Figure 6 ( A ) Expression relative to GAPDH of pro - inflammatory cytokines ( TNFA , IL1B , IL6 ) , interferon genes ( IFNB , IFNL1 , IFNL3 ) and interferon stimulating genes ( IP10 , ISG15 ) in the AT and endothelial layer of infected LoCs at 1 dpi reconstituted without macrophages ( n = 3 biological replicates for IFNB , IFNL1 and IFNL3 in the AT layer , n = 4 biological replicates for all other data ) . ( B ) Fold - change in the markers in ( A ) relative to uninfected controls ( n = 3 biological replicates ) ( data in Appendix Fig . S12 ) at the same timepoint . The bar represents the mean , and the error bars the standard deviation . ( Data information : The bar represents the mean , and the error bars the standard deviation . P - values are calculated using a Kruskal - Wallis One - Way ANOVA Test , * represents p ≤ 0 . 05 , * * represents p ≤ 0 . 01 , * * represents p ≤ 0 . 001 . ( D ) Expression of IL6R and ADAM17 relative to GAPDH in the AT and endothelial layer of infected LoCs at 1 dpi ( n = 3 biological replicates for the AT layer and n = 4 biological replicates for endothelial layer ) . ( E ) Fold - change in expression of IL6 and ADAM17 in the epithelial and endothelial layers of infected LoCs at 1 dpi relative to uninfected controls ( n = 3 biological replicates for both layers ) . ( Data information : The bar represents the mean , and the error bars the standard deviation . P - values are calculated using a Kruskal - Wallis One - Way ANOVA Test , * represents p ≤ 0 . 05 , * * represents p ≤ 0 . 01 , * * represents p ≤ 0 . 001 . ( F - I ) 3D views of representative 232 x 232 μm2 fields of view of the AT ( F , H ) and endothelial layer ( G , I ) from infected LoCs reconstituted with macrophages at 1 ( F , G ) and 3 dpi ( H , I ) . IL6 mRNA , and nuclear staining are indicated with the chartreuse and electric indigo LUTs , respectively .

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 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 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

Figure 6(A) Expression relative to GAPDH of pro-inflammatory cytokines (TNFA, IL1B, IL6), interferon genes (IFNB, IFNL1, IFNL3) and interferon stimulating genes (IP10, ISG15) in the AT and endothelial layer of infected LoCs at 1 dpi reconstituted without macrophages (n=3 biological replicates for IFNB, IFNL1 and IFNL3 in the AT layer, n=4 biological replicates for all other data). (B) Fold-change in the markers in (A) relative to uninfected controls (n=3 biological replicates) (data in Appendix Fig. S12) at the same timepoint. The bar represents the mean, and the error bars the standard deviation. ( Data information: The bar represents the mean, and the error bars the standard deviation. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001.(D) Expression of IL6R and ADAM17 relative to GAPDH in the AT and endothelial layer of infected LoCs at 1 dpi (n=3 biological replicates for the AT layer and n=4 biological replicates for endothelial layer). (E) Fold-change in expression of IL6 and ADAM17 in the epithelial and endothelial layers of infected LoCs at 1 dpi relative to uninfected controls (n=3 biological replicates for both layers). ( Data information: The bar represents the mean, and the error bars the standard deviation. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001.(F-I) 3D views of representative 232 x 232 μm2 fields of view of the AT (F, H) and endothelial layer (G, I) from infected LoCs reconstituted with macrophages at 1 (F, G) and 3 dpi (H, I). IL6 mRNA, and nuclear staining are indicated with the chartreuse and electric indigo LUTs, respectively.


Figure 7 ( A - F ) Maximum intensity projections of representative 232 x 232 μm2 fields of view of the endothelial layer from an infected LoC reconstituted without macrophages at 2 dpi with ( A ) and without Tocilizumab treatment ( D ) . Actin and nuclear staining are indicated with the azure and electric indigo LUTs respectively , the actin staining is saturated for the endothelial cell clusters to highlight cells with lower levels of actin . ( B , C ) Zooms corresponding to the region in ( A ) highlighted with white and yellow boxes , respectively . ( B ) A region with normal vascular profile , ( C ) A region of endothelial cell cluster formation with increased actin staining . ( E , F ) Zooms corresponding to the regions in ( D ) representing regions highlighted with white and yellow boxes , respectively . ( E ) An example of reduced tight junctions between endothelial cells ; bare patches are indicated with arrows . ( F ) A region of endothelial cell clusters . Scale bar = 20 μm . ( G , H ) 3D views from two 232 x 232 μm2 field of views of the endothelial layer of a Tocilizumab treated infected LoC at 2 dpi , immunostaining for the tight junction protein CD31 is shown in the Spring Green LUT . Endothelial cell clusters with low CD31 expression and altered actin staining are also visible and indicated by yellow arrows .

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 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

Figure 7(A-F) Maximum intensity projections of representative 232 x 232 μm2 fields of view of the endothelial layer from an infected LoC reconstituted without macrophages at 2 dpi with (A) and without Tocilizumab treatment (D). Actin and nuclear staining are indicated with the azure and electric indigo LUTs respectively, the actin staining is saturated for the endothelial cell clusters to highlight cells with lower levels of actin. (B, C) Zooms corresponding to the region in (A) highlighted with white and yellow boxes, respectively. (B) A region with normal vascular profile, (C) A region of endothelial cell cluster formation with increased actin staining. (E, F) Zooms corresponding to the regions in (D) representing regions highlighted with white and yellow boxes, respectively. (E) An example of reduced tight junctions between endothelial cells; bare patches are indicated with arrows. (F) A region of endothelial cell clusters. Scale bar = 20 μm.(G, H) 3D views from two 232 x 232 μm2 field of views of the endothelial layer of a Tocilizumab treated infected LoC at 2 dpi, immunostaining for the tight junction protein CD31 is shown in the Spring Green LUT. Endothelial cell clusters with low CD31 expression and altered actin staining are also visible and indicated by yellow arrows.


Figure 1A Immunoblot analysis of HeLa cells using the indicated antibodies that were synchronized in G1 / S phase using a double thymidine block or in mitosis using sequential thymidine and nocodazole treatment and collected at the given time points . B Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides as indicated and left untreated or synchronized in mitosis using sequential thymidine and nocodazole treatment . C Co - immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were transfected with FLAG - tagged XIAP or empty vector ( EV ) and synchronized in mitosis using nocodazole or left untreated . D Co - immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were treated as in ( C ) , but with additional transfection of the FLAG - tagged XIAP single amino acid mutants G188R and G188E . Only mitotic samples are shown . The asterisk denotes ubiquitylated forms of XIAP . E In vivo ubiquitylation of XIAP in HeLa cells that were infected with the indicated expression constructs carrying FLAG - tagged XIAP and transfected with siRNA oligonucleotides as specified . Cells were synchronized in mitosis using sequential thymidine / nocodazole treatment , as indicated . Subsequent to treatment with MG132 , whole cell extracts ( WCE ) were prepared and ubiquitylated XIAP was isolated by anti - FLAGimmunoprecipitation ( IP ) under denaturing conditions . F Immunoblot analysis of NIH 3T3 cells that were transfected with expression constructs for USP9XWT or the catalytically inactive mutant USP9XC1556S . The band in the EV control lane of the anti - V5 panel marks an unspecific band produced by the antibody . G Immunoblot analysis of HeLa cells using antibodies to the indicated endogenous proteins that were synchronized in mitosis using thymidine / nocodazole and treated with DMSO or the USP9X inhibitor WP1130 as indicated .

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 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 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 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 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 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 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

Figure 1A Immunoblot analysis of HeLa cells using the indicated antibodies that were synchronized in G1/S phase using a double thymidine block or in mitosis using sequential thymidine and nocodazole treatment and collected at the given time points.B Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides as indicated and left untreated or synchronized in mitosis using sequential thymidine and nocodazole treatment.C Co-immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were transfected with FLAG-tagged XIAP or empty vector (EV) and synchronized in mitosis using nocodazole or left untreated.D Co-immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were treated as in (C), but with additional transfection of the FLAG-tagged XIAP single amino acid mutants G188R and G188E. Only mitotic samples are shown. The asterisk denotes ubiquitylated forms of XIAP.E In vivo ubiquitylation of XIAP in HeLa cells that were infected with the indicated expression constructs carrying FLAG-tagged XIAP and transfected with siRNA oligonucleotides as specified. Cells were synchronized in mitosis using sequential thymidine/nocodazole treatment, as indicated. Subsequent to treatment with MG132, whole cell extracts (WCE) were prepared and ubiquitylated XIAP was isolated by anti-FLAGimmunoprecipitation (IP) under denaturing conditions.F Immunoblot analysis of NIH 3T3 cells that were transfected with expression constructs for USP9XWT or the catalytically inactive mutant USP9XC1556S. The band in the EV control lane of the anti-V5 panel marks an unspecific band produced by the antibody.G Immunoblot analysis of HeLa cells using antibodies to the indicated endogenous proteins that were synchronized in mitosis using thymidine/nocodazole and treated with DMSO or the USP9X inhibitor WP1130 as indicated.


Figure 2A Immunoblot analysis of WT , Xiap - / - , Mcl1 - / - or cIap2 - / - mouse embryonic fibroblasts that were lentivirally infected with shRNA constructs directed against a non - relevant mRNA ( Ctrl ) or against Usp9X mRNA and treated with taxol as specified . B Two - dimensional cell - cycle analysis ( BrdU / PI ) of cells described in ( A ) . Sub G1 and G2 / M fractions of cells were quantified and averaged with two additional , independent experiments ( n = 3 , ± SD ; WT : * * , P = 0 . 0016 ; * * * , P = 0 . 0004 ; Mcl1 - / - : * * * , P = 0 . 0003 ; * * , P = 0 . 0017 ; cIap2 - / - : * * , P = 0 . 0043 , Student ' s t test ) . Black bars exemplify shRNA Ctrl , white bars shRNA Usp9X samples . C Immunoblot analysis of HeLa cells transfected with a FLAG - tagged USP9X expression construct or empty vector ( EV ) and treated with taxol for the indicated times . D Immunoblot analysis of HeLa cells that were transfected with a FLAG - tagged XIAP expression construct or empty vector and treated as in ( C ) . E Two dimensional cell - cycle analysis ( BrdU / PI ) of cells described in ( C ) and ( D ) . Apoptotic indices represent ratios of sub G1 to G1 / S cells and are shown for analyses at the indicated timepoints . * * , P = 0 . 0020 ; * * * , P = 0 . 0004 ; Student ' s t test . F Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides directed against a non - relevant mRNA ( Ctrl ) or against Xiap mRNA and treated with taxol as specified .

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 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 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 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 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 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

Figure 2A Immunoblot analysis of WT, Xiap-/-, Mcl1-/- or cIap2-/- mouse embryonic fibroblasts that were lentivirally infected with shRNA constructs directed against a non-relevant mRNA (Ctrl) or against Usp9X mRNA and treated with taxol as specified.B Two-dimensional cell-cycle analysis (BrdU/PI) of cells described in (A). Sub G1 and G2/M fractions of cells were quantified and averaged with two additional, independent experiments (n=3, ± SD; WT: **, P = 0.0016; ***, P = 0.0004; Mcl1-/-: ***, P = 0.0003; **, P = 0.0017; cIap2-/-: **, P = 0.0043, Student's t test). Black bars exemplify shRNA Ctrl, white bars shRNA Usp9X samples.C Immunoblot analysis of HeLa cells transfected with a FLAG-tagged USP9X expression construct or empty vector (EV) and treated with taxol for the indicated times.D Immunoblot analysis of HeLa cells that were transfected with a FLAG-tagged XIAP expression construct or empty vector and treated as in (C).E Two dimensional cell-cycle analysis (BrdU/PI) of cells described in (C) and (D). Apoptotic indices represent ratios of sub G1 to G1/S cells and are shown for analyses at the indicated timepoints. **, P = 0.0020; ***, P = 0.0004; Student's t test.F Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides directed against a non-relevant mRNA (Ctrl) or against Xiap mRNA and treated with taxol as specified.


Figure 3A Immunoblot analysis of sorted Eμ - Myc lymphoma cells that were retrovirally transduced with the indicated IRES - GFP shRNA constructs . B Kaplan Meier survival curves of mice that were injected intravenously with syngeneic Eμ - Myc lymphoma cells transduced with the indicated shRNA constructs as described in ( A ) . Prior to injection , cells were sorted for viability ( propidium iodide negative ) and GFP positivity ( sh _ Ctrl , black , n = 6 ; sh _ USP9X , gray , n = 5 ) . * P = 0 . 0293 ; Mantel - Cox test . C Kaplan - Meier survival curves of mice that were injected with syngeneic Eμ - Myc lymphoma cells transduced with the indicated shRNA constructs as described in ( A ) and treated as in ( B ) ( sh _ Ctrl , black , n = 6 ; sh _ XIAP , gray , n = 6 ) . * * , P = 0 . 0020 ; Mantel - Cox test . D Histopathology of representative lymph nodes derived from mice shown in ( B ) to visualize histomorphology by haematoxylin / eosin ( H & amp ; amp ; E ) , B - cell origin ( B220 ) , and spontaneous apoptosis ( cleaved Caspase 3 ) in situ . Scale bars denote 50 μm and 20 μm for insets . E Histopathology of representative lymph nodes derived from mice shown in ( C ) which were stained as described in ( D ) . F Mean tumor burden in mice injected with Eμ - Myc lymphoma cells modified as in ( B ) and ( C ) , 15 days after injection , assessed by PET imaging ( 4 animals per group ) . * * , P = 0 . 0025 ; * * * , P = 0 . 0003 , student ' s t - testG Representative PET images of animals , injected with Eμ - Myc lymphoma cells modified as in ( B ) and ( C ) , 15 days after injection ( pre treatment ) and three days following intraperitoneal vincristine administration ( post treatment ) . Bone marrow infiltration , representative for systemic lymphoma manifestation , is indicated by a blue asterisk . H Mean change in standardized 18FDG uptake of ROI between pre and post treatment imaging corrected for initial tumor burden ( sh _ Ctrl , black , n = 6 ; sh _ Usp9X , white , n = 8 ; sh _ Xiap , grey , n = 8 ) . Within the sh _ Usp9X and sh _ Xiap groups all animals lived up to control imaging , while two of eight animals of the sh _ ctrl group had to be sacrificed prematurely because they had reached the predefined criteria of maximum tumor burden . These animals had to be excluded from final analysis , suggesting an underestimation of the actual effect . Data was obtained from two independent experiments . * * * * , P < 0 . 0001 ; * , P = 0 . 0167 , student ' s t -

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 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 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 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 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 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

Figure 3A Immunoblot analysis of sorted Eμ-Myc lymphoma cells that were retrovirally transduced with the indicated IRES-GFP shRNA constructs.B Kaplan Meier survival curves of mice that were injected intravenously with syngeneic Eμ-Myc lymphoma cells transduced with the indicated shRNA constructs as described in (A). Prior to injection, cells were sorted for viability (propidium iodide negative) and GFP positivity (sh_Ctrl, black, n = 6; sh_USP9X, gray, n = 5). *P = 0.0293; Mantel-Cox test.C Kaplan-Meier survival curves of mice that were injected with syngeneic Eμ-Myc lymphoma cells transduced with the indicated shRNA constructs as described in (A) and treated as in (B) (sh_Ctrl, black, n = 6; sh_XIAP, gray, n = 6). **, P = 0.0020; Mantel-Cox test.D Histopathology of representative lymph nodes derived from mice shown in (B) to visualize histomorphology by haematoxylin/eosin (H&amp;E), B-cell origin (B220), and spontaneous apoptosis (cleaved Caspase 3) in situ. Scale bars denote 50 μm and 20 μm for insets.E Histopathology of representative lymph nodes derived from mice shown in (C) which were stained as described in (D).F Mean tumor burden in mice injected with Eμ-Myc lymphoma cells modified as in (B) and (C), 15 days after injection, assessed by PET imaging (4 animals per group). **, P = 0.0025; ***, P = 0.0003, student's t-testG Representative PET images of animals, injected with Eμ-Myc lymphoma cells modified as in (B) and (C), 15 days after injection (pre treatment) and three days following intraperitoneal vincristine administration (post treatment). Bone marrow infiltration, representative for systemic lymphoma manifestation, is indicated by a blue asterisk.H Mean change in standardized 18FDG uptake of ROI between pre and post treatment imaging corrected for initial tumor burden (sh_Ctrl, black, n=6; sh_Usp9X, white, n=8; sh_Xiap, grey, n=8). Within the sh_Usp9X and sh_Xiap groups all animals lived up to control imaging, while two of eight animals of the sh_ctrl group had to be sacrificed prematurely because they had reached the predefined criteria of maximum tumor burden. These animals had to be excluded from final analysis, suggesting an underestimation of the actual effect. Data was obtained from two independent experiments. ****, P < 0.0001; *, P = 0.0167, student's t-test


Figure 4A Immunoblot analyses of different human DLBCL lines using antibodies to the indicated proteins . B FACS analysis ( propidium iodide ( PI ) ) of USP9X high and low expressing DLBCL cells shown in ( A ) to determine cell survival . Cells were treated with taxol and collected at the specified time points . PI negative cells are indicated relative to time point 0 ( n = 3 ± SD ) . C Immunoblot analyses of the indicated DLBCL cell lines that were lentivirally transduced with IRES - GFP shRNA constructs against USP9X or a non - relevant mRNA , FACS sorted for GFP + PI - cells and exposed to taxol for the indicated periods of time . The quantification of cleaved caspase 3 ( CC3 ) at the 24 hr post taxol treatment timepoint from four independent experiments is quantified in the graph on the right side and normalized to control shRNA . * * , P = 0 . 0099 , student ' s t - testD Immunoblot analyses of the indicated DLBCL cell lines that were exposed to taxol for the indicated periods of time . Three hours before collecting , WP1130 at a concentration of 5μM or DMSO was added as specified . E FACS analysis ( propidium iodide ( PI ) uptake ) of DLBCL cell lines treated with taxol and / or the SMAC mimetic BV6 as indicated . Results displayed are from three independent experiments ( n = 3 ± SD ) . * * * , P = 0 . 00085 ; * * * , P = 0 . 00529 ; Student ' s t test . F Collective assessment of USP9X and XIAP expression determined by immunohistochemistry in tissue microarrays ( TMA ) derived from a patient cohort representing 121 evaluable human CD20 + aggressive B - cell lymphomas . High USP9X expression was identified in 37 cases ( 31 % ) and significantly correlated with high XIAP expression levels ( * * , P = 0 . 004 ; Student ' s t test ) . G Expression data derived from ( E ) was correlated with clinical follow - up . Within a cohort of 58 patients receiving vincristine - containing chemotherapy ( CHOP regimen : cyclophosphamide , doxorubicin , vincristine , prednisone ) without the addition of the anti - CD20 antibody rituximab ( left panel ) overexpression of USP9X and XIAP ( n = 14 , straight line ) was associated with a significantly shortened event - free survival ( EFS ) as compared to all remaining patients ( n = 44 , dotted line ) ( * , P = 0 . 050 ; log rank test ) . Within a cohort of 63 patients receiving the CHOP regimen with addition of the anti - CD20 antibody rituximab overexpression of USP9X and XIAP ( n = 14 , straight line ) was not associated with a shortened event - free survival ( EFS ) as compared to all remaining patients ( n = 49 , dotted line ) ( P = 0 . 454 ) .

O B-PANEL_START 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 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 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 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 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

Figure 4A Immunoblot analyses of different human DLBCL lines using antibodies to the indicated proteins.B FACS analysis (propidium iodide (PI)) of USP9X high and low expressing DLBCL cells shown in (A) to determine cell survival. Cells were treated with taxol and collected at the specified time points. PI negative cells are indicated relative to time point 0 (n=3 ± SD).C Immunoblot analyses of the indicated DLBCL cell lines that were lentivirally transduced with IRES-GFP shRNA constructs against USP9X or a non-relevant mRNA, FACS sorted for GFP+ PI- cells and exposed to taxol for the indicated periods of time. The quantification of cleaved caspase 3 (CC3) at the 24 hr post taxol treatment timepoint from four independent experiments is quantified in the graph on the right side and normalized to control shRNA. **, P = 0.0099, student's t-testD Immunoblot analyses of the indicated DLBCL cell lines that were exposed to taxol for the indicated periods of time. Three hours before collecting, WP1130 at a concentration of 5μM or DMSO was added as specified.E FACS analysis (propidium iodide (PI) uptake) of DLBCL cell lines treated with taxol and/or the SMAC mimetic BV6 as indicated. Results displayed are from three independent experiments (n=3 ± SD). ***, P = 0.00085; ***, P = 0.00529; Student's t test.F Collective assessment of USP9X and XIAP expression determined by immunohistochemistry in tissue microarrays (TMA) derived from a patient cohort representing 121 evaluable human CD20+ aggressive B-cell lymphomas. High USP9X expression was identified in 37 cases (31 %) and significantly correlated with high XIAP expression levels (**, P = 0.004; Student's t test).G Expression data derived from (E) was correlated with clinical follow-up. Within a cohort of 58 patients receiving vincristine-containing chemotherapy (CHOP regimen: cyclophosphamide, doxorubicin, vincristine, prednisone) without the addition of the anti-CD20 antibody rituximab (left panel) overexpression of USP9X and XIAP (n = 14, straight line) was associated with a significantly shortened event-free survival (EFS) as compared to all remaining patients (n = 44, dotted line) (*, P = 0.050; log rank test). Within a cohort of 63 patients receiving the CHOP regimen with addition of the anti-CD20 antibody rituximab overexpression of USP9X and XIAP (n = 14, straight line) was not associated with a shortened event-free survival (EFS) as compared to all remaining patients (n = 49, dotted line) (P = 0.454).


Figure 1Representative composite confocal image of single cultured hippocampal mouse neurons , labeled with MAP2 ( green ) on islands of glial cells , labeled with GFAP ( red ) . Scale bar : 100 µm . Representative confocal image of single cultured hippocampal mouse neuron labeled with endogenous DCV cargo ChgB ( green ) and ß3 - tubulin ( red ) as neurite marker . Scale bar : 20 µm . Analysis of hippocampal neurons ( DIV14 ) immunostained for ChgB as DCV marker and ß3 - tubulin as morphological marker . DCV pool size measured as number of ChgB puncta per neuron . Analysis of hippocampal neurons ( DIV14 ) immunostained for ChgB as DCV marker and ß3 - tubulin as morphological marker . Total neurite length ( mm ) per neuron . Analysis of hippocampal neurons ( DIV14 ) immunostained for ChgB as DCV marker and ß3 - tubulin as morphological marker . ChgB puncta per μm neurite . Analysis of striatal neurons ( DIV14 ) immunostained for ChgB as DCV marker and ß3 - tubulin as morphological marker . DCV pool size measured as number of ChgB puncta per neuron . Total neurite length ( mm ) per neuron . Analysis of striatal neurons ( DIV14 ) immunostained for ChgB as DCV marker and ß3 - tubulin as morphological marker . ChgB puncta per μm neurite . Correlation between ChgB puncta per neuron and total neurite length for hippocampal ( green ) and striatal ( red ) neurons . Analysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post - hoc immunostained for ChgA as DCV marker . Representative mouse brain slice with labeled axonal projections in cortical region ( mCherry - filler , red ) and immunostained for ChgA ( green ) . Scale bar : 1 mm . ( a - b ) Zoom of axonal projections in ( L ) , scale bars : 100 µm . Analysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post - hoc immunostained for ChgA as DCV marker . Representative confocal image and zoom of cortex tissue containing labeled axonal projections . Measurements of ChgA puncta ( black ) were performed within axonal projections ( mask , yellow ) . Zoomed areas are indicated in top , right image ( M ) . Scale bars : 10 µm ( M ) , 5 µm ( NAnalysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post - hoc immunostained for ChgA as DCV marker . Representative confocal image and zoom of cortex tissue containing labeled axonal projections . Measurements of ChgA puncta ( black ) were performed within axonal projections ( mask , yellow ) . Zoomed areas Arrows ( O ) indicate ChgA staining measured as individual ChgA puncta . Scale bars : , 5 µmAnalysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post - hoc immunostained for ChgA as DCV marker . ChgA puncta per μm axon from mouse tissue , compared to the distribution of ChgB puncta in axons of cultured hippocampal

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

Figure 1Representative composite confocal image of single cultured hippocampal mouse neurons, labeled with MAP2 (green) on islands of glial cells, labeled with GFAP (red). Scale bar: 100 µm. Representative confocal image of single cultured hippocampal mouse neuron labeled with endogenous DCV cargo ChgB (green) and ß3-tubulin (red) as neurite marker. Scale bar: 20 µm.Analysis of hippocampal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. DCV pool size measured as number of ChgB puncta per neuron.Analysis of hippocampal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. Total neurite length (mm) per neuron.Analysis of hippocampal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. ChgB puncta per μm neurite.Analysis of striatal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. DCV pool size measured as number of ChgB puncta per neuron.Total neurite length (mm) per neuron.Analysis of striatal neurons (DIV14) immunostained for ChgB as DCV marker and ß3-tubulin as morphological marker. ChgB puncta per μm neurite.Correlation between ChgB puncta per neuron and total neurite length for hippocampal (green) and striatal (red) neurons.Analysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. Representative mouse brain slice with labeled axonal projections in cortical region (mCherry-filler, red) and immunostained for ChgA (green). Scale bar: 1 mm. (a-b) Zoom of axonal projections in (L), scale bars: 100 µm.Analysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. Representative confocal image and zoom of cortex tissue containing labeled axonal projections. Measurements of ChgA puncta (black) were performed within axonal projections (mask, yellow). Zoomed areas are indicated in top, right image (M). Scale bars: 10 µm (M), 5 µm (NAnalysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. Representative confocal image and zoom of cortex tissue containing labeled axonal projections. Measurements of ChgA puncta (black) were performed within axonal projections (mask, yellow). Zoomed areas Arrows (O) indicate ChgA staining measured as individual ChgA puncta. Scale bars: , 5 µmAnalysis of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. ChgA puncta per μm axon from mouse tissue, compared to the distribution of ChgB puncta in axons of cultured hippocampal neurons


Figure 2Representation of resolution limit in confocal imaging and dSTORM strategy to increase resolution . Electron micrograph of axon ( green ) and dendrite ( red ) containing DCVs , defined based on morphology . In conventional immunofluorescence confocal microscopy , individual DCVs cannot be separated ( right zoom , green signal representing fluorescent signal using confocal ) . Super - resolution dSTORM imaging resolves the point - spread function ( PSF ) resulting in single DCV localizations . Scale bars : 100 nm . Representative confocal image and zoom of a single isolated hippocampal neuron ( DIV14 ) immunostained for the endogenous DCV marker ChgB ( green ) , axonal marker SMI312 ( red ) and dendritic marker MAP2 ( blue ) . Areas of zoom ( C ) is indicated in ( B ) . Scale bars : 20 µm ( B ) , 5 µm ( C ) . ChgB immunoreactivity of zoomed area indicated in ( C ) ( top panel ) . Reconstructed ChgB localizations of dSTORM imaging ( middle panel ) . Lower panel shows overlay of dSTORM ChgB localizations and ChgB immunoreactivity ( confocal , 40x ) . Calibration bar represents no ( 0 ) to high ( 255 ) clustering of dSTORM localizations . Scale bar : 2 µm . Fluorescence intensity of ChgB immunoreactivity puncta ( confocal ) with corresponding number of dSTORM puncta for hippocampal ( green ) or striatal ( red ) cultured neurons . Squares show average ± SEM , dots represent individual observations . Hippocampus : N = 2 cells ( 89 puncta ) , striatum : N = 3 cells ( 37 puncta ) . Average histogram of fluorescence intensity of ChgB puncta ( 40x confocal ) in neurons used for dSTORM imaging . Dotted lines indicate cut - off values of fluorescence intensity corresponding 1 , 2 , 3 or 4 dSTORM puncta . Data shows average ± SEM , Hippocampus : N = 2 cells , striatum : N = 3 cells . Percentage of ChgB puncta per neuron with corresponding fluorescence value of 1 , 2 , 3 or 4 dSTORM puncta . Data shows average ± SEM . Hippocampus : N = 2 cells , striatum : N = 3 cells . Analysis of STED microscopy of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post - hoc immunostained for ChgA as DCV marker . ( H ) Representative confocal ( 100x magnification ) image of mCherry labeled axon ( blue ) with ChgA confocal signal ( red ) and corresponding super - resolution STED ChgA signal ( green ) . Zoomed areas are indicated by arrows . Scale bar merged image ( left ) : 1 µm , scale bars zooms : 100 nm . ( I ) Percentage of single ChgA puncta within mCherry labeled axons with corresponding number of overlaying STED puncta . Data shows average ± SEM . N = 2 mice ( 7 STED images , 70 single observations ) .

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 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 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 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 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

Figure 2Representation of resolution limit in confocal imaging and dSTORM strategy to increase resolution. Electron micrograph of axon (green) and dendrite (red) containing DCVs, defined based on morphology. In conventional immunofluorescence confocal microscopy, individual DCVs cannot be separated (right zoom, green signal representing fluorescent signal using confocal). Super-resolution dSTORM imaging resolves the point-spread function (PSF) resulting in single DCV localizations. Scale bars: 100 nm.Representative confocal image and zoom of a single isolated hippocampal neuron (DIV14) immunostained for the endogenous DCV marker ChgB (green), axonal marker SMI312 (red) and dendritic marker MAP2 (blue). Areas of zoom (C) is indicated in (B). Scale bars: 20 µm (B), 5 µm (C).ChgB immunoreactivity of zoomed area indicated in (C) (top panel). Reconstructed ChgB localizations of dSTORM imaging (middle panel). Lower panel shows overlay of dSTORM ChgB localizations and ChgB immunoreactivity (confocal, 40x). Calibration bar represents no (0) to high (255) clustering of dSTORM localizations. Scale bar: 2 µm.Fluorescence intensity of ChgB immunoreactivity puncta (confocal) with corresponding number of dSTORM puncta for hippocampal (green) or striatal (red) cultured neurons. Squares show average ± SEM, dots represent individual observations. Hippocampus: N = 2 cells (89 puncta), striatum: N = 3 cells (37 puncta).Average histogram of fluorescence intensity of ChgB puncta (40x confocal) in neurons used for dSTORM imaging. Dotted lines indicate cut-off values of fluorescence intensity corresponding 1, 2, 3 or 4 dSTORM puncta. Data shows average ± SEM, Hippocampus: N = 2 cells, striatum: N = 3 cells.Percentage of ChgB puncta per neuron with corresponding fluorescence value of 1, 2, 3 or 4 dSTORM puncta. Data shows average ± SEM. Hippocampus: N = 2 cells, striatum: N = 3 cells.Analysis of STED microscopy of mouse cortex tissue expressing mCherry in axonal projections after stereotaxic viral injections in the thalamus and post-hoc immunostained for ChgA as DCV marker. (H) Representative confocal (100x magnification) image of mCherry labeled axon (blue) with ChgA confocal signal (red) and corresponding super-resolution STED ChgA signal (green). Zoomed areas are indicated by arrows. Scale bar merged image (left): 1 µm, scale bars zooms: 100 nm. (I) Percentage of single ChgA puncta within mCherry labeled axons with corresponding number of overlaying STED puncta. Data shows average ± SEM. N = 2 mice (7 STED images, 70 single observations).


Figure 3Representative confocal images and zooms of a single isolated hippocampal neuron ( DIV14 ) immunostained for the endogenous DCV marker ChgB ( green ) , dendritic marker MAP2 ( red ) and ß3 - tubulin ( blue ) as neurite marker . Areas of zooms are indicated in ( A ) . Closed arrows : dendritic ChgB staining , open arrows : axonal ChgB staining . Scale bars : 20 µm ( A ) , 5 µm ( B , C ) . Zoom of immunostaining for the excitatory synaptic vesicle protein VGLUT1 and DCV marker ChgB . Closed arrows : synaptic ChgB staining , open arrows : extra - synaptic ChgB staining . Scale bar : 5 µm . Zoom of immunostaining for two synaptic proteins VGLUT1 and Synapsin I / II . Scale bar : 5 µm . Manders ' coefficients of colocalization analysis between ChgB , ß3 - tubulin and MAP2 . Manders ' coefficients of colocalization analysis between ChgB and VGLUT1 , with VGLUT1 - Synapsin I / II colocalization as positive control and VGLUT1 - Synapsin I / II rotated 90 ° as negative control . Number of ChgB puncta per neuron in dendrites , axons and the total pool ( dendrites + axons ) . Wilcoxon matched - pairs signed rank test : n . s . = not significant , p = 0 . 75 . Total dendritic and axonal length ( mm ) per neuron . Wilcoxon matched - pairs signed rank test : * * * * p = < 0 . 0001 . ChgB puncta per μm dendrite and axon per neuron . Lines next to individual data points show mean ± SEM . Wilcoxon matched - pairs signed rank test : * * * * p = < 0 . 0001 . Mean ChgB fluorescence intensity per puncta in the total neuron ( dendrites + axons ) and in dendrites only . Wilcoxon matched - pairs signed rank test : * * * * p = < 0 . 0001 . Histogram of fluorescence intensity of ChgB puncta in the total neuron ( dendrites + axons ) and in dendrites only . ChgB puncta per μm dendrite and axon per neuron . Lines next to individual data points show mean ± SEM . Wilcoxon matched - pairs signed rank test : * * * * p = < 0 . 0001 . Mean ChgB fluorescence intensity per puncta in dendrites and axons . Wilcoxon matched - pairs signed rank test : * * * * p = < 0 . 0001 . Distribution of DCVs in dendrites and axons of sparse - labeled network cultures . ( P ) ChgB puncta per μm dendrite or axon . Lines next to individual data points show mean ± SEM . Mann - Whitney U test : * * * * p = < 0 . 0001 . ( Q ) Mean ChgB fluorescence intensity per puncta in dendrites and axons . Unpaired t - test : * * * p = < 0 . 0001 . Analysis of DCVs in axons and dendrites of mCherry labeled mouse Dentate Gyrus ( DG ) granule cell tissue after stereotaxic viral injections and post - hoc immunostained for ChgA as DCV marker . ( R ) Representative mouse brain slice with labeled DG granule cells ( mCherry - filler , red ) and immunostained for ChgA ( green ) . ChgA puncta were measured in dendrites and axons at indicated areas . Scale bar : 250 µm . ( S ) ChgA puncta per μm dendrite or axon in DG granule cells . N = 1 mouse ( 6 dendrite regions , 7 axonal regions ) .

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 B-PANEL_START 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 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 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 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 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 B-PANEL_START 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 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 O O O O O 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 3Representative confocal images and zooms of a single isolated hippocampal neuron (DIV14) immunostained for the endogenous DCV marker ChgB (green), dendritic marker MAP2 (red) and ß3-tubulin (blue) as neurite marker. Areas of zooms are indicated in (A). Closed arrows: dendritic ChgB staining, open arrows: axonal ChgB staining. Scale bars: 20 µm (A), 5 µm (B, C). Zoom of immunostaining for the excitatory synaptic vesicle protein VGLUT1 and DCV marker ChgB. Closed arrows: synaptic ChgB staining, open arrows: extra-synaptic ChgB staining. Scale bar: 5 µm. Zoom of immunostaining for two synaptic proteins VGLUT1 and Synapsin I/II. Scale bar: 5 µm.Manders' coefficients of colocalization analysis between ChgB, ß3-tubulin and MAP2.Manders' coefficients of colocalization analysis between ChgB and VGLUT1, with VGLUT1-Synapsin I/II colocalization as positive control and VGLUT1-Synapsin I/II rotated 90° as negative control.Number of ChgB puncta per neuron in dendrites, axons and the total pool (dendrites + axons). Wilcoxon matched-pairs signed rank test: n.s. = not significant, p = 0.75.Total dendritic and axonal length (mm) per neuron. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001.ChgB puncta per μm dendrite and axon per neuron. Lines next to individual data points show mean ± SEM. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001.Mean ChgB fluorescence intensity per puncta in the total neuron (dendrites + axons) and in dendrites only. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001.Histogram of fluorescence intensity of ChgB puncta in the total neuron (dendrites + axons) and in dendrites only.ChgB puncta per μm dendrite and axon per neuron. Lines next to individual data points show mean ± SEM. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001. Mean ChgB fluorescence intensity per puncta in dendrites and axons. Wilcoxon matched-pairs signed rank test: **** p = < 0.0001.Distribution of DCVs in dendrites and axons of sparse-labeled network cultures. (P) ChgB puncta per μm dendrite or axon. Lines next to individual data points show mean ± SEM. Mann-Whitney U test: **** p = < 0.0001. (Q) Mean ChgB fluorescence intensity per puncta in dendrites and axons. Unpaired t-test: *** p = < 0.0001.Analysis of DCVs in axons and dendrites of mCherry labeled mouse Dentate Gyrus (DG) granule cell tissue after stereotaxic viral injections and post-hoc immunostained for ChgA as DCV marker. (R) Representative mouse brain slice with labeled DG granule cells (mCherry-filler, red) and immunostained for ChgA (green). ChgA puncta were measured in dendrites and axons at indicated areas. Scale bar: 250 µm. (S) ChgA puncta per μm dendrite or axon in DG granule cells. N = 1 mouse (6 dendrite regions, 7 axonal regions).


Figure 4Representative confocal image and zoom of a single isolated hippocampal neuron ( DIV14 ) immunostained for the endogenous DCV marker ChgB ( green ) , synaptic protein VGLUT1 ( red ) and dendritic marker MAP2 ( blue ) . Area of zoom ( B ) is indicated in ( A ) . Scale bars : 20 µm ( A ) , 2 µm ( B ) . Reconstructed ChgB localizations of dSTORM imaging of zoomed area in ( B ) ( top ) . Middle panel shows overlay between dSTORM ChgB localizations and VGLUT1 immunofluorescence ( confocal , 40x ) . Scale bar : 2 µm . Zooms of boxed areas containing synapses are visualized in lower panels ( a - c ) . Arrows indicate extra - synaptic DCVs . Scale bar ( a - c ) : 1 µm . Calibration bar represents n : o ( 0 ) to high ( 255 ) clustering of dSTORM localizations . Representative electron micrographs of synaptic sections containing DCVs . AZ indicates the active zone , dashed lines represent DCV diameter measurements . Arrow indicates extra - synaptic DCV . Scale bars : 100 nm . Histogram of number of DCVs in percentage of synapses . Inset : number of DCVs per synaptic section . Average diameter of DCVs ( nm ) . Distance of DCVs to the active zone ( nm ) , measured from center of DCV to the middle of the active zone . Histogram of distance of DCVs to the middle of the active zone ( nm ) . Inset : shortest distance of DCVs to the active zone ( nm ) . Histogram of normalized distance of percentage of synaptic vesicles ( grey ) to the active zone and corresponding distance of DCVs ( black ) to the active zone . Example electron micrograph zooms of synapses containing DCVs outside ( top ) or inside ( bottom ) the synaptic vesicle cluster . Scale bars : 100 nm . Percentage of DCVs outside or inside the synaptic vesicle cluster . Histogram of distance ( nm ) of DCVs to the border of the synaptic vesicle cluster , negative values are inside , positive values outside .

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 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 B-PANEL_START 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 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 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 O O O O O O O O O O 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 4Representative confocal image and zoom of a single isolated hippocampal neuron (DIV14) immunostained for the endogenous DCV marker ChgB (green), synaptic protein VGLUT1 (red) and dendritic marker MAP2 (blue). Area of zoom (B) is indicated in (A). Scale bars: 20 µm (A), 2 µm (B). Reconstructed ChgB localizations of dSTORM imaging of zoomed area in (B) (top). Middle panel shows overlay between dSTORM ChgB localizations and VGLUT1 immunofluorescence (confocal, 40x). Scale bar: 2 µm. Zooms of boxed areas containing synapses are visualized in lower panels (a-c). Arrows indicate extra-synaptic DCVs. Scale bar (a-c): 1 µm. Calibration bar represents n:o (0) to high (255) clustering of dSTORM localizations.Representative electron micrographs of synaptic sections containing DCVs. AZ indicates the active zone, dashed lines represent DCV diameter measurements. Arrow indicates extra-synaptic DCV. Scale bars: 100 nm.Histogram of number of DCVs in percentage of synapses. Inset: number of DCVs per synaptic section.Average diameter of DCVs (nm).Distance of DCVs to the active zone (nm), measured from center of DCV to the middle of the active zone. Histogram of distance of DCVs to the middle of the active zone (nm). Inset: shortest distance of DCVs to the active zone (nm).Histogram of normalized distance of percentage of synaptic vesicles (grey) to the active zone and corresponding distance of DCVs (black) to the active zone.Example electron micrograph zooms of synapses containing DCVs outside (top) or inside (bottom) the synaptic vesicle cluster. Scale bars: 100 nm. Percentage of DCVs outside or inside the synaptic vesicle cluster. Histogram of distance (nm) of DCVs to the border of the synaptic vesicle cluster, negative values are inside, positive values outside.


Figure 5Schematic representation of NPY - pHluorin as optical reporter for DCV fusion . Inset below shows F / F0 of single DCV fusion event . Scale bar : 1 µm . Zoomed confocal image of NPY - pHluorin expressing ( green ) hippocampal neuron ( DIV14 ) immunostained for ChgB ( red ) and corresponding fluorescence intensity plot showing overlap between NPY - pHluorin and ChgB . Scale bar : 2 µm . Manders ' coefficients of colocalization between NPY - pHluorin and ChgB per neuron . Total ChgB puncta per neuron in uninfected ( control ) and NPY - pHluorin infected neurons ( NPY - pH ) . Student ' s t - test : n . s . = not significant , p = 0 . 90 . Mean ChgB fluorescence intensity per puncta per neuron in uninfected ( control ) and NPY - pHluorin infected neurons ( NPY - pH ) . Student ' s t - test : n . s . = not significant , p = 0 . 71 . Correlation between ChgB puncta per neuron and total neurite length ( mm ) for uninfected control ( grey ) and NPY - pHluorin infected ( green ) neurons . Linear regression , inset shows goodness of fit ( r2 ) and significance ( p - value ) per group . Slopes are not significantly different , ANCOVA : p = 0 . 19 . Single isolated hippocampal neurons ( DIV14 ) were labeled with NPY - pHluorin . DCV fusion events were quantified per neuron upon different stimulation paradigms . Histogram of DCV fusion events per stimulation per time point . Stimulations include 1 action potential ( AP ) ( grey ) , 16 bursts of 50 AP at 50 Hz ( light - green ) , superfusion with 60 mM KCl Tyrode solution for 30 s . ( dark - green ) and superfusion with 5 μM Ionomycin for 30 s . ( blue ) . Inset : Typical ∆ F / F0 trace of rise in intracellular calcium ( Fluo5 - AM ) upon different stimulations . Traces are corrected for baseline ( first 10 frames ) and normalized . Single isolated hippocampal neurons ( DIV14 ) were labeled with NPY - pHluorin . DCV fusion events were quantified per neuron upon different stimulation paradigms . Cumulative plot of fusion events per neuron for different stimulations . Single isolated hippocampal neurons ( DIV14 ) were labeled with NPY - pHluorin . DCV fusion events were quantified per neuron upon different stimulation paradigms . DCV fusion events per neuron per stimulation . Kruskal - Wallis with Dunn ' s correction : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . 1AP vs . 5 μM Ionomycin , 16x50 AP , 50Hz vs . 60 mM KCl and 60 mM KCl vs 5 μM Ionomycin were non - significant , p > 0 . 05 . DCV release probability per neuron per stimulation . Kruskal - Wallis with Dunn ' s correction : * * p < 0 . 01 , * * * p < 0 . 001 . 1AP vs . 5 μM Ionomycin , 16x50 AP , 50Hz vs . 60 mM KCl and 60 mM KCl vs 5 μM Ionomycin were non - significant , p > 0 . 05 . DCV release probability per neuron in CaMKII - positive and negative hippocampus neurons ( DIV14 ) upon repetitive electrical stimulation ( 16x50AP at 50 Hz ) . Mann - Whitney U test : n . s . = not significant , p = 0 . 12 .

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 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 B-PANEL_START 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 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 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 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 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

Figure 5Schematic representation of NPY-pHluorin as optical reporter for DCV fusion. Inset below shows F/F0 of single DCV fusion event. Scale bar: 1 µm.Zoomed confocal image of NPY-pHluorin expressing (green) hippocampal neuron (DIV14) immunostained for ChgB (red) and corresponding fluorescence intensity plot showing overlap between NPY-pHluorin and ChgB. Scale bar: 2 µm.Manders' coefficients of colocalization between NPY-pHluorin and ChgB per neuron.Total ChgB puncta per neuron in uninfected (control) and NPY-pHluorin infected neurons (NPY-pH). Student's t-test: n.s. = not significant, p = 0.90. Mean ChgB fluorescence intensity per puncta per neuron in uninfected (control) and NPY-pHluorin infected neurons (NPY-pH). Student's t-test: n.s. = not significant, p = 0.71.Correlation between ChgB puncta per neuron and total neurite length (mm) for uninfected control (grey) and NPY-pHluorin infected (green) neurons. Linear regression, inset shows goodness of fit (r2) and significance (p-value) per group. Slopes are not significantly different, ANCOVA: p = 0.19.Single isolated hippocampal neurons (DIV14) were labeled with NPY-pHluorin. DCV fusion events were quantified per neuron upon different stimulation paradigms. Histogram of DCV fusion events per stimulation per time point. Stimulations include 1 action potential (AP) (grey), 16 bursts of 50 AP at 50 Hz (light-green), superfusion with 60 mM KCl Tyrode solution for 30 s. (dark-green) and superfusion with 5 μM Ionomycin for 30 s. (blue). Inset: Typical ∆F/F0 trace of rise in intracellular calcium (Fluo5-AM) upon different stimulations. Traces are corrected for baseline (first 10 frames) and normalized.Single isolated hippocampal neurons (DIV14) were labeled with NPY-pHluorin. DCV fusion events were quantified per neuron upon different stimulation paradigms. Cumulative plot of fusion events per neuron for different stimulations.Single isolated hippocampal neurons (DIV14) were labeled with NPY-pHluorin. DCV fusion events were quantified per neuron upon different stimulation paradigms. DCV fusion events per neuron per stimulation. Kruskal-Wallis with Dunn's correction: *p < 0.05, **p < 0.01, ***p < 0.001. 1AP vs. 5 μM Ionomycin, 16x50 AP,50Hz vs. 60 mM KCl and 60 mM KCl vs 5 μM Ionomycin were non-significant, p > 0.05. DCV release probability per neuron per stimulation. Kruskal-Wallis with Dunn's correction: **p < 0.01, ***p < 0.001. 1AP vs. 5 μM Ionomycin, 16x50 AP,50Hz vs. 60 mM KCl and 60 mM KCl vs 5 μM Ionomycin were non-significant, p>0.05.DCV release probability per neuron in CaMKII-positive and negative hippocampus neurons (DIV14) upon repetitive electrical stimulation (16x50AP at 50 Hz). Mann-Whitney U test: n.s. = not significant, p = 0.12.


Figure 6Schematic representation of NPY - mCherry as optical reporter for DCV fusion with full cargo release ( top ) . Zoom of axonal and dendritic areas of ( B ) , with location of fusion events ( middle ) . Average fluorescence decrease upon NPY - mCherry labeled DCV fusion in axons and dendrites ( below ) . Representative neuron labeled with membrane - targeted EGFP ( mEGFP ) ( black ) and localization of axonal ( blue dots ) or dendritic ( purple dots ) events upon stimulation . Scale bar : 20 µm . Histogram of axonal ( blue ) and dendritic ( purple ) DCV fusion events upon repetitive electrical stimulation ( 16x50AP at 50 Hz ) . Cumulative plot of axonal or dendritic fusion events per neuron . Normalized cumulative plot of axonal and dendritic fusion events per neuron . Percentage of axonal fusion events per cell .

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Figure 6Schematic representation of NPY-mCherry as optical reporter for DCV fusion with full cargo release (top). Zoom of axonal and dendritic areas of (B), with location of fusion events (middle). Average fluorescence decrease upon NPY-mCherry labeled DCV fusion in axons and dendrites (below).Representative neuron labeled with membrane-targeted EGFP (mEGFP) (black) and localization of axonal (blue dots) or dendritic (purple dots) events upon stimulation. Scale bar: 20 µm.Histogram of axonal (blue) and dendritic (purple) DCV fusion events upon repetitive electrical stimulation (16x50AP at 50 Hz).Cumulative plot of axonal or dendritic fusion events per neuron. Normalized cumulative plot of axonal and dendritic fusion events per neuron. Percentage of axonal fusion events per cell.


Figure 1C . Phage displaying specific peptides for mATG8 are identified by phage ELISA on immobilized GST - mATG8s and negative control proteins BSA and GST . Binding signal is shown as absorbance at 450 nm in arbitrary units [ AU ] . D . Pull - down of mTurquoise - peptide fusions from cell lysates on GST - mATG8s beads followed by immuno - blotting with α - GFP . As loading control , the protein staining with amido black is shown .

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Figure 1C. Phage displaying specific peptides for mATG8 are identified by phage ELISA on immobilized GST-mATG8s and negative control proteins BSA and GST. Binding signal is shown as absorbance at 450 nm in arbitrary units [AU].D. Pull-down of mTurquoise-peptide fusions from cell lysates on GST-mATG8s beads followed by immuno-blotting with α-GFP. As loading control, the protein staining with amido black is shown.


Figure 2B , C , E . Annexin A4 based co - translocation assays of peptide binders and EGFP - mATG8s . In comparison to single peptides ( B ) , triplication of peptides ( C ) or introduction of negative charges adjacent to the binding motif ( E ) increases the co - translocation extent of the peptide - sensors with their target mATG8 . Control assays with non - target mATG8 are shown in expanded view figure 2 . B , C , E . Annexin A4 based co - translocation assays of peptide binders and EGFP - mATG8s . In comparison to single peptides ( B ) , triplication of peptides ( C ) or introduction of negative charges adjacent to the binding motif ( E ) increases the co - translocation extent of the peptide - sensors with their target mATG8 . Control assays with non - target mATG8 are shown in expanded view figure 2 . D . Biotinylated octameric peptides representing a single amino acid walk - through along the complete 16mer peptide AS _ 67 and AS _ M67 are immobilized on streptavidin . Binding of GST - LC3C fusion - protein to the immobilized peptides is detected by a GST specific antibody ( HRP - conjugate ) and shown as absorbance at 450 nm . AU = arbitrary unitB , C , E . Annexin A4 based co - translocation assays of peptide binders and EGFP - mATG8s . In comparison to single peptides ( B ) , triplication of peptides ( C ) or introduction of negative charges adjacent to the binding motif ( E ) increases the co - translocation extent of the peptide - sensors with their target mATG8 . Control assays with non - target mATG8 are shown in expanded view figure 2 . F . Translocation efficiency of the unmodified peptide AS _ 67 , triplicate AS3 _ 67 and charge optimized peptide AS _ M67 is classified as % of cells that show no translocation ' - ' to very good ' + + + + ' translocation with EGFP - LC3C ; n = total number of analyzed cells . Data are presented as mean of three biological replicates ± SD .

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

Figure 2B, C, E. Annexin A4 based co-translocation assays of peptide binders and EGFP-mATG8s. In comparison to single peptides (B), triplication of peptides (C) or introduction of negative charges adjacent to the binding motif (E) increases the co-translocation extent of the peptide-sensors with their target mATG8. Control assays with non-target mATG8 are shown in expanded view figure 2.B, C, E. Annexin A4 based co-translocation assays of peptide binders and EGFP-mATG8s. In comparison to single peptides (B), triplication of peptides (C) or introduction of negative charges adjacent to the binding motif (E) increases the co-translocation extent of the peptide-sensors with their target mATG8. Control assays with non-target mATG8 are shown in expanded view figure 2.D. Biotinylated octameric peptides representing a single amino acid walk-through along the complete 16mer peptide AS_67 and AS_M67 are immobilized on streptavidin. Binding of GST-LC3C fusion-protein to the immobilized peptides is detected by a GST specific antibody (HRP-conjugate) and shown as absorbance at 450 nm. AU=arbitrary unitB, C, E. Annexin A4 based co-translocation assays of peptide binders and EGFP-mATG8s. In comparison to single peptides (B), triplication of peptides (C) or introduction of negative charges adjacent to the binding motif (E) increases the co-translocation extent of the peptide-sensors with their target mATG8. Control assays with non-target mATG8 are shown in expanded view figure 2.F. Translocation efficiency of the unmodified peptide AS_67, triplicate AS3_67 and charge optimized peptide AS_M67 is classified as % of cells that show no translocation '-' to very good '++++' translocation with EGFP-LC3C; n = total number of analyzed cells. Data are presented as mean of three biological replicates ±SD.


Figure 3A , B . The recruitment of the mCherry fused p62 derived sensor AS3 _ p62 to the autophagosomes positive for overexpressed EGFP - LC3B ( A ) or endogenous LC3B ( B ) visualized by immunofluorescence upon autophagy induction for 3h by KU + Baf treatment . C . Recruitment of AS3 _ p62 in Atg5 + / + or Atg5 - / - MEFs to endogenous Lc3b . D . Lack of recruitment of the p62 - derived sensor in the absence of the hydrophobic core residues of the LIR motif ( ΔLIR ) to EGFP - LC3B positive autophagosomes . E , F . ATG8 sensors are recruited to autophagosomes decorated with respective target mATG8 proteins . E , F . ATG8 sensors are recruited to autophagosomes decorated with respective target mATG8 proteins .

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 B-PANEL_START 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 B-PANEL_START 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

Figure 3A, B. The recruitment of the mCherry fused p62 derived sensor AS3_p62 to the autophagosomes positive for overexpressed EGFP-LC3B (A) or endogenous LC3B (B) visualized by immunofluorescence upon autophagy induction for 3h by KU + Baf treatment.C. Recruitment of AS3_p62 in Atg5+/+ or Atg5-/- MEFs to endogenous Lc3b.D. Lack of recruitment of the p62-derived sensor in the absence of the hydrophobic core residues of the LIR motif (ΔLIR) to EGFP-LC3B positive autophagosomes.E, F. ATG8 sensors are recruited to autophagosomes decorated with respective target mATG8 proteins.E, F. ATG8 sensors are recruited to autophagosomes decorated with respective target mATG8 proteins.


Figure 4A - C . Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry - fusion proteins of indicated ATG8 sensor upon doxycycline ( DOX ) induction : Autophagy flux is monitored in basal conditions ( A ) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62 . A - C . Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry - fusion proteins of indicated ATG8 sensor upon doxycycline ( DOX ) induction : Autophagy flux is monitored upon starvation ( EBSS ) ( B ) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62 . A - C . Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry - fusion proteins of indicated ATG8 sensor upon doxycycline ( DOX ) induction : Autophagy flux is monitored after autophagy induction by Torin I ( C ) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62 . D . FACS based mitophagy flux analysis performed in HeLa cell line stably expressing HA - Parkin , mt - mKeima , and a GFP fusion of the indicated sensor . Cells were treated with OAQ ( oligomycin , antimycin , Q - VD ) for indicated times and analyzed by FACS for lysosomal - positive mt - mKeima ( 561nm ) . The average of three independent experiments is presented . Representative FACS data from experiments ( GFP control and AS3 _ 67 ) are shown in the lower panels . Error bars indicate standard deviation . Significance calculation : 2 - way anova of one - way anova . n . s . = not significant . * * * indicates p - value of 0 . 0001 .

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 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 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 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

Figure 4A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored in basal conditions (A) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62.A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored upon starvation (EBSS) (B) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62.A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored after autophagy induction by Torin I (C) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62.D. FACS based mitophagy flux analysis performed in HeLa cell line stably expressing HA-Parkin, mt-mKeima, and a GFP fusion of the indicated sensor. Cells were treated with OAQ (oligomycin, antimycin, Q-VD) for indicated times and analyzed by FACS for lysosomal-positive mt-mKeima (561nm). The average of three independent experiments is presented. Representative FACS data from experiments (GFP control and AS3_67) are shown in the lower panels. Error bars indicate standard deviation. Significance calculation: 2-way anova of one-way anova. n.s.= not significant. *** indicates p-value of 0.0001.


Figure 5A . LC3C specific sensor mCh - AS3 _ 67 is recruited to Salmonella ( SFH2 ) escaping SCV . Salmonella SFH2 strain is carrying a GFP - reporter gene activated by availability of glucose - 6 - phosphate in the host ' s cytosol . B . Salmonella with prolonged cytosolic residence and strong activation of the GFP - reporter gene ( green arrow ) are not recognized by the LC3C specific sensor mCh - AS3 _ 67 . C . A subpopulation of hyper proliferative Salmonella in the cytosol is coated with endogenous LC3C . In contrast , endogenous LC3B is enriched in spots in close proximity to the cytosolic bacteria . D . Sensitivity of LC3C specific sensor mCh - AS3 _ 67 is increased upon fusion of membrane - tethering FYVE domain or PB1 oligomerization domain . E . Introduction of the PB1 domain to mCh - AS3 _ p62 increases its performance in recognizing endogenous LC3B . F . High - performance sensor mCh - PB1 - AS3 _ 67 was transiently expressed in Salmonella infected HeLa cells to monitor xenophagy events .

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Figure 5A. LC3C specific sensor mCh-AS3_67 is recruited to Salmonella (SFH2) escaping SCV. Salmonella SFH2 strain is carrying a GFP-reporter gene activated by availability of glucose-6-phosphate in the host's cytosol.B. Salmonella with prolonged cytosolic residence and strong activation of the GFP-reporter gene (green arrow) are not recognized by the LC3C specific sensor mCh-AS3_67.C. A subpopulation of hyper proliferative Salmonella in the cytosol is coated with endogenous LC3C. In contrast, endogenous LC3B is enriched in spots in close proximity to the cytosolic bacteria.D. Sensitivity of LC3C specific sensor mCh-AS3_67 is increased upon fusion of membrane-tethering FYVE domain or PB1 oligomerization domain.E. Introduction of the PB1 domain to mCh-AS3_p62 increases its performance in recognizing endogenous LC3B.F. High-performance sensor mCh-PB1-AS3_67 was transiently expressed in Salmonella infected HeLa cells to monitor xenophagy events.


Figure 6A , B . LC3C specific sensor mCh - PB1 - AS3 _ 67 is recruited to a subset of mitochondria undergoing mitophagy . Mitochondria were labeled with MitoTracker Deep Red ( ThermoFisher ) . U2OS cells were transiently co - transfected for indicated sensor and mATG8 proteins . Mitophagy was induced by CCCP treatment over three hours . Lysosomal degradation of mitochondria undergoing mitophagy was blocked by the addition of BafilomycinA1 ( Baf ) . C . Upon depolarization , endogenous LC3C is localized to mitochondria labeled with mt - mCherry in U2OS cells . An example of rare events with nearly complete overlap of signals for endogenous LC3C and mitochondria ( upper panel ) and a representative image ( lower panel ) is given . D . Overlap of LC3C specific sensor signal with endogenous LC3C and endogenous LAMP1 in U2OS cells induced for mitophagy .

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Figure 6A,B. LC3C specific sensor mCh-PB1-AS3_67 is recruited to a subset of mitochondria undergoing mitophagy. Mitochondria were labeled with MitoTracker Deep Red (ThermoFisher). U2OS cells were transiently co-transfected for indicated sensor and mATG8 proteins. Mitophagy was induced by CCCP treatment over three hours. Lysosomal degradation of mitochondria undergoing mitophagy was blocked by the addition of BafilomycinA1 (Baf).C. Upon depolarization, endogenous LC3C is localized to mitochondria labeled with mt-mCherry in U2OS cells. An example of rare events with nearly complete overlap of signals for endogenous LC3C and mitochondria (upper panel) and a representative image (lower panel) is given.D. Overlap of LC3C specific sensor signal with endogenous LC3C and endogenous LAMP1 in U2OS cells induced for mitophagy.


figf1a , Etoposide - treated Atg5 - / - MEFs were assessed by EM ( left ) and by Lamp2immunofluorescence ( IF ; middle ) . Right : reverse Lamp2 immunofluorescence image merged with the electron microscopy image . Arrows indicate Lamp2 - positive autolysosomes . Magnified photos are provided in Supplementary Fig . 5b , The size of each autophagic vacuole ( AV ) ( n = 30 cells ) in MEFs treated with etoposide . The total number of AVs in 30 cells was 483 in WT cells and 494 in Atg5 - / - cells . c , Percentage autophagic area ( n ≥ 20 cells ) in MEFs treated with etoposide . d , e , Representative macroautophagy in etoposide - treated Atg5 - / - MEFs . Conventional ( d ) and quick freezing and freeze - substitution ( e ) techniques were used . The autolysosomes ( arrows ) contain multilamellar bodies ( arrowheads ) . f , The number of autophagosomes ( white columns ) and autolysosomes ( black columns ) in MEFs ( n = 10 cells ) treated with etoposide . g , Reduction of percentage autophagic area by 3 - methyladenine ( 3 - MA ; n = 22 cells ) . . h , Time - course analysis of long - lived protein degradation ( n = 4 ) . i , Inhibition of long - lived protein degradation by bafilomycin A1 , protease inhibitor ( PI ) cocktail and 3 - MA ( * P 0 . 05 ; n = 4 ) . j , k , Similar experiments to those in c and i were performed by starvation . Red and blue lines in b , c , g and j indicate means and s . e . m . , respectively . The dashed line in c , g and j indicates the autophagic cell border ( 6 % ) . Black error bars indicate s . d .

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figf1a, Etoposide-treated Atg5 -/- MEFs were assessed by EM (left) and by Lamp2immunofluorescence (IF; middle). Right: reverse Lamp2 immunofluorescence image merged with the electron microscopy image. Arrows indicate Lamp2-positive autolysosomes. Magnified photos are provided in Supplementary Fig. 5b, The size of each autophagic vacuole (AV) (n = 30 cells) in MEFs treated with etoposide. The total number of AVs in 30 cells was 483 in WT cells and 494 in Atg5-/- cells.c, Percentage autophagic area (n ≥ 20 cells) in MEFs treated with etoposide.d, e, Representative macroautophagy in etoposide-treated Atg5-/- MEFs. Conventional (d) and quick freezing and freeze-substitution (e) techniques were used. The autolysosomes (arrows) contain multilamellar bodies (arrowheads).f, The number of autophagosomes (white columns) and autolysosomes (black columns) in MEFs (n = 10 cells) treated with etoposide.g, Reduction of percentage autophagic area by 3-methyladenine (3-MA; n = 22 cells).. h, Time-course analysis of long-lived protein degradation (n = 4).i, Inhibition of long-lived protein degradation by bafilomycin A1, protease inhibitor (PI) cocktail and 3-MA (*P 0.05; n = 4).j, k, Similar experiments to those in c and i were performed by starvation. Red and blue lines in b, c, g and j indicate means and s.e.m., respectively. The dashed line in c, g and j indicates the autophagic cell border (6%). Black error bars indicate s.d.


figf2a , The production of LC3 - II . Gapdh was used as a loading control . b , c , Punctate GFP - LC3 fluorescence in WT MEFs but not in Atg5 - / - MEFs . b , Representative photographs . c , Graphical presentation of the percentages of WT MEFs and Atg5 - / - MEFs with punctate GFP - LC3 fluorescence . b , c , Punctate GFP - LC3 fluorescence in WT MEFs but not in Atg5 - / - MEFs . b , Representative photographs . c , Graphical presentation of the percentages of WT MEFs and Atg5 - / - MEFs with punctate GFP - LC3 fluorescence . d , The same GFP - LC3 - transfected Atg5 - / - MEFs were assessed by IF and by EM . The magnified photo is the area indicated by the squares . Arrows indicate AVs . e , Atg5 - / - MEFs were incubated without ( NT ) or with etoposide and immunostained with an anti - Lamp2 antibody ( green ) . Nuclei were counterstained ( red ) . The percentages of cells with punctate Lamp2 immunostaining are shown ( means ± s . d . , n = 4 ) . f , Microarray analysis of the Atg5 - / - MEFs during treatment with etoposide . g , h , Induction of Ulk1 in the etoposide - treated Atg5 - / - MEFs was assessed by qPCR ( g ) and by immunoblotting ( h ) . i - l , Atg5 - / - MEFs were transfected with the siRNAs related to the Ulk1 complex ( i , j ) for 24 h and treated with etoposide for 18 h . Induction of macroautophagy was assessed by Lamp2 IF ( i , k ) i - l , Atg5 - / - MEFs were transfected with the siRNAs related to the Ulk1 complex ( i , j ) for 24 h and treated with etoposide for 18 h . Induction of macroautophagy was assessed by EM ( j , l ) . i - l , Atg5 - / - MEFs were transfected with the siRNAs related to the PI ( 3 ) K complex ( k , l ) and a beclin 1 plasmid ( k ) for 24 h and treated with etoposide for 18 h . Induction of macroautophagy was assessed by Lamp2 IF ( i , k ) . i - l , Atg5 - / - MEFs were transfected with the siRNAs related to the PI ( 3 ) K complex ( k , l ) for 24 h and treated with etoposide for 18 h . Induction of macroautophagy was assessed by EM ( j , l ) .

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figf2a, The production of LC3-II. Gapdh was used as a loading control.b, c, Punctate GFP-LC3 fluorescence in WT MEFs but not in Atg5-/- MEFs. b, Representative photographs. c, Graphical presentation of the percentages of WT MEFs and Atg5-/- MEFs with punctate GFP-LC3 fluorescence.b, c, Punctate GFP-LC3 fluorescence in WT MEFs but not in Atg5-/- MEFs. b, Representative photographs. c, Graphical presentation of the percentages of WT MEFs and Atg5-/- MEFs with punctate GFP-LC3 fluorescence.d, The same GFP-LC3-transfected Atg5-/- MEFs were assessed by IF and by EM. The magnified photo is the area indicated by the squares. Arrows indicate AVs.e, Atg5-/- MEFs were incubated without (NT) or with etoposide and immunostained with an anti-Lamp2 antibody (green). Nuclei were counterstained (red). The percentages of cells with punctate Lamp2 immunostaining are shown (means ± s.d., n = 4).f, Microarray analysis of the Atg5-/- MEFs during treatment with etoposide.g, h, Induction of Ulk1 in the etoposide-treated Atg5-/- MEFs was assessed by qPCR (g) and by immunoblotting (h).i-l, Atg5-/- MEFs were transfected with the siRNAs related to the Ulk1 complex (i, j) for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by Lamp2 IF (i, k)i-l, Atg5-/- MEFs were transfected with the siRNAs related to the Ulk1 complex (i, j) for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by EM (j, l).i-l, Atg5-/- MEFs were transfected with the siRNAs related to the PI(3)K complex (k, l) and a beclin 1 plasmid (k) for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by Lamp2 IF (i, k).i-l, Atg5-/- MEFs were transfected with the siRNAs related to the PI(3)K complex (k, l)for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by EM (j, l).


figf3a - d , Electron micrographs of etoposide - treated Atg5 - / - MEFs . a , AVs were observed near the Golgi apparatus ( G ) . Inset , the isolation membrane ( I ) was extended from the Golgi stack . b , Developing autophagosome . rER , rough endoplasmic reticulum . c , d , Isolation membrane fusing with vesicles containing thick membrane ( d ) and a complete autophagosome ( c ) is generated . e , f , Co - localization of Lamp2 with GFP - M6PR , GFP - Stx7 or GFP - Rab9 in etoposide - treated Atg5 - / - MEFs . Arrows indicate co - localized dots . Original magnification , × 200 . g - i , Atg5 - / - MEFs were transfected with the indicated siRNAs for 24 h and treated with etoposide . Induction of macroautophagy was assessed by Lamp2 IF ( g ) and by EM ( h ) , and the number of isolation membranes was calculated ( i ) . In g , error bars indicate s . d . ( n = 4 ) . In h and i , red and blue lines indicate means and s . e . m . , respectively ; n = 35 cells each . j , No effect of silencing of Rab9 ( siRab9 ) on the production of LC3 - II in etoposide - treated WT MEFs .

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figf3a-d, Electron micrographs of etoposide-treated Atg5-/- MEFs. a, AVs were observed near the Golgi apparatus (G). Inset, the isolation membrane (I) was extended from the Golgi stack. b, Developing autophagosome. rER, rough endoplasmic reticulum. c, d, Isolation membrane fusing with vesicles containing thick membrane (d) and a complete autophagosome (c) is generated.e, f, Co-localization of Lamp2 with GFP-M6PR, GFP-Stx7 or GFP-Rab9 in etoposide-treated Atg5-/- MEFs. Arrows indicate co-localized dots. Original magnification, × 200.g-i, Atg5-/- MEFs were transfected with the indicated siRNAs for 24 h and treated with etoposide. Induction of macroautophagy was assessed by Lamp2 IF (g) and by EM (h), and the number of isolation membranes was calculated (i). In g, error bars indicate s.d. (n = 4). In h and i, red and blue lines indicate means and s.e.m., respectively; n = 35 cells each.j, No effect of silencing of Rab9 (siRab9) on the production of LC3-II in etoposide-treated WT MEFs.


figf4a - e , Typical autophagic structures in the tissues of an Atg5 - / - embryo ( E14 . 5 ) : a , midbrain ; b - d , liver ; c , d , developing autophagosomes ; e , heart . Arrows indicate the autophagic vacuoles . f , g , k , l , Electron micrographs of wild - type ( WT ) and Atg5 - / - ( KO ) reticulocytes ( f , g ) and erythrocytes ( k , l ) . An isolation membrane ( asterisk ) , autophagosomes ( arrows ) and autolysosomes ( arrowheads ) are shown . Inset , engulfed mitochondria . h - j , m - o , The numbers of autophagic vacuoles ( h , m ) and autophagic vacuoles containing mitochondria ( i , n ) and persisting mitochondria ( j , o ) in reticulocytes ( h - j ) and erythrocytes ( m - o ) . Error bars indicate s . d . ( * P 0 . 05 ; n = 55 cells ) . f , g , k , l , Electron micrographs of wild - type ( WT ) and Atg5 - / - ( KO ) reticulocytes ( f , g ) and erythrocytes ( k , l ) . An isolation membrane ( asterisk ) , autophagosomes ( arrows ) and autolysosomes ( arrowheads ) are shown . Inset , engulfed mitochondria .

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figf4a-e, Typical autophagic structures in the tissues of an Atg5-/- embryo (E14.5): a, midbrain; b-d, liver; c, d, developing autophagosomes; e, heart. Arrows indicate the autophagic vacuoles.f, g, k, l, Electron micrographs of wild-type (WT) and Atg5-/- (KO) reticulocytes (f, g) and erythrocytes (k, l). An isolation membrane (asterisk), autophagosomes (arrows) and autolysosomes (arrowheads) are shown. Inset, engulfed mitochondria.h-j, m-o, The numbers of autophagic vacuoles (h, m) and autophagic vacuoles containing mitochondria (i, n) and persisting mitochondria (j, o) in reticulocytes (h-j) and erythrocytes (m-o). Error bars indicate s.d. (*P  0.05; n = 55 cells).f, g, k, l, Electron micrographs of wild-type (WT) and Atg5-/- (KO) reticulocytes (f, g) and erythrocytes (k, l). An isolation membrane (asterisk), autophagosomes (arrows) and autolysosomes (arrowheads) are shown. Inset, engulfed mitochondria.


Figure 1Chromatin - enriched fractions of HeLa cells exposed to the indicated genotoxic agents for 1 h were subjected to immunoblot analysis using SUMO2 / 3 antibody . except that cells were preextracted in 0 . 2 % Triton X - 100 , fixed and immunostained with SUMO2 / 3 antibody . Representative images are shown . Scale bar , 5 μm . Relative DPC levels in cells treated were quantified using a KCl / SDS precipitation assay ( mean ± SD ; n = 3 independent experiments ) . HeLa cells were treated with formaldehyde for 1 h , and where indicated propagated for an additional h in the absence of formaldehyde ( recovery ) . Cells were then fractionated into soluble and chromatin - enriched fractions and immunoblotted with SUMO2 / 3 and SUMO1 antibodies . using HeLa cells transfected with non - targeting control ( CTRL ) or SPRTN siRNAs . Mass spectrometry - based analysis of formaldehyde - induced SUMOylation changes . His10 - SUMO2 conjugates from HeLa / His10 - SUMO2 cells subjected or not to formaldehyde were purified on Ni - NTA under stringent conditions and analyzed by mass spectrometry . All proteins displaying significant upregulation of SUMOylation in response to formaldehyde treatment were subjected to network analysis using the STRING database , at the default interaction confidence setting of 0 . 4 . Proteins not connected to the network were omitted . SUMO2 conjugates from HeLa / His10 - SUMO2 cells subjected or not to formaldehyde or heat stress for 1 h were purified and immunoblotted with indicated antibodies .

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Figure 1Chromatin-enriched fractions of HeLa cells exposed to the indicated genotoxic agents for 1 h were subjected to immunoblot analysis using SUMO2/3 antibody.except that cells were preextracted in 0.2% Triton X-100, fixed and immunostained with SUMO2/3 antibody. Representative images are shown. Scale bar, 5 μm.Relative DPC levels in cells treated were quantified using a KCl/SDS precipitation assay (mean±SD; n=3 independent experiments).HeLa cells were treated with formaldehyde for 1 h, and where indicated propagated for an additional h in the absence of formaldehyde (recovery). Cells were then fractionated into soluble and chromatin-enriched fractions and immunoblotted with SUMO2/3 and SUMO1 antibodies.using HeLa cells transfected with non-targeting control (CTRL) or SPRTN siRNAs.Mass spectrometry-based analysis of formaldehyde-induced SUMOylation changes. His10-SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to formaldehyde were purified on Ni-NTA under stringent conditions and analyzed by mass spectrometry. All proteins displaying significant upregulation of SUMOylation in response to formaldehyde treatment were subjected to network analysis using the STRING database, at the default interaction confidence setting of 0.4. Proteins not connected to the network were omitted.SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to formaldehyde or heat stress for 1 h were purified and immunoblotted with indicated antibodies.