cell carrying β'-COPVN•Dsl3pVC (green) and RFPHDEL (red). Arrowheads mark BiFC signals which may correspond to the localizations found in (A) and (B). Scale bar 2 µm.D. COPI-positive, electron-dense structures in close apposition to perinuclear ER. 10 nm gold particles mark COPI. Scale bar 100 nm.E. Accumulation of COPI-positive vesicles. 10 nm gold particles mark COPI. PM: plasma membrane, nuc: nucleus. Scale bar 100 nm.


Figure 1 ( A ) Immunofluorescence of HEK ‐ 293T cells that express TFEB - 3 × FLAG , subjected to the indicated treatments and stained with antibodies against FLAG and the lysosomal marker LAMP2 . The FLAG and LAMP2 channels are in green and red , respectively , in the merge . DAPI ( blue ) is included in the merge . Scale bars represent 10 μm . ( B ) Quantification of the number of cells with nuclear TFEB - 3 × FLAG in the four conditions in ( A ) . Each value represents mean ± s . d . from three independent fields with N = 300 . ( C ) Immunofluorescence of HEK ‐ 293T cells treated as indicated and stained with antibodies against endogenous TFEB and the lysosomal protein RagC ( green and red , respectively , in the merge ) . DAPI is included in the merge . Scale bars represent 10 μm . ( D ) Immunoblotting of proteins extracted from HeLa cells that express TFEB - 3 × FLAG treated with DMSO , chloroquine ( CQ ) or SalA , subjected to nuclear / cytosolic fractionation and blotted with antibody against FLAG to detect TFEB . H3 and tubulin were used as nuclear and cytosolic markers , respectively . Blots are representative of triplicate experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG, subjected to the indicated treatments and stained with antibodies against FLAG and the lysosomal marker LAMP2. The FLAG and LAMP2 channels are in green and red, respectively, in the merge. DAPI (blue) is included in the merge. Scale bars represent 10 μm.(B) Quantification of the number of cells with nuclear TFEB-3 × FLAG in the four conditions in (A). Each value represents mean±s.d. from three independent fields with N=300.(C) Immunofluorescence of HEK‐293T cells treated as indicated and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μm.(D) Immunoblotting of proteins extracted from HeLa cells that express TFEB-3 × FLAG treated with DMSO, chloroquine (CQ) or SalA, subjected to nuclear/cytosolic fractionation and blotted with antibody against FLAG to detect TFEB. H3 and tubulin were used as nuclear and cytosolic markers, respectively. Blots are representative of triplicate experiments.


Figure 2 ( A ) Lysosomal stress inhibits mTOR signalling . Immunoblotting of protein extracts isolated from HeLa cells treated overnight as indicated . Membranes were probed with antibodies against p ‐ T202 / Y204 ‐ ERK1 / 2 , ERK1 / 2 , p ‐ T389 ‐ S6K , and S6K to measure ERK and mTORC1 activities . ( B ) Torin 1 induces TFEB dephosphorylation and nuclear translocation . FLAG immunoblotting of cytosolic and nuclear fractions isolated from TFEB - 3 × FLAG HeLa cells starved in amino acid ‐ free media and subsequently stimulated as indicated for at least 3 h . Correct subcellular fractionation was verified with H3 and tubulin antibodies . ( C ) Effects of ERK and mTOR inhibitors on TFEB nuclear translocation . TFEB - GFP HeLa cells were seeded in 96 ‐ well plates , cultured for 12 h , and then treated with the indicated concentrations of the ERK inhibitor U0126 , or the mTOR inhibitors Rapamycin , Torin 1 , and Torin 2 . After 3 h at 37 ° C , cells were processed and images were acquired using the OPERA automated confocal microscope ( Perkin ‐ Elmer ) . Scale bars represent 30 μm . ( D ) Dose - response curves of the effects of ERK and mTOR inhibitors on TFEBnuclear translocation . TFEB - GFPHeLa cells were seeded in 384 ‐ well plates , cultured for 12 h , and treated with 10 different concentrations of the ERK inhibitor U0126 , or the mTOR inhibitors Rapamycin , Torin 1 , and Torin 2 ranging from 2 . 54 nM to 50 μM . The graph shows the percentage of nuclear translocation at the different concentrations of each compound ( in log of the concentration ) . The EC50 for each compound was calculated using Prism software ( see Materials and methods for details ) . ( E ) Immunofluorescence of HEK ‐ 293T cells treated with DMSO or Torin 1 and stained with antibodies against endogenous TFEB and the lysosomal protein RagC ( green and red , respectively , in the merge ) . DAPI is included in the merge . Scale bars represent 10 μM . ( F ) Rag GTPase knockdown induces TFEB nuclear translocation . HeLa cells stably expressing TFEB - 3 × FLAG were infected with lentiviruses encoding Short hairpin ( Sh ‐ ) RNAs targeting luciferase ( control ) or RagC and RagD mRNAs . In all samples , 96 h post infection , cells were left untreated ( N = normal media ) , starved ( S = starved media ) or treated with Torin 1 ( T = Torin 1 ) for 4 h and then subjected to nuclear / cytosolic fractionation . TFEB localization was detected with a FLAG antibody , whereas tubulin and H3 were used as controls for the cytosolic and nuclear fraction , respectively ; levels of S6K phosphorylation were used to test RagC and RagD knockdown efficiency . ( G ) Loss of mTORC2 does not affect TFEB phosphorylation . Mouse embryonic fibroblasts ( MEFs ) isolated from Sin1 − / − or control embryos ( E14 . 5 ) were infected with a retrovirus encoding TFEB - 3 × FLAG ; 48 h post infection , cells were treated with Torin 1 ( T ) for 4 h where indicated , subjected to nuclear / cytosolic fractionation and immunoblotted for FLAG , tubulin , and H3 . ( H ) Binding of TFEB to mTORC1 . HEK ‐ 293T cells that express TFEB - 3 × FLAG were lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR , the mTORC1 subunit raptor and the mTORC2 components rictor and Sin1 . FLAG - Rap2A served as negative control .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Lysosomal stress inhibits mTOR signalling. Immunoblotting of protein extracts isolated from HeLa cells treated overnight as indicated. Membranes were probed with antibodies against p‐T202/Y204‐ERK1/2, ERK1/2, p‐T389‐S6K, and S6K to measure ERK and mTORC1 activities.(B) Torin 1 induces TFEB dephosphorylation and nuclear translocation. FLAG immunoblotting of cytosolic and nuclear fractions isolated from TFEB-3 × FLAG HeLa cells starved in amino acid‐free media and subsequently stimulated as indicated for at least 3 h. Correct subcellular fractionation was verified with H3 and tubulin antibodies.(C) Effects of ERK and mTOR inhibitors on TFEB nuclear translocation. TFEB-GFP HeLa cells were seeded in 96‐well plates, cultured for 12 h, and then treated with the indicated concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2. After 3 h at 37°C, cells were processed and images were acquired using the OPERA automated confocal microscope (Perkin‐Elmer). Scale bars represent 30 μm.(D) Dose-response curves of the effects of ERK and mTOR inhibitors on TFEBnuclear translocation. TFEB-GFPHeLa cells were seeded in 384‐well plates, cultured for 12 h, and treated with 10 different concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2 ranging from 2.54 nM to 50 μM. The graph shows the percentage of nuclear translocation at the different concentrations of each compound (in log of the concentration). The EC50 for each compound was calculated using Prism software (see Materials and methods for details).(E) Immunofluorescence of HEK‐293T cells treated with DMSO or Torin 1 and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μM.(F) Rag GTPase knockdown induces TFEB nuclear translocation. HeLa cells stably expressing TFEB-3 × FLAG were infected with lentiviruses encoding Short hairpin (Sh‐) RNAs targeting luciferase (control) or RagC and RagD mRNAs. In all samples, 96 h post infection, cells were left untreated (N=normal media), starved (S=starved media) or treated with Torin 1 (T=Torin 1) for 4 h and then subjected to nuclear/cytosolic fractionation. TFEB localization was detected with a FLAG antibody, whereas tubulin and H3 were used as controls for the cytosolic and nuclear fraction, respectively; levels of S6K phosphorylation were used to test RagC and RagD knockdown efficiency.(G) Loss of mTORC2 does not affect TFEB phosphorylation. Mouse embryonic fibroblasts (MEFs) isolated from Sin1−/− or control embryos (E14.5) were infected with a retrovirus encoding TFEB-3 × FLAG; 48 h post infection, cells were treated with Torin 1 (T) for 4 h where indicated, subjected to nuclear/cytosolic fractionation and immunoblotted for FLAG, tubulin, and H3.(H) Binding of TFEB to mTORC1. HEK‐293T cells that express TFEB-3 × FLAG were lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR, the mTORC1 subunit raptor and the mTORC2 components rictor and Sin1. FLAG-Rap2A served as negative control.


Figure 3 ( A ) Torin 1 induces S142 dephosphorylation . HeLa cells were treated as indicated and total and nuclear extracts were probed with a TFEB p ‐ S142 phosphoantibody and with anti ‐ FLAG antibody . Disappearance of TFEB S142 phosphorylation upon starvation or Torin 1 treatment correlates with accumulation of TFEB in the nuclear fraction . ( B ) mTORC1 in ‐ vitro kinase assays . Highly purified FLAG - S6K1 , TFEB - 3 × FLAG , or TFEBS142A - 3 × FLAG were incubated with radiolabelled ATP without kinase , with purified mTORC1 or with mTORC1 + Torin 1 , and analysed by autoradiography . The lower panel shows a FLAG immunoblot of the substrates . ( D ) Sequence conservation scores of the phosphosites and quantitative agreement between mTOR consensus motif and the sequence around the phosphosites of TFEB . ( E ) S142 and S211 regulate TFEB localization . FLAGimmunostaining ( red ) of HeLa cells expressing serine ‐ to ‐ alanine mutated versions of TFEB - 3 × FLAG . Nuclei were stained with DAPI ( blue ) . Values are means of five fields containing at least 50 transfected cells . Student ' s t ‐ test ( unpaired ) * * * P0 . 001 . Scale bars represent 30 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Torin 1 induces S142 dephosphorylation. HeLa cells were treated as indicated and total and nuclear extracts were probed with a TFEB p‐S142 phosphoantibody and with anti‐FLAG antibody. Disappearance of TFEB S142 phosphorylation upon starvation or Torin 1 treatment correlates with accumulation of TFEB in the nuclear fraction.(B) mTORC1 in‐vitro kinase assays. Highly purified FLAG-S6K1, TFEB-3 × FLAG, or TFEBS142A-3 × FLAG were incubated with radiolabelled ATP without kinase, with purified mTORC1 or with mTORC1+Torin 1, and analysed by autoradiography. The lower panel shows a FLAG immunoblot of the substrates.(D) Sequence conservation scores of the phosphosites and quantitative agreement between mTOR consensus motif and the sequence around the phosphosites of TFEB.(E) S142 and S211 regulate TFEB localization. FLAGimmunostaining (red) of HeLa cells expressing serine‐to‐alanine mutated versions of TFEB-3 × FLAG. Nuclei were stained with DAPI (blue). Values are means of five fields containing at least 50 transfected cells. Student's t‐test (unpaired) ***P0.001. Scale bars represent 30 μm.