Figure 4Longitudinal associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the effect of time plus the biomarker by time interactions , capturing the longitudinal effects of the biomarkers . For each model , the " average " effect of time is also shown for comparison . Effects were significant ( * ) , meaning that biomarker levels affected the slopes of the outcome , for MMSE : T - tau ( p = . 021 ) and NFL ( p < . 0001 ) in A - negative and for T - tau ( p < . 0001 ) , Ng ( p = . 0031 ) and NFL ( p < . 0001 ) in A - positive . Biomarkers and outcomes were standardized to z - scores . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) . Longitudinal associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the effect of time plus the biomarker by time interactions , capturing the longitudinal effects of the biomarkers . For each model , the " average " effect of time is also shown for comparison . Effects were significant ( * ) , meaning that biomarker levels affected the slopes of the outcome , for ADAS - cog : NFL in A - negative ( p < . 0001 ) and for T - tau ( p < . 0001 ) , Ng ( p = . 0015 ) and NFL ( p = . 0027 ) in A - positive . Biomarkers and outcomes were standardized to z - scores . Note that the y - axes for ADAS - cog are flipped , so that the lower ranges constantly reflect " worse " outcomes . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) . Longitudinal associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the effect of time plus the biomarker by time interactions , capturing the longitudinal effects of the biomarkers . For each model , the " average " effect of time is also shown for comparison . Effects were significant ( * ) , meaning that biomarker levels affected the slopes of the outcome , for hippocampal volume : NFL in A - negative ( p = . 0048 ) and for T - tau ( p = . 0015 ) and Ng ( p = . 0027 ) in A - positive . Biomarkers and outcomes were standardized to z - scores . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) . Longitudinal associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the effect of time plus the biomarker by time interactions , capturing the longitudinal effects of the biomarkers . For each model , the " average " effect of time is also shown for comparison . Effects were significant ( * ) , meaning that biomarker levels affected the slopes of the outcome , for lateral ventricles : Ng ( p = . 0051 ) and NFL ( p < . 0001 ) in A - negative and NFL ( p < . 0001 ) in A - positive . Biomarkers and outcomes were standardized to z - scores . Note that the y - axes for ventricle size are flipped , so that the lower ranges constantly reflect " worse " outcomes . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) . Longitudinal associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the effect of time plus the biomarker by time interactions , capturing the longitudinal effects of the biomarkers . For each model , the " average " effect of time is also shown for comparison . Effects were significant ( * ) , meaning that biomarker levels affected the slopes of the outcome , for FDG - PET : T - tau ( p = . 00038 ) and Ng ( p = . 0019 ) in A - positive . Biomarkers and outcomes were standardized to z - scores . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) . Longitudinal associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the effect of time plus the biomarker by time interactions , capturing the longitudinal effects of the biomarkers . For each model , the " average " effect of time is also shown for comparison . Biomarkers and outcomes were standardized to z - scores . Note that the y - axes for WMH are flipped , so that the lower ranges constantly reflect " worse " outcomes . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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 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 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 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 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 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

Figure 4Longitudinal associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the effect of time plus the biomarker by time interactions, capturing the longitudinal effects of the biomarkers. For each model, the "average" effect of time is also shown for comparison. Effects were significant (*), meaning that biomarker levels affected the slopes of the outcome, for MMSE: T-tau (p=.021) and NFL (p<.0001) in A-negative and for T-tau (p<.0001), Ng (p=.0031) and NFL (p<.0001) in A-positive. Biomarkers and outcomes were standardized to z-scores. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).Longitudinal associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the effect of time plus the biomarker by time interactions, capturing the longitudinal effects of the biomarkers. For each model, the "average" effect of time is also shown for comparison. Effects were significant (*), meaning that biomarker levels affected the slopes of the outcome, for ADAS-cog: NFL in A-negative (p<.0001) and for T-tau (p<.0001), Ng (p=.0015) and NFL (p=.0027) in A-positive. Biomarkers and outcomes were standardized to z-scores. Note that the y-axes for ADAS-cog are flipped, so that the lower ranges constantly reflect "worse" outcomes. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).Longitudinal associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the effect of time plus the biomarker by time interactions, capturing the longitudinal effects of the biomarkers. For each model, the "average" effect of time is also shown for comparison. Effects were significant (*), meaning that biomarker levels affected the slopes of the outcome, for hippocampal volume: NFL in A-negative (p=.0048) and for T-tau (p=.0015) and Ng (p=.0027) in A-positive. Biomarkers and outcomes were standardized to z-scores. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).Longitudinal associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the effect of time plus the biomarker by time interactions, capturing the longitudinal effects of the biomarkers. For each model, the "average" effect of time is also shown for comparison. Effects were significant (*), meaning that biomarker levels affected the slopes of the outcome, for lateral ventricles: Ng (p=.0051) and NFL (p<.0001) in A-negative and NFL (p<.0001) in A-positive. Biomarkers and outcomes were standardized to z-scores. Note that the y-axes for ventricle size are flipped, so that the lower ranges constantly reflect "worse" outcomes. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).Longitudinal associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the effect of time plus the biomarker by time interactions, capturing the longitudinal effects of the biomarkers. For each model, the "average" effect of time is also shown for comparison. Effects were significant (*), meaning that biomarker levels affected the slopes of the outcome, for FDG-PET: T-tau (p=.00038) and Ng (p=.0019) in A-positive. Biomarkers and outcomes were standardized to z-scores. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).Longitudinal associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the effect of time plus the biomarker by time interactions, capturing the longitudinal effects of the biomarkers. For each model, the "average" effect of time is also shown for comparison. Biomarkers and outcomes were standardized to z-scores. Note that the y-axes for WMH are flipped, so that the lower ranges constantly reflect "worse" outcomes. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Figure 2A - B Clearance of a target with a consensus PAM by direct interference . Overlay of fluorescent and phase contrast time - lapse images . Presence of the target plasmid is tracked by its YFP production ( A ) . Reconstructed lineage traces of the imaged population ( A ) from induction of the CRISPR - Cas system over time ( grey ) lineages show some variation in plasmid clearance times ( coloured ) ( B ) . E - F Clearance of a matching target with a mutated PAM via priming . Overlay of fluorescent and phase contrast time - lapse images as in ( A ) . Reconstructed lineage traces of the imaged population ( E ) ( grey ) . Lineages show large variations in the time taken to clear the plasmid ( coloured ) ( F ) .

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

Figure 2A-B Clearance of a target with a consensus PAM by direct interference. Overlay of fluorescent and phase contrast time-lapse images. Presence of the target plasmid is tracked by its YFP production (A). Reconstructed lineage traces of the imaged population (A) from induction of the CRISPR-Cas system over time (grey) lineages show some variation in plasmid clearance times (coloured) (B).E-F Clearance of a matching target with a mutated PAM via priming. Overlay of fluorescent and phase contrast time-lapse images as in (A). Reconstructed lineage traces of the imaged population (E) (grey). Lineages show large variations in the time taken to clear the plasmid (coloured) (F).


Figure 1e . Retrospective immunohistochemistry DAB staining of IgG4 and IgG on archival temporal lobe resection tissue . As suggested by the consensus statement on the pathology of IgG4 - RD ( Deshpande et al , 2012 ) , three 40x fields with the highest number of IgG4 + and IgG + cells were selected , counted and averaged within these fields . Cell counts as indicated . f . C - X - C motif chemokine 13 ( CXCL13 ) concentrations measured by ELISA from inflammatory pseudotumor ( IPT ) CSF , control CSF , and MS patient - derived CSF . Individual values , mean ± SEM ; n = 4 experiment repeats with technical replicates . g . Stacked bar chart depicting mean relative expression levels of T helper cell - associated indicated cytokines in T cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF as in ( a ) . TEM , T effector memory ; TCM , T central memory ; Treg , regulatory T cell . h . Violin plot depicting relative expression levels of IL4R ( left ) and IL10RA ( right ) in B cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF .

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 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

Figure 1e. Retrospective immunohistochemistry DAB staining of IgG4 and IgG on archival temporal lobe resection tissue. As suggested by the consensus statement on the pathology of IgG4-RD (Deshpande et al, 2012), three 40x fields with the highest number of IgG4+ and IgG+ cells were selected, counted and averaged within these fields. Cell counts as indicated.f. C-X-C motif chemokine 13 (CXCL13) concentrations measured by ELISA from inflammatory pseudotumor (IPT) CSF, control CSF, and MS patient-derived CSF. Individual values, mean ± SEM; n=4 experiment repeats with technical replicates.g. Stacked bar chart depicting mean relative expression levels of T helper cell-associated indicated cytokines in T cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF as in (a). TEM, T effector memory; TCM, T central memory; Treg, regulatory T cell.h. Violin plot depicting relative expression levels of IL4R (left) and IL10RA (right) in B cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF.


Figure 2a . Relative abundances of cell types identified by Seurat v4 reference mapping as in Figure 1a in IPT CSF compared to control or MS patient - derived CSF . Boxplot depicting 25th - 75th percentiles with median shown as central band and whiskers extending from minimum to maximum values .

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

Figure 2a. Relative abundances of cell types identified by Seurat v4 reference mapping as in Figure 1a in IPT CSF compared to control or MS patient-derived CSF. Boxplot depicting 25th-75th percentiles with median shown as central band and whiskers extending from minimum to maximum values.


figf1 . ( b ) Conjugation of LC3 and two other mammalian homologues ( GL1 and GL2 ) with phosphatidylethanolamine ( PE ) was performed as described in ref . . Complete reactions contain 1 . 5 μM Atg7 , 2 . 5 μM Atg3 , 8 - 12 μM LC3 / GL1 / GL2 , 2 mM lipid ( 30 nm liposomes composed of 10 mol % bl - PI , 55 mol % DOPE , 0 . 15 mol % Rh - DOPE and 34 . 85 mol % POPC ; see Methods for full definitions ) , 1 mM dithiothreitol and 1 mM ATP and were run at 30 ° C for 90 min . Each reaction was run on a 12 % SDS - PAGE gel and visualized by Coomassie blue stain . The mobility of the lipidated protein is faster than the corresponding non - lipidated forms . Intermediates in the reaction are also detectable , including the Atg7 - GRL1 conjugate , the Atg3 - GRL1 conjugate and a modified form of GRL1 , GRL2 and LC3 that is labelled simply as GRL1 / GRL2 / LC3 - intermediate and is probably the adenylated form of each LC3 - related protein . ( c ) The lipidation reactions are highly dependent on the molar percentage of DOPE in the liposomes . Reactions are as in b , but run on 400 nm liposomes . Liposomes are composed of the indicated amount of DOPE , 10 mol % bl - PI , 0 . 15 mol % Rh - DOPE and POPC . ( d ) Completed reactions in c were subjected to density gradient flotation to isolate only the lipidated material . Floated samples were run on SDS - PAGE and imaged by Coomassie . For c and d , the samples were prepared at the same time from the same materials and run simultaneously on multiple gels , as indicated by the vertical lines . Uncropped versions of all gels are included in Supplementary Fig . 7 .

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 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

figf1. (b) Conjugation of LC3 and two other mammalian homologues (GL1 and GL2) with phosphatidylethanolamine (PE) was performed as described in ref. . Complete reactions contain 1.5 μM Atg7, 2.5 μM Atg3, 8-12 μM LC3/GL1/GL2, 2 mM lipid (30 nm liposomes composed of 10 mol% bl-PI, 55 mol% DOPE, 0.15 mol% Rh-DOPE and 34.85 mol% POPC; see Methods for full definitions), 1 mM dithiothreitol and 1 mM ATP and were run at 30 °C for 90 min. Each reaction was run on a 12% SDS-PAGE gel and visualized by Coomassie blue stain. The mobility of the lipidated protein is faster than the corresponding non-lipidated forms. Intermediates in the reaction are also detectable, including the Atg7-GRL1 conjugate, the Atg3-GRL1 conjugate and a modified form of GRL1, GRL2 and LC3 that is labelled simply as GRL1/GRL2/LC3-intermediate and is probably the adenylated form of each LC3-related protein.(c) The lipidation reactions are highly dependent on the molar percentage of DOPE in the liposomes. Reactions are as in b, but run on 400 nm liposomes. Liposomes are composed of the indicated amount of DOPE, 10 mol% bl-PI, 0.15 mol% Rh-DOPE and POPC.(d) Completed reactions in c were subjected to density gradient flotation to isolate only the lipidated material. Floated samples were run on SDS-PAGE and imaged by Coomassie. For c and d, the samples were prepared at the same time from the same materials and run simultaneously on multiple gels, as indicated by the vertical lines. Uncropped versions of all gels are included in Supplementary Fig. 7.


figf2 ( b ) Filling the defects with inverted cone - shaped lipids inhibits lipidation . In vitro coupling reactions of GL1 were run on sonicated liposomes containing 30 mol % DOPE ( 2 mM total lipid ) as in Fig . 1 . To determine whether inverted cones influenced the reaction , increasing amounts of stearoyl - CoA were added immediately before initiating the reaction with ATP . Quantification is of n = 3 samples ; error bars represent standard deviation . P values represent a comparison with samples without stearoyl - CoA . * * P 0 . 1 ; * P 0 . 05 . ( c ) Altering the shape of PE to reduce local membrane defects inhibits the lipidation reaction . In vitro coupling reactions of GL1 were run on liposomes of three different sizes ( 400 nm and 50 nm extruded liposomes and sonicated liposomes ) and of three different lipid compositions . Lipid compositions include 30 mol % DPPE , DSPE or DOPE ( see Methods for full definitions ) , 10 % bl - PI , and POPC . Total lipid = 2 mM . Quantification is of n = 3 samples ; error bars represent standard deviation . The extent of lipidation in b and c is plotted as a percentage of total GL1 as determined by densitometry . P values represent comparison with most active liposomes ( sonicated DOPE ) . * * P 0 . 1 ; * P 0 . 05 .

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 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

figf2(b) Filling the defects with inverted cone-shaped lipids inhibits lipidation. In vitro coupling reactions of GL1 were run on sonicated liposomes containing 30 mol% DOPE (2 mM total lipid) as in Fig. 1. To determine whether inverted cones influenced the reaction, increasing amounts of stearoyl-CoA were added immediately before initiating the reaction with ATP. Quantification is of n = 3 samples; error bars represent standard deviation. P values represent a comparison with samples without stearoyl-CoA. **P 0.1; *P 0.05.(c) Altering the shape of PE to reduce local membrane defects inhibits the lipidation reaction. In vitro coupling reactions of GL1 were run on liposomes of three different sizes (400 nm and 50 nm extruded liposomes and sonicated liposomes) and of three different lipid compositions. Lipid compositions include 30 mol% DPPE, DSPE or DOPE (see Methods for full definitions), 10% bl-PI, and POPC. Total lipid = 2 mM. Quantification is of n = 3 samples; error bars represent standard deviation. The extent of lipidation in b and c is plotted as a percentage of total GL1 as determined by densitometry. P values represent comparison with most active liposomes (sonicated DOPE). **P 0.1; *P 0.05.


figf3 ( b ) To determine the impact of curvature on lipidation , we compared liposomes with unphysiologically high surface densities of PE ( 55 mol % ) as have been used in other Atg8 or LC3 lipidation publications ( for example , refs , , ) , with liposomes having the highest densities of liposomes observed in mammalian organelle membranes ( 30 mol % ) . At physiologically relevant concentrations of liposomes ( 30 mol % ) , lipidation becomes membrane curvature dependent . In vitro coupling reactions were performed as in Fig . 1 , except that liposomes were made by extrusion through membranes with different pore diameters ( as indicated ) . Actual diameters of these liposomes were determined by dynamic light scattering ( Supplementary Table 1 ) . Top : the lipidation of GL2 , GL2 and PE proceeds efficiently and with relatively little curvature dependence on liposomes composed of 55 % PE . However , when the liposomes is reduced to 30 % , flat liposomes are no longer suitable for lipidation , while highly curved liposomes ( 100 nm diameter or less ) continue to couple effectively . Bottom : to confirm that this curvature dependence reveals the lipidated product and not the intermediate , the 30 % reactions were each run on Nycodenz flotation gradients . Only the lipidated product is recovered at the top of the gradient . ( c ) The lipidation reaction is largely insensitive to PE concentrations when run on highly curved liposomes . Coupling of GL1 and LC3 to PE was assessed on liposomes of two sizes ( 400 nm , black squares ; sonicated , red circles ) and varying molar percentage of DOPE . The extent of lipidation is determined as in Fig . 2 . ( n = 2 for 30 and 55 % PE , n = 4 for all the other points , and error bars represent standard deviation . ) P values were determined by comparing sonicated with 400 nm samples at each PE concentration . * * P 0 . 1 ; * P 0 . 01 . Uncropped versions of all gels are included in Supplementary Fig . 7 .

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 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

figf3(b) To determine the impact of curvature on lipidation, we compared liposomes with unphysiologically high surface densities of PE (55 mol%) as have been used in other Atg8 or LC3 lipidation publications (for example, refs , , ), with liposomes having the highest densities of liposomes observed in mammalian organelle membranes (30 mol%). At physiologically relevant concentrations of liposomes (30 mol%), lipidation becomes membrane curvature dependent. In vitro coupling reactions were performed as in Fig. 1, except that liposomes were made by extrusion through membranes with different pore diameters (as indicated). Actual diameters of these liposomes were determined by dynamic light scattering (Supplementary Table 1). Top: the lipidation of GL2, GL2 and PE proceeds efficiently and with relatively little curvature dependence on liposomes composed of 55% PE. However, when the liposomes is reduced to 30%, flat liposomes are no longer suitable for lipidation, while highly curved liposomes (100 nm diameter or less) continue to couple effectively. Bottom: to confirm that this curvature dependence reveals the lipidated product and not the intermediate, the 30% reactions were each run on Nycodenz flotation gradients. Only the lipidated product is recovered at the top of the gradient.(c) The lipidation reaction is largely insensitive to PE concentrations when run on highly curved liposomes. Coupling of GL1 and LC3 to PE was assessed on liposomes of two sizes (400 nm, black squares; sonicated, red circles) and varying molar percentage of DOPE. The extent of lipidation is determined as in Fig. 2. (n = 2 for 30 and 55% PE, n = 4 for all the other points, and error bars represent standard deviation.) P values were determined by comparing sonicated with 400 nm samples at each PE concentration. **P 0.1; *P 0.01. Uncropped versions of all gels are included in Supplementary Fig. 7.


figf4 ( a ) The accumulation of the Atg3 - GL1 conjugate depends on the extent of membrane curvature . In vitro GL1 coupling reactions were run as in Fig . 1 . The reactions were visualized by immunoblot against Atg3 ( top ) and by Coomassie staining of GRL1 ( bottom ) . The accumulation of Atg3 - GL1 is inversely related to the formation of the lipidated GRL1 , suggesting that the membrane - curvature - dependent step may involve Atg3 recognition of the membrane surface . ( b ) Atg3 binding to liposomes . Atg3 ( 10 μM ) was incubated at 30 ° C for 90 min with liposomes containing 0 , 30 or 55 mol % DOPE ( 5 mM lipids ) and of varying sizes ( extrusion membrane dimensions are shown on the figure ; actual final sizes were determined by dynamic light scattering ( Supplementary Table 1 ) ) . The liposome - associated Atg3 was recovered by Nycodenz density gradient centrifugation and analysed by SDS - PAGE . ( c ) Quantification of liposome - associated Atg3 . Densitometric plot of the amount of Atg3 recovered in the top fraction in flotation assays as in b . Error bars indicate s . e . m . from three independent experiments . P values calculated as compared with putative negative control ( 0 % PE , 400 nm ) . * P 0 . 05 . Uncropped versions of all gels are included in Supplementary Fig . 7 .

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 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

figf4(a) The accumulation of the Atg3-GL1 conjugate depends on the extent of membrane curvature. In vitro GL1 coupling reactions were run as in Fig. 1. The reactions were visualized by immunoblot against Atg3 (top) and by Coomassie staining of GRL1 (bottom). The accumulation of Atg3-GL1 is inversely related to the formation of the lipidated GRL1, suggesting that the membrane-curvature-dependent step may involve Atg3 recognition of the membrane surface.(b) Atg3 binding to liposomes. Atg3 (10 μM) was incubated at 30 °C for 90 min with liposomes containing 0, 30 or 55 mol% DOPE (5 mM lipids) and of varying sizes (extrusion membrane dimensions are shown on the figure; actual final sizes were determined by dynamic light scattering (Supplementary Table 1)). The liposome-associated Atg3 was recovered by Nycodenz density gradient centrifugation and analysed by SDS-PAGE.(c) Quantification of liposome-associated Atg3. Densitometric plot of the amount of Atg3 recovered in the top fraction in flotation assays as in b. Error bars indicate s.e.m. from three independent experiments. P values calculated as compared with putative negative control (0% PE, 400 nm). *P 0.05. Uncropped versions of all gels are included in Supplementary Fig. 7.


figf5 ( b ) Analysis of reaction efficiency for point mutants of the amphipathic helix . GL1 lipidation reactions were run on 400 nm and sonicated liposomes ( 30 mol % DOPE ) with the indicated Atg3 mutants . The extent of GL1 - PE formation ( top gel ) was determined from densitometry and is plotted as percentage of total GL1 ( bar graph ) . The ability to form the Atg3 - GL1 conjugate was also assessed for each mutant ( bottom immunoblot ) . Note that this conjugate forms in every case , indicating that no mutant inhibited a step upstream in the reaction . Further , this conjugate specifically accumulates in reactions where lipidation is impaired . In both panels , the samples were prepared at the same time from the same materials and run simultaneously on multiple gels , as indicated by the lines . ( c ) Curvature dependence of K11L mutant binding to liposomes compared with wild - type Atg3 . The binding reaction was performed as in Fig . 4b on 30 DOPE % liposomes . F , float - up ; T , total input . ( d ) Lys 11 represents a tunable node through which curvature dependence of the lipidation reaction can be controlled . Lipidation assays were run as in b . Total GL1 lipidation was assessed from reactions with wild - type Atg3 or mutant forms of Atg3 in which position Lys 11 was changed as indicated . Lipidation efficiency on low PE / low curvature liposomes ( 30 % PE , 400 nm ) increases with increasing hydrophobicity of the new amino acid , while liposomes with high PE ( 55 % ) or high curvature ( sonicated ) are good substrates for all active forms of Atg3 . Left : Coomassie gels of individual lipidation reactions . Right : densitometry of Coomassie - stained gels revealed the ratio of GL1 - PE to the sum of all three GL1 species as in Fig . 2 . Light grey bars are sonicated liposomes ; dark grey bars are 400 nm extruded liposomes . For b , c , P values were calculated between sonicated and 400 nm samples of the same mutant . For d , P values represent comparison with WT protein on the same liposome size and composition . For all panels , n = 3 independent experiments , error bars represent standard deviation and * * P 0 . 05 ; * P 0 . 01 . Uncropped gels in Supplementary Fig . 7 .

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 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

figf5(b) Analysis of reaction efficiency for point mutants of the amphipathic helix. GL1 lipidation reactions were run on 400 nm and sonicated liposomes (30 mol% DOPE) with the indicated Atg3 mutants. The extent of GL1-PE formation (top gel) was determined from densitometry and is plotted as percentage of total GL1 (bar graph). The ability to form the Atg3-GL1 conjugate was also assessed for each mutant (bottom immunoblot). Note that this conjugate forms in every case, indicating that no mutant inhibited a step upstream in the reaction. Further, this conjugate specifically accumulates in reactions where lipidation is impaired. In both panels, the samples were prepared at the same time from the same materials and run simultaneously on multiple gels, as indicated by the lines.(c) Curvature dependence of K11L mutant binding to liposomes compared with wild-type Atg3. The binding reaction was performed as in Fig. 4b on 30 DOPE% liposomes. F, float-up; T, total input.(d) Lys 11 represents a tunable node through which curvature dependence of the lipidation reaction can be controlled. Lipidation assays were run as in b. Total GL1 lipidation was assessed from reactions with wild-type Atg3 or mutant forms of Atg3 in which position Lys 11 was changed as indicated. Lipidation efficiency on low PE/low curvature liposomes (30% PE, 400 nm) increases with increasing hydrophobicity of the new amino acid, while liposomes with high PE (55%) or high curvature (sonicated) are good substrates for all active forms of Atg3. Left: Coomassie gels of individual lipidation reactions. Right: densitometry of Coomassie-stained gels revealed the ratio of GL1-PE to the sum of all three GL1 species as in Fig. 2. Light grey bars are sonicated liposomes; dark grey bars are 400 nm extruded liposomes. For b,c, P values were calculated between sonicated and 400 nm samples of the same mutant. For d, P values represent comparison with WT protein on the same liposome size and composition. For all panels, n = 3 independent experiments, error bars represent standard deviation and **P 0.05; *P 0.01. Uncropped gels in Supplementary Fig. 7.


figf6Atg3 - knockout MEFs ( Atg3 − / − ) are incapable of forming LC3 - II and do not accumulate LC3 puncta . Instead , they accumulate autophagic intermediates that are positive for Atg16 . To determine whether the amphipathic helix is critical for in vivo function , Atg3 with wild - type or mutant forms of the amphipathic helix was introduced into Atg3 − / − MEFs by lentiviral infection and rescue of the three phenotypes was tested . ( a ) Immunoblot analysis of LC3 - II , GL2 - II and GABARAP - II formation . GABARAP lipidation is significantly observed only in the presence of bafilomycin A1 ( BafA1 : 100 nM ) . Asterisk indicates a nonspecific band recognized by the Atg3 antibody . ( b ) Cells were infected as in a and transduced cells were selected with puromycin for 1 - 2 days before starvation and immunolabelling with the anti - LC3B antibody . Representative images are shown in b ( c ) The number of LC3 puncta per cell was counted manually from 22 - 96 cells per replicate . Raw data can be found in Supplementary Fig . 5 . P values represent difference from vector negative control . n = 2 - 5 independent experiments ; the precise n values and number of cells per experiment are listed in the Methods . d ) Cells as in b were kept in full media before being subjected to fixation and immunolabelling with the anti - Atg16L antibody . Atg16L puncta area as a fraction of total cellular area was obtained as described in Methods . This ratio was plotted as arbitrary units . Grey dashed line indicates the value for Atg3 − / − cells rescued with wild - type Atg3 . n = 2 ( for V15K ) or 3 ( for all others ) independent experiments ; the precise n values and number of fields visualized per experiment are listed in the Methods . Raw data can be found in Supplementary Fig . 4 . Atg3 variants that support lipidation of LC3 family proteins in vitro ( Fig . 5 ) are indicated with black bars , while those that do not support lipidation are indicated with white bars . P values represent difference from wild - type rescue . * * P 0 . 1 ; * P 0 . 05 . Error bars in c , d represent s . d . for all samples of n > 2 . Uncropped versions of all gels are included in Supplementary Fig . 7 .

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

figf6Atg3-knockout MEFs (Atg3−/−) are incapable of forming LC3-II and do not accumulate LC3 puncta. Instead, they accumulate autophagic intermediates that are positive for Atg16. To determine whether the amphipathic helix is critical for in vivo function, Atg3 with wild-type or mutant forms of the amphipathic helix was introduced into Atg3−/− MEFs by lentiviral infection and rescue of the three phenotypes was tested. (a) Immunoblot analysis of LC3-II, GL2-II and GABARAP-II formation. GABARAP lipidation is significantly observed only in the presence of bafilomycin A1 (BafA1: 100 nM). Asterisk indicates a nonspecific band recognized by the Atg3 antibody.(b) Cells were infected as in a and transduced cells were selected with puromycin for 1-2 days before starvation and immunolabelling with the anti-LC3B antibody. Representative images are shown in b(c) The number of LC3 puncta per cell was counted manually from 22-96 cells per replicate. Raw data can be found in Supplementary Fig. 5. P values represent difference from vector negative control. n = 2-5 independent experiments; the precise n values and number of cells per experiment are listed in the Methods.d) Cells as in b were kept in full media before being subjected to fixation and immunolabelling with the anti-Atg16L antibody. Atg16L puncta area as a fraction of total cellular area was obtained as described in Methods. This ratio was plotted as arbitrary units. Grey dashed line indicates the value for Atg3−/− cells rescued with wild-type Atg3. n = 2 (for V15K) or 3 (for all others) independent experiments; the precise n values and number of fields visualized per experiment are listed in the Methods. Raw data can be found in Supplementary Fig. 4. Atg3 variants that support lipidation of LC3 family proteins in vitro (Fig. 5) are indicated with black bars, while those that do not support lipidation are indicated with white bars. P values represent difference from wild-type rescue. **P 0.1; *P 0.05. Error bars in c,d represent s.d. for all samples of n > 2. Uncropped versions of all gels are included in Supplementary Fig. 7.


Figure 1 ( A ) The indicated yeast strains ( wild type , pex19Δ , pex3Δ , msp1Δ , pex19Δmsp1Δ , and pex3Δmsp1Δ ) expressing RFP - SKL ( peroxisomal marker ) and mitochondria - targeted green ﬂuorescent protein ( mGFP ) were grown to mid - log phase and imaged by ﬂuorescence microscopy . Representative images are shown , scale bar , 2 μm . DIC , differential interference contrast . Enhanced : the red signal intensity was set to ' best - fit ' in ZENPro microscopy software analysis .

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

Figure 1(A) The indicated yeast strains (wild type, pex19Δ, pex3Δ, msp1Δ, pex19Δmsp1Δ, and pex3Δmsp1Δ) expressing RFP-SKL (peroxisomal marker) and mitochondria-targeted green ﬂuorescent protein (mGFP) were grown to mid-log phase and imaged by ﬂuorescence microscopy. Representative images are shown, scale bar, 2 μm. DIC, differential interference contrast. Enhanced: the red signal intensity was set to 'best-fit' in ZENPro microscopy software analysis.


Figure 2 ( B ) Volcano plot showing the most enriched / decreased proteins in the mitochondrial proteome of pex19Δ versus pex19Δmsp1Δ strains as detected by quantitative mass spectrometry ( n = 7 , biological replicates ; n = 2 , technical replicates ) . Effect sizes were calculated for Cohen ' s d A protein ' s relevance was determined using a p - value threshold of < 0 . 05 with an absolute effect size threshold of 1 or greater ( large effect ) . Gray dots represent all measured proteins and red dots highlight peroxisomal - associated proteins . ( D ) The indicated yeast strains expressing PEX13 - RFP and mitochondria - targeted green ﬂuorescent protein ( mGFP ) were grown to mid - log phase and visualized by ﬂuorescence microscopy . Representative images are shown , scale bar , 2 μm . DIC , differential interference contrast . ( F ) Total yeast cell lysate , post mitochondrial supernatant , and HistodenzTM - purified mitochondria from the indicated strain ( wild type , msp1Δ , pex19Δ , pex19Δmsp1Δ , pex3Δ , and pex3Δmsp1Δ ) expressing Pex13 - V5 ( under its endogenous promotor ) were separated by SDS - PAGE and immunoblotted for Pex13 ( α - V5 ) , cytochrome c ( α - cyt c ) , and HSP70 ( α - hsp70 ) . ( G ) Total yeast cell lysate , post mitochondrial supernatant , and HistodenzTM - purified mitochondria from the indicated strain ( wild type , msp1Δ , pex19Δ , pex19Δmsp1Δ , pex3Δ , and pex3Δmsp1Δ ) expressing Pex11 - V5 ( under its endogenous promotor ) were separated by SDS - PAGE and immunoblotted for Pex11 ( α - V5 ) and porin ( α - porin ) , cytochrome c ( α - cyt c ) , and HSP70 ( α - hsp70 ) .

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 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 O O O O O O O O O O O O O O O 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(B) Volcano plot showing the most enriched/decreased proteins in the mitochondrial proteome of pex19Δ versus pex19Δmsp1Δ strains as detected by quantitative mass spectrometry (n=7, biological replicates; n=2, technical replicates). Effect sizes were calculated for Cohen's d A protein's relevance was determined using a p-value threshold of < 0.05 with an absolute effect size threshold of 1 or greater (large effect). Gray dots represent all measured proteins and red dots highlight peroxisomal-associated proteins.(D) The indicated yeast strains expressing PEX13-RFP and mitochondria-targeted green ﬂuorescent protein (mGFP) were grown to mid-log phase and visualized by ﬂuorescence microscopy. Representative images are shown, scale bar, 2 μm. DIC, differential interference contrast.(F) Total yeast cell lysate, post mitochondrial supernatant, and HistodenzTM-purified mitochondria from the indicated strain (wild type, msp1Δ, pex19Δ, pex19Δmsp1Δ, pex3Δ, and pex3Δmsp1Δ) expressing Pex13-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for Pex13 (α-V5), cytochrome c (α-cyt c), and HSP70 (α-hsp70). (G) Total yeast cell lysate, post mitochondrial supernatant, and HistodenzTM-purified mitochondria from the indicated strain (wild type, msp1Δ, pex19Δ, pex19Δmsp1Δ, pex3Δ, and pex3Δmsp1Δ) expressing Pex11-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for Pex11 (α-V5) and porin (α-porin), cytochrome c (α-cyt c), and HSP70 (α-hsp70).


Figure 3 ( A ) Volcano plot ( log10 ) of the most enriched / decreased proteins in the mitochondrial proteome of pex3Δ versus pex3Δmsp1Δ strains as detected by quantitative mass spectrometry ( n = 3 , biological replicates , n = 1 , technical replicate ) . 2010 ) . Effect sizes were calculated for Cohen ' s d A protein ' s relevance to the model was determined using a p - value threshold of < 0 . 05 with an absolute effect size threshold of 1 or greater ( large effect ) . Gray dots represent all measured proteins . Red dots highlight peroxisomal - associated proteins . ( C ) Mitochondria from the indicated yeast strains ( wild type , pex19Δ , pex19Δmsp1Δ ) , expressing Pex13 - V5 ( under its endogenous promotor ) were either left untreated ( lane 1 , 2 , 3 ) or exposed to Na2CO3 and separated into the membrane pellet ( 4 , 5 , 6 ) or supernatant fraction ( 7 , 8 , 9 ) . After separation by SDS - PAGE the fractions were immunoblotted for Pex13 - V5 ( α - V5 ) and porin ( α - porin ) .

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 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

Figure 3(A) Volcano plot (log10) of the most enriched/decreased proteins in the mitochondrial proteome of pex3Δ versus pex3Δmsp1Δ strains as detected by quantitative mass spectrometry (n=3, biological replicates, n=1, technical replicate). 2010). Effect sizes were calculated for Cohen's d A protein's relevance to the model was determined using a p-value threshold of < 0.05 with an absolute effect size threshold of 1 or greater (large effect). Gray dots represent all measured proteins. Red dots highlight peroxisomal-associated proteins.(C) Mitochondria from the indicated yeast strains (wild type, pex19Δ, pex19Δmsp1Δ), expressing Pex13-V5 (under its endogenous promotor) were either left untreated (lane 1,2,3) or exposed to Na2CO3 and separated into the membrane pellet (4,5,6) or supernatant fraction (7,8,9). After separation by SDS-PAGE the fractions were immunoblotted for Pex13-V5 (α-V5) and porin (α-porin).


Figure 4 ( B ) Digitonin - solubilized ( 1 or 4 g / g digitonin / protein ratio , as indicated ) mitochondrial membranes from the indicated yeast strains ( wild type , pex19Δ , pex19Δmsp1Δ ) expressing Pex13 - V5 ( under its endogenous promotor ) were separated by BN - PAGE with a 3 - 18 % gradient and stained with Coomassie brilliant blue . BHM , bovine heart mitochondria ; V2 , complex V - dimer ; SL , supercomplex III2IV2 ; Ss , supercomplex III2IV1 ; V1 , complex V - monomer . ( F ) Mitochondria from the indicated yeast strains ( pex19Δ , pex19Δmsp1Δ , and pex13Δmsp1Δpex19Δ ) expressing Pex22 - V5 ( under its endogenous promotor ) ( HistodenzTM - purified ) were separated by SDS - PAGE and immunoblotted for Pex22 - V5 ( α - Pex22 - V5 ) and porin ( α - porin ) . ( G ) Mitochondria from the indicated yeast strains ( pex19Δ , pex19Δmsp1Δ , and pex13Δmsp1Δpex19Δ ) expressing Pot1 - V5 ( under its endogenous promotor ) were separated by SDS - PAGE and immunoblotted for Pot1 - V5 ( α - Pot1 - V5 ) and porin ( α - porin ) . ( H ) Mitochondria from the indicated yeast strains ( pex19Δ , pex19Δmsp1 Δ , and pex13Δmsp1Δpex19Δ ) expressing Mdh3 - V5 ( under its endogenous promotor ) ( HistodenzTM - purified ) were separated by SDS - PAGE and immunoblotted for Mdh3 - V5 ( α - Mdh3 - V5 ) and porin ( α - porin ) . ( I ) Mitochondria from the indicated yeast strains ( pex19Δ , pex19Δmsp1Δ , and pex13Δmsp1Δpex19Δ ) expressing Pot1 - V5 ( under its endogenous promotor ) were either left untreated ( nt ) ( lane 1 , 4 , 7 ) or were treated with proteinase K in high fidelity buffer ( PK - HF ) ( cutting control , lane 2 , 5 , 8 ) or low fidelity buffer ( PK - LF ) ( protease protection assay , lane 3 , 6 , 9 ) and separated by SDS - PAGE and immunoblotted for Mia40 ( intermembrane space ) ( α - Mia40 ) , Pot1 - V5 ( α - V5 ) , porin ( outer membrane ) ( α - porin ) and Tom22 ( outer membrane but partially proteinase exposed ) ( α - Tom22 ) .

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 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 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

Figure 4(B) Digitonin-solubilized (1 or 4 g/g digitonin/protein ratio, as indicated) mitochondrial membranes from the indicated yeast strains (wild type, pex19Δ, pex19Δmsp1Δ) expressing Pex13-V5 (under its endogenous promotor) were separated by BN-PAGE with a 3-18% gradient and stained with Coomassie brilliant blue. BHM, bovine heart mitochondria; V2, complex V-dimer; SL,supercomplex III2IV2; Ss, supercomplex III2IV1; V1, complex V-monomer.(F) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1Δ, and pex13Δmsp1Δpex19Δ) expressing Pex22-V5 (under its endogenous promotor) (HistodenzTM-purified) were separated by SDS-PAGE and immunoblotted for Pex22-V5 (α-Pex22-V5) and porin (α-porin). (G) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1Δ, and pex13Δmsp1Δpex19Δ ) expressing Pot1-V5 (under its endogenous promotor) were separated by SDS-PAGE and immunoblotted for Pot1-V5 (α-Pot1-V5) and porin (α-porin). (H) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1 Δ, and pex13Δmsp1Δpex19Δ) expressing Mdh3-V5 (under its endogenous promotor) (HistodenzTM-purified) were separated by SDS-PAGE and immunoblotted for Mdh3-V5 (α-Mdh3-V5) and porin (α-porin).(I) Mitochondria from the indicated yeast strains (pex19Δ, pex19Δmsp1Δ, and pex13Δmsp1Δpex19Δ) expressing Pot1-V5 (under its endogenous promotor) were either left untreated (nt) (lane 1,4,7) or were treated with proteinase K in high fidelity buffer (PK-HF) (cutting control, lane 2,5,8) or low fidelity buffer (PK-LF) (protease protection assay, lane 3,6,9) and separated by SDS-PAGE and immunoblotted for Mia40 (intermembrane space) (α-Mia40), Pot1-V5 (α-V5), porin (outer membrane) (α-porin) and Tom22 (outer membrane but partially proteinase exposed) (α-Tom22).


Figure 5 ( A ) Fluorescence microscopy of human fibroblast cell lines derived from ZSD patients - cWT , cWT + ATAD1 , PEX3 - / - , and PEX3 - / - + ATAD1 - expressing PEX13 - GFP and stained with Mitotracker  Deep Red FM . Representative images are shown , scale bar , 10 μm . ( B ) Electron microscopy of human fibroblast cell lines cWT , cWT + ATAD1 , PEX3 - / - , and PEX3 - / - + ATAD1 . Representative images are shown , scale bar 500 nm .

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 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

Figure 5(A) Fluorescence microscopy of human fibroblast cell lines derived from ZSD patients-cWT, cWT+ATAD1, PEX3-/-, and PEX3-/-+ATAD1-expressing PEX13-GFP and stained with Mitotracker Deep Red FM. Representative images are shown, scale bar, 10 μm.(B) Electron microscopy of human fibroblast cell lines cWT, cWT+ATAD1, PEX3-/-, and PEX3-/-+ATAD1. Representative images are shown, scale bar 500 nm.


Figure 6 ( A ) Bioenergetic profile of human fibroblast cell lines . OCR ( pmol / min / norm . unit ) for cWT , cWT + ATAD1 , PEX3 - / - , PEX3 - / - + ATAD1 , cWT - ATAD1 - / - , and PEX3 - / - atad1 - / - cells plotted against time ( XF96e Extracellular Flux Analyzer , Mito - Stress - Test ) . 1 μM Oligomycin A , 0 . 15 μM FCCP , and 1 μM rotenone + 1 μM antimycin A ( final concentrations ) were sequentially delivered to the XF96e assay medium through injection ports in the sensor cartridge . The graph is the compilation of two independent assays , error bars show mean + / - s . d . ( n = 4 , biological replicates ) . ( E ) Scatter plot representation of cardiolipin lipidomics . The average ( n = 4 , biological replicates ) normalized peak intensity of each of the 11 detected cardiolipin species , log10 pareto - scaled is visualized . The horizontal lines represent the mean peak intesity of all 11 caridolipin species in each cell line . ( F ) Scatter plot representation of a subpopulation of the phosphoethanolamine ( PE ) lipidomics . The average ( n = 4 , biological replicates ) normalized peak intensity of each of the 20 phosphoethanolamine ( PE ) species ( decreased in PEX3 - / - ) , log10 pareto - scaled , is visualized . The horizontal lines represent the mean peak intesity of all 20 PE species in each cell line . ( G ) Scatter plot representation of ether - phospholipid lipidomics . The average ( n = 4 , biological replicates ) normalized peak intensity of each of the 21 detected ether - phospholipid ( plasmalogen ) species , log10 pareto - scaled , is visualized . The horizontal lines represent the mean peak intesity of all 21 plasmalogen species in each cell line .

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Figure 6(A) Bioenergetic profile of human fibroblast cell lines. OCR (pmol/min/norm.unit) for cWT, cWT+ATAD1, PEX3-/-, PEX3-/-+ATAD1, cWT-ATAD1-/-, and PEX3-/- atad1-/- cells plotted against time (XF96e Extracellular Flux Analyzer, Mito-Stress-Test). 1 μM Oligomycin A, 0.15 μM FCCP, and 1 μM rotenone + 1 μM antimycin A (final concentrations) were sequentially delivered to the XF96e assay medium through injection ports in the sensor cartridge. The graph is the compilation of two independent assays, error bars show mean +/- s.d. (n=4, biological replicates).(E) Scatter plot representation of cardiolipin lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 11 detected cardiolipin species, log10 pareto-scaled is visualized. The horizontal lines represent the mean peak intesity of all 11 caridolipin species in each cell line. (F) Scatter plot representation of a subpopulation of the phosphoethanolamine (PE) lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 20 phosphoethanolamine (PE) species (decreased in PEX3-/-), log10 pareto-scaled, is visualized. The horizontal lines represent the mean peak intesity of all 20 PE species in each cell line. (G) Scatter plot representation of ether-phospholipid lipidomics. The average (n = 4, biological replicates) normalized peak intensity of each of the 21 detected ether-phospholipid (plasmalogen) species, log10 pareto-scaled, is visualized. The horizontal lines represent the mean peak intesity of all 21 plasmalogen species in each cell line.


Figure 1B 18F - FDG PET - MRI at the time of the diagnosis and in the interim staging after one cycle of FOLFIRINOX . C H & E staining of FNB and the resection specimen . Scale bars represent 50 µm . D Phase - contrast images of organoids ( upper panel ) and respective 2D cultures ( isolated from the biopsy before FOLFIRINOX ( ID188 ) or after the neoadjuvant therapy from the resected cancer ( ID211 ) . Scale bar represents 200µm . E IF staining of the actin filaments ( red ) and nuclei ( blue ) of organoids in the left panel and H & E staining of embedded and sectioned organoids in the right panel ( ID188 p30 , ID211 p30 ) . F Normalized expression ( log2 scale ) for gene pairs evaluated during Purity independent subtyping of tumors ( PurIST ) . Normalized expression ( log2 scale ) , gene 1 = basal - like , gene 2 = classical . G Gene Set Enrichment Analysis ( GSEA ) of pre - ( ID188 , p7 ) and post - treatment ( ID211 , p6 ) PDO lines . Normalized enrichment score ( NES ) and the adjusted p - value is depicted . H Whole exome sequencing - based copy number profiles for ID188 ( p7 ) and ID211 ( p6 ) organoids . I Allele frequencies of miss - and nonsense SNVs and Indels which are shared between both organoid lines ( purple ) , private to ID188 ( red ) or private to ID211 ( blue ) .

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Figure 1B 18F-FDG PET-MRI at the time of the diagnosis and in the interim staging after one cycle of FOLFIRINOX.C H&E staining of FNB and the resection specimen. Scale bars represent 50 µm.D Phase-contrast images of organoids (upper panel) and respective 2D cultures (isolated from the biopsy before FOLFIRINOX (ID188) or after the neoadjuvant therapy from the resected cancer (ID211). Scale bar represents 200µm.E IF staining of the actin filaments (red) and nuclei (blue) of organoids in the left panel and H&E staining of embedded and sectioned organoids in the right panel (ID188 p30, ID211 p30).F Normalized expression (log2 scale) for gene pairs evaluated during Purity independent subtyping of tumors (PurIST). Normalized expression (log2 scale), gene 1 = basal-like, gene 2 = classical.G Gene Set Enrichment Analysis (GSEA) of pre-(ID188, p7) and post-treatment (ID211, p6) PDO lines. Normalized enrichment score (NES) and the adjusted p-value is depicted.H Whole exome sequencing-based copy number profiles for ID188 (p7) and ID211 (p6) organoids.I Allele frequencies of miss- and nonsense SNVs and Indels which are shared between both organoid lines (purple), private to ID188 (red) or private to ID211 (blue).


Figure 2A Dose response curves of ID188 ( p27 - 29 ) and ID211 ( p26 - 28 ) organoids treated with FOLFIRINOX over three days . ATP was measured with CellTiter - Glo assays . Shown is the mean ± SD of three independent experiments . C Plot of the delta AUC in ID188 ( p5 + 27 ) versus ID211 ( p9 + 11 ) cells over 415 drugs . Dashed lines demark the threshold of delta AUC > 0 . 3 or < - 0 . 3 . D Heatmap of the screening hits as defined in C . AUC is color coded . E - J Dose response curves of ID188 and ID211 organoids ( ID188 p27 - 29 , ID211 p26 - 28 ) or 2D lines ( ID188 p5 + 29 , ID211 p9 + 13 ) treated with ( E and F ) : Cobimetinib or ( H and I ) : Biminetinib . ( G ) Cobimetinib and ( J ) Biminetinib : GI50 values . Shown is the mean ± SD of three independent experiments . K Illustration of a cohort of patients ( n = 15 ) with available primary 2D PDAC cell lines . This cohort includes the ID188 ( pre - FFX ( FOLFIRINOX ) , red ) and ID211 ( post - FFX , blue ) lines . These lines were screened for sensitivity towards three MEKi . L 15 primary human PDAC 2D cell lines were screened for MEKi ( as indicated ) sensitivity . The determined AUC was variance scaled and the z - scores are depicted . The ID188 and ID211 identity is color coded .

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Figure 2A Dose response curves of ID188 (p27-29) and ID211 (p26-28) organoids treated with FOLFIRINOX over three days. ATP was measured with CellTiter-Glo assays. Shown is the mean ± SD of three independent experiments.C Plot of the delta AUC in ID188 (p5+27) versus ID211 (p9+11) cells over 415 drugs. Dashed lines demark the threshold of delta AUC >0.3 or <-0.3. D Heatmap of the screening hits as defined in C. AUC is color coded.E - J Dose response curves of ID188 and ID211 organoids (ID188 p27-29, ID211 p26-28) or 2D lines (ID188 p5+29, ID211 p9+13) treated with (E and F): Cobimetinib or (H and I): Biminetinib. (G) Cobimetinib and (J) Biminetinib: GI50 values. Shown is the mean ± SD of three independent experiments.K Illustration of a cohort of patients (n=15) with available primary 2D PDAC cell lines. This cohort includes the ID188 (pre-FFX (FOLFIRINOX), red) and ID211 (post-FFX, blue) lines. These lines were screened for sensitivity towards three MEKi. L 15 primary human PDAC 2D cell lines were screened for MEKi (as indicated) sensitivity. The determined AUC was variance scaled and the z-scores are depicted. The ID188 and ID211 identity is color coded.


Figure 1A . A positive control experiment . SARS - CoV - 2 readily targets ciliated human respiratory epithelial cells ( hRECs ) . Acetylated α - tubulin labels cilia . Arrows point SARS - CoV - 2 - positive cells labeled by AB4 ( green ) . Figures display scale bars . Bar diagram at right quantifies frequencies of SARS - CoV - 2 - positive cells in hRECs . At least six hREC sections from three ( n = 3 ) independent samples were examined . Data presented as mean + SEM . B . Mock organoids of two age groups Day - 15 ( i ) and - 60 ( ii ) display typical cytoarchitecture of brain organoids . L , Lumen , VZ , Ventricular Zone is containing compact and palisade - like elongated nuclei of Neural Progenitor Cells ( NPCs , blue ) and CP , a cortical plate containing TUJ - 1 - positive neurons ( magenta ) . Note a distinct difference TUJ - 1 labeling pattern between younger ( Day - 15 ) and older ( Day - 60 ) brain organoid . Figures display scale bars . Representative images from eight organoids cultured in at least three independent batches ( n = 3 ) derived from donor - 1 ( IMR90 ) iPSC line . C . Compared to mock organoids ( i ) , SARS - CoV - 2 - exposed Day - 15 organoids display SARS - CoV - 2 - positive cells ( AB4 , green ) in their outer periphery , a region of the cortical plate ( ii ) that is specified by TUJ - 1 - positive neurons ( magenta ) . L , the lumen of a VZ , the inner area of an organoid where NPCs are located , is free from SARS - CoV - 2 - positive cells . Magnified region ( dotted while box ) is given below . At least ten organoids from five different batches ( n = 5 ) are tested . Figures display scale bars . D . SARS - CoV - 2 - exposed Day - 60 organoids . Compared to Day 15 organoids and mock ( i ) , Day - 60 organoids display an increased number of SARS - CoV - 2 - positive cells ( AB4 , green ) in their cortical plate that is specified by TUJ - 1 - positive neurons ( magenta ) ( ii ) . Magnified region ( dotted while box ) is given below , showing the peri - nuclear location of SARS - CoV - 2 in cortical neurons . At least ten organoids from five different batches ( n = 5 ) are tested . Figures display scale bars . E . The bar diagram quantifies frequencies of SARS - CoV - 2 - positive cells in different brain organoid sections derived from two donor iPSC lines ( IMR90 and Crx - iPS , see methods ) . Please note that each point represents one organoid section . SARS - CoV - 2 shows an enhanced tropism for Day - 60 organoids . Note , comparative statistics are shown between different age groups and respective days post infection ( dpi ) of organoids , and the significance is given as Asterisks in Day - 60 groups . There is no significant difference in SARS - CoV - 2 - positive cells between 2 - and 4 - dpi within each age groups . At least twelve organoids sections from four ( n = 4 ) independent batches , from each donor and day post infections ( dpi ) were analyzed . One - way ANOVA , followed by Tukey ' s multiple comparisons test , * * * P < 0 . 001 . Data presented as mean + SEM . F . Subcellular localization SARS - CoV - 2 in cortical neurons . High - resolution imaging and de - convolution show peri - nuclear localization of SARS - CoV - 2 . SARS - CoV - 2 ( AB4 , green ) and nucleus ( grey ) . Figures display scale bars . Representative images from at least 200 cells are examined . White line surrounds perinuclear border and red line encircles the nucleus . G . Determination of viral progeny . Supernatants of SARS - CoV - 2 exposed Vero cells , and brain organoids were analyzed for viral RNA assessed by qRT - PCR . While an increase in viral RNA was detected in the supernatants of Vero cells , no apparent increase was identified in brain organoid supernatants . Data are obtained from five technical replicates from four ( n = 4 ) independent batches of organoids . Data presented as mean + SEM .

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 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 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 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

Figure 1A. A positive control experiment. SARS-CoV-2 readily targets ciliated human respiratory epithelial cells (hRECs). Acetylated α-tubulin labels cilia. Arrows point SARS-CoV-2-positive cells labeled by AB4 (green). Figures display scale bars. Bar diagram at right quantifies frequencies of SARS-CoV-2-positive cells in hRECs. At least six hREC sections from three (n=3) independent samples were examined. Data presented as mean + SEM.B. Mock organoids of two age groups Day-15 (i) and-60 (ii) display typical cytoarchitecture of brain organoids. L, Lumen, VZ, Ventricular Zone is containing compact and palisade-like elongated nuclei of Neural Progenitor Cells (NPCs, blue) and CP, a cortical plate containing TUJ-1-positive neurons (magenta). Note a distinct difference TUJ-1 labeling pattern between younger (Day-15) and older (Day-60) brain organoid. Figures display scale bars. Representative images from eight organoids cultured in at least three independent batches (n=3) derived from donor-1 (IMR90) iPSC line. C. Compared to mock organoids (i), SARS-CoV-2-exposed Day-15 organoids display SARS-CoV-2-positive cells (AB4, green) in their outer periphery, a region of the cortical plate (ii) that is specified by TUJ-1-positive neurons (magenta). L, the lumen of a VZ, the inner area of an organoid where NPCs are located, is free from SARS-CoV-2-positive cells. Magnified region (dotted while box) is given below. At least ten organoids from five different batches (n=5) are tested. Figures display scale bars. D. SARS-CoV-2-exposed Day-60 organoids. Compared to Day 15 organoids and mock (i), Day-60 organoids display an increased number of SARS-CoV-2-positive cells (AB4, green) in their cortical plate that is specified by TUJ-1-positive neurons (magenta) (ii). Magnified region (dotted while box) is given below, showing the peri-nuclear location of SARS-CoV-2 in cortical neurons. At least ten organoids from five different batches (n=5) are tested. Figures display scale bars.E. The bar diagram quantifies frequencies of SARS-CoV-2-positive cells in different brain organoid sections derived from two donor iPSC lines (IMR90 and Crx-iPS, see methods). Please note that each point represents one organoid section. SARS-CoV-2 shows an enhanced tropism for Day-60 organoids. Note, comparative statistics are shown between different age groups and respective days post infection (dpi) of organoids, and the significance is given as Asterisks in Day-60 groups. There is no significant difference in SARS-CoV-2-positive cells between 2- and 4-dpi within each age groups. At least twelve organoids sections from four (n=4) independent batches, from each donor and day post infections (dpi) were analyzed. One-way ANOVA, followed by Tukey's multiple comparisons test, ***P<0.001. Data presented as mean + SEM.F. Subcellular localization SARS-CoV-2 in cortical neurons. High-resolution imaging and de-convolution show peri-nuclear localization of SARS-CoV-2. SARS-CoV-2 (AB4, green) and nucleus (grey). Figures display scale bars. Representative images from at least 200 cells are examined. White line surrounds perinuclear border and red line encircles the nucleus.G. Determination of viral progeny. Supernatants of SARS-CoV-2 exposed Vero cells, and brain organoids were analyzed for viral RNA assessed by qRT-PCR. While an increase in viral RNA was detected in the supernatants of Vero cells, no apparent increase was identified in brain organoid supernatants. Data are obtained from five technical replicates from four (n=4) independent batches of organoids. Data presented as mean + SEM.


Figure 2A . Tau immunoreactivity ( magenta ) specifies the cortical plate ( CP ) surrounding the luman ( L ) ( i ) . Selected optic sections at high magnification ( ii and iii ) and highresolution imaging ( iv ) show Tau localization only in axons of cortical neurons . Note the somas of neurons are free from Tau protein . At least eight organoids from four different batches ( n = 4 ) are tested . Figures display scale bars . B . Tau localization in SARS - CoV - 2 - positive neurons ( AB4 , green ) in selected optic sections ( i ) . Note , in contrast , to control groups , SARS - CoV - 2 - exposed organoids display mislocalized Tau ( magenta ) majorly into the somas of neurons ( arrowheads ) . Selected confocal slices are shown to distinguish Tau mislocalization into neuronal soma ( arrowheads ) . At high magnification , neuronal soma is further specified by the perinuclear localization of SARS - CoV - 2 ( green ) ( ii and iii ) . Bar diagrams at right quantifies the percentage of neurons ( Mock and SARS - CoV - 2 exposed ) exhibiting mislocalized Tau ( iv ) and the fraction of SARS - CoV - 2 - positive neurons exhibiting Tau - positive axons spanning different cortical areas ( v ) . For statistics , at least 300 cells from six organoids from four different batches ( n = 4 ) were tested . Figures display scale bars . Unpaired t test with Welch ' s correction , * * * P < 0 . 001 . Data presented as mean + SD . D . In contrast to controls ( i ) , Tau AT180 antibody ( magenta ) that specifically recognizes the phosphorylated Threonine 231 of Tau protein distinctly localizes at the somas of SARS - CoV - 2 - positive neurons ( AB4 , green ) ( ii ) . At least four organoids from two different batches ( n = 2 ) are tested . Figures display scale bars . E . Co - localization of SARS - CoV - 2 ( AB4 , green ) and phosphorylated Tau protein ( magenta ) at somas of cortical neurons revealed by high - resolution imaging and de - convolution . Representative images from at least 300 cells examined . Figures display scale bars . F . The bar diagram quantifies the fraction of Tau AT180 - positive neurons that co - localize with SARS - CoV - 2 - positive neurons . For statistics , at least 250 cells from four organoids and two independent batches ( n = 2 ) were examined . Unpaired t test with Welch ' s correction . Data presented as mean + SEM .

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 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 O O O O O O O O O O O O O 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. Tau immunoreactivity (magenta) specifies the cortical plate (CP) surrounding the luman (L) (i). Selected optic sections at high magnification (ii and iii) and highresolution imaging (iv) show Tau localization only in axons of cortical neurons. Note the somas of neurons are free from Tau protein. At least eight organoids from four different batches (n=4) are tested. Figures display scale bars. B. Tau localization in SARS-CoV-2-positive neurons (AB4, green) in selected optic sections (i). Note, in contrast, to control groups, SARS-CoV-2-exposed organoids display mislocalized Tau (magenta) majorly into the somas of neurons (arrowheads). Selected confocal slices are shown to distinguish Tau mislocalization into neuronal soma (arrowheads). At high magnification, neuronal soma is further specified by the perinuclear localization of SARS-CoV-2 (green) (ii and iii). Bar diagrams at right quantifies the percentage of neurons (Mock and SARS-CoV-2 exposed) exhibiting mislocalized Tau (iv) and the fraction of SARS-CoV-2-positive neurons exhibiting Tau-positive axons spanning different cortical areas (v). For statistics, at least 300 cells from six organoids from four different batches (n=4) were tested. Figures display scale bars. Unpaired t test with Welch's correction, ***P<0.001. Data presented as mean + SD. D. In contrast to controls (i), Tau AT180 antibody (magenta) that specifically recognizes the phosphorylated Threonine 231 of Tau protein distinctly localizes at the somas of SARS-CoV-2-positive neurons (AB4, green) (ii). At least four organoids from two different batches (n=2) are tested. Figures display scale bars.E. Co-localization of SARS-CoV-2 (AB4, green) and phosphorylated Tau protein (magenta) at somas of cortical neurons revealed by high-resolution imaging and de-convolution. Representative images from at least 300 cells examined. Figures display scale bars. F. The bar diagram quantifies the fraction of Tau AT180-positive neurons that co-localize with SARS-CoV-2-positive neurons. For statistics, at least 250 cells from four organoids and two independent batches (n=2) were examined. Unpaired t test with Welch's correction. Data presented as mean + SEM.


Figure 3A . Cells from mock - organoids display a healthy nucleus labeled by DAPI ( blue ) ( i ) . SARS - CoV - 2 - positive cells ( green ) display condensed ( middle panel , ii ) and fragmented DNA ( bottom panel , iii , arrows ) . At least 75 cells from two ( n = 2 ) independent batches of organoids were examined . Figures display scale bars . B . Compare to mock organoids ( i ) SARS - CoV - 2 - exposed organoids ( ii ) display increased TUNEL - positive cells ( displayed as inverted LUT ) at the cortical plate that is specified by TUJ - 1 ( magenta ) . At least four organoids from two ( n = 2 ) independent batches of organoids were examined . Figures display scale bars . The bar diagram below quantifies the frequencies of TUNEL - positive cells between mock and SARS - CoV - 2 - exposed organoids . Four organoids from two ( n = 2 ) independent batches were examined . Unpaired t test , * P < 0 . 05 . Data presented as mean + SD . C . Most of the SARS - CoV - 2 - positive cells ( AB4 , green ) are TUNEL - positive ( i ) and some of the SARS - CoV - 2 - positive cells are caspase - positive ( ii ) . Caspase - positive cells in SARS - CoV - 2 - exposed organoids display pT231Tau localization at the cell soma specified by AT - 180 ( ii ) , which are not observed in in mock organoids ( iii ) . Arrowheads point SARS - CoV - 2 - positive cells ( AB4 , green ) that are also positive for TUNNEL ( red ) , caspase 3 ( yellow ) and Tau AT - 180 ( magenta ) . Figures display scale bars . Bar diagrams at right quantifies proportions of TUNEL and caspase - positive cells among SARS - CoV - 2 - positive cells . The second graph below quantifies the proportions of pT231Tau - positive cells among caspase positive cells between control and virus exposed groups . At least 400 cells from 4 organoids and two independent ( n = 2 ) batches were examined . Unpaired t test , * * P < 0 . 01 . Data presented as mean + SEM . TUNEL - positive cells in control un - exposed organoids do not contain pT231Tau ( iii ) . Figures display scale bars . Bar diagrams at right quantifies proportions of pT231Tau - positive cells among TUNNEL - positive cells between control and virus exposed groups . At least 350 cells from 4 organoids and two independent ( n = 2 ) batches were examined . Unpaired t test , * * * P < 0 . 001 . Data presented as mean + SEM .

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

Figure 3A. Cells from mock-organoids display a healthy nucleus labeled by DAPI (blue) (i). SARS-CoV-2-positive cells (green) display condensed (middle panel, ii) and fragmented DNA (bottom panel, iii, arrows). At least 75 cells from two (n=2) independent batches of organoids were examined. Figures display scale bars.B. Compare to mock organoids (i) SARS-CoV-2-exposed organoids (ii) display increased TUNEL-positive cells (displayed as inverted LUT) at the cortical plate that is specified by TUJ-1 (magenta). At least four organoids from two (n=2) independent batches of organoids were examined. Figures display scale bars. The bar diagram below quantifies the frequencies of TUNEL-positive cells between mock and SARS-CoV-2-exposed organoids. Four organoids from two (n=2) independent batches were examined. Unpaired t test, *P<0.05. Data presented as mean + SD.C. Most of the SARS-CoV-2-positive cells (AB4, green) are TUNEL-positive (i) and some of the SARS-CoV-2-positive cells are caspase-positive (ii). Caspase-positive cells in SARS-CoV-2-exposed organoids display pT231Tau localization at the cell soma specified by AT-180 (ii), which are not observed in in mock organoids (iii). Arrowheads point SARS-CoV-2-positive cells (AB4, green) that are also positive for TUNNEL (red), caspase 3 (yellow) and Tau AT-180 (magenta). Figures display scale bars. Bar diagrams at right quantifies proportions of TUNEL and caspase-positive cells among SARS-CoV-2-positive cells. The second graph below quantifies the proportions of pT231Tau-positive cells among caspase positive cells between control and virus exposed groups. At least 400 cells from 4 organoids and two independent (n=2) batches were examined. Unpaired t test, **P<0.01. Data presented as mean + SEM. TUNEL-positive cells in control un-exposed organoids do not contain pT231Tau (iii). Figures display scale bars. Bar diagrams at right quantifies proportions of pT231Tau-positive cells among TUNNEL-positive cells between control and virus exposed groups. At least 350 cells from 4 organoids and two independent (n=2) batches were examined. Unpaired t test, ***P<0.001. Data presented as mean + SEM.


Figure 1 ( B ) Western blot analysis of the generated isogenic cell lines described in A after inducing GFP - TDP - 43 expression for 48 h showing the tightness of the doxycycline ( DOX ) - modulated expression system . Note also the different protein levels of the expressed variants . endTDP - 43 : endogenous TDP - 43 . ( C ) Representative images of widefield fluorescence microscopy of the isogenic cell lines depicted in B . GFP brightness is adjusted in each condition for optimal visualization of GFP - TDP - 43 localization . Original intensity values are represented in the right column using grayscale . Cell nuclei are stained with DAPI . Scale bar : 20 µm . ( D ) GFP - TDP - 43 expression was induced with DOX for 24 h before cycloheximide ( CHX ) treatment for the indicated times and western blot analysis . ( E ) Quantification of the GFP signal from D . N = 3 independent experiments . Two - way ANOVA with Tukey ' s multiple comparisons post hoc test .

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 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

Figure 1(B) Western blot analysis of the generated isogenic cell lines described in A after inducing GFP-TDP-43 expression for 48 h showing the tightness of the doxycycline (DOX)-modulated expression system. Note also the different protein levels of the expressed variants. endTDP-43: endogenous TDP-43.(C) Representative images of widefield fluorescence microscopy of the isogenic cell lines depicted in B. GFP brightness is adjusted in each condition for optimal visualization of GFP-TDP-43 localization. Original intensity values are represented in the right column using grayscale. Cell nuclei are stained with DAPI. Scale bar: 20 µm.(D) GFP-TDP-43 expression was induced with DOX for 24 h before cycloheximide (CHX) treatment for the indicated times and western blot analysis. (E) Quantification of the GFP signal from D. N=3 independent experiments. Two-way ANOVA with Tukey's multiple comparisons post hoc test.


Figure 2 ( B ) Representative maximum intensity Z - projections from confocal fluorescence imaging of human neurons transduced with TDP - 43 - HA variants and immunolabeled for the HA tag and the neuron - specific marker MAP2 . Scale bar : 10 µm . Cell nuclei are stained with DAPI ( D ) GFP - TDP - 43 expression was induced with doxycycline ( DOX ) for 4 h before nucleocytoplasmic fractionation and subsequent analysis of GFP - TDP - 43 and endogenous TDP - 43 ( endTDP - 43 ) levels by western blot . T : total lysate , C : cytoplasmic fraction , N : nuclear fraction . ( E ) Quantification of the GFP signal from D . Repeated measures one - way ANOVA with Greenhouse - Geisser correction and Tukey ' s multiple comparisons post hoc test . Cyt : cytoplasm , Nuc : nucleus . ( F ) HEK293 cells were treated with ActD to inhibit transcription or subjected to protein - protein cross - linking with DSG followed by nucleocytoplasmic fractionation and western blot analysis . T : total lysate , C : cytoplasmic fraction , N : nuclear fraction , H3 : histone H3 . ( G ) Quantification of endTDP - 43 signal from F . N = 3 independent experiments . Repeated measures one - way ANOVA with Greenhouse - Geisser correction and Dunnett ' s multiple comparisons post hoc test . ( H ) Representative images of widefield fluorescence microscopy of HEK293 cells treated with ActD for 4 h and immunolabeled for TDP - 43 . Scale bar : 20 µm . ( I ) Quantification of nucleocytoplasmic levels of endTDP - 43 in the immunocytochemistry images shown in H . N = 3 independent experiments . Unpaired two - tailed t - test . Cell nuclei are stained with DAPI ( J ) Representative images of confocal fluorescence microscopy of human neural cultures treated with ActD and ivermectin ( IVM ) and immunolabeled for TDP - 43 and the neuron - specific marker MAP2 . Scale bar : 20 µm . ( K ) Quantification of nucleocytoplasmic levels of endTDP - 43 in the immunocytochemistry images shown in J . Kruskal - Wallis test with Dunn ' s multiple comparisons post hoc test . N = 23 - 48 fields corresponding to a total of 351 - 569 neurons per condition . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Graph bars represent mean ± SD . Violin plots show mean and quartiles . Cell nuclei are stained with

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 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 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 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

Figure 2(B) Representative maximum intensity Z-projections from confocal fluorescence imaging of human neurons transduced with TDP-43-HA variants and immunolabeled for the HA tag and the neuron-specific marker MAP2. Scale bar: 10 µm. Cell nuclei are stained with DAPI(D) GFP-TDP-43 expression was induced with doxycycline (DOX) for 4 h before nucleocytoplasmic fractionation and subsequent analysis of GFP-TDP-43 and endogenous TDP-43 (endTDP-43) levels by western blot. T: total lysate, C: cytoplasmic fraction, N: nuclear fraction. (E) Quantification of the GFP signal from D. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Tukey's multiple comparisons post hoc test. Cyt: cytoplasm, Nuc: nucleus.(F) HEK293 cells were treated with ActD to inhibit transcription or subjected to protein-protein cross-linking with DSG followed by nucleocytoplasmic fractionation and western blot analysis. T: total lysate, C: cytoplasmic fraction, N: nuclear fraction, H3: histone H3. (G) Quantification of endTDP-43 signal from F. N=3 independent experiments. Repeated measures one-way ANOVA with Greenhouse-Geisser correction and Dunnett's multiple comparisons post hoc test.(H) Representative images of widefield fluorescence microscopy of HEK293 cells treated with ActD for 4 h and immunolabeled for TDP-43. Scale bar: 20 µm. (I) Quantification of nucleocytoplasmic levels of endTDP-43 in the immunocytochemistry images shown in H. N=3 independent experiments. Unpaired two-tailed t-test. Cell nuclei are stained with DAPI(J) Representative images of confocal fluorescence microscopy of human neural cultures treated with ActD and ivermectin (IVM) and immunolabeled for TDP-43 and the neuron-specific marker MAP2. Scale bar: 20 µm. (K) Quantification of nucleocytoplasmic levels of endTDP-43 in the immunocytochemistry images shown in J. Kruskal-Wallis test with Dunn's multiple comparisons post hoc test. N=23-48 fields corresponding to a total of 351-569 neurons per condition. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Graph bars represent mean ± SD. Violin plots show mean and quartiles. Cell nuclei are stained with DAPI