figf4 ( a , b ) Constructs identical to those described in Figure 2a , but lacking mRFP ( representations of S , HS , Q , HQ are shown in Supplementary Fig . 3a ) were co - transfected with tNHTT - 16Q - EGFP ( a ) and tNHTT - 150Q - EGFP ( b ) constructs into Neuro2a cells ( bar , 4 μm ) . ( c ) Dot blot analysis of the lumenal fraction of purified lysosomes from Neuro2a cells co - transfected with tNHTT - 60Q - EGFP and R , RS , RHS , RQ or RHQ . PNS , post - nuclear supernatant . ( d ) Quantification of HTT translocation into lysosomes . Levels of HTT detected by EM48 ( anti - huntingtin antibody ) were normalized to levels of β - tubulin ( in PNS ) and levels of cathepsin D ( in lysosomes ) . ( e ) Compound images generated by ArrayScan illustrating inclusion formation in 150Q Neuro2a cells transfected with R , RS , RHS , RQ and RHQ and shRNA specific for HSC70 and / or Lamp2a . ( blue , Hoechst 33258 ; green , tNHTT - 150Q - EGFP ; red , tested molecule ) . ( f ) RNAi of HSC70 , Lamp2a or both alleviated the inhibitory effect of RHQ on the formation of inclusions containing tNHTT - 150Q - EGFP inclusions . ( g ) Western blot analysis of soluble polyQ protein in 150Q Neuro2a cells transfected with tested constructs and shRNA for HSC70 and / or Lamp2a . Full - length western blots are presented in Supplementary Figure 13 . ( h ) Silencing of HSC70 , Lamp2a or both alleviated the inhibitory effect of RHQ on levels of soluble tNHTT - 150Q - EGFP . Bars in d and f represent mean values ± s . e . m . from three independent experiments . Bars in h represent relative mean values ± s . e . m . from three independent experiments . For h , values for the R construct were arbitrarily set to 1 .

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figf4(a,b) Constructs identical to those described in Figure 2a, but lacking mRFP (representations of S, HS, Q, HQ are shown in Supplementary Fig. 3a) were co-transfected with tNHTT-16Q-EGFP (a) and tNHTT-150Q-EGFP (b) constructs into Neuro2a cells (bar, 4 μm).(c) Dot blot analysis of the lumenal fraction of purified lysosomes from Neuro2a cells co-transfected with tNHTT-60Q-EGFP and R, RS, RHS, RQ or RHQ. PNS, post-nuclear supernatant.(d) Quantification of HTT translocation into lysosomes. Levels of HTT detected by EM48 (anti-huntingtin antibody) were normalized to levels of β-tubulin (in PNS) and levels of cathepsin D (in lysosomes).(e) Compound images generated by ArrayScan illustrating inclusion formation in 150Q Neuro2a cells transfected with R, RS, RHS, RQ and RHQ and shRNA specific for HSC70 and/or Lamp2a. (blue, Hoechst 33258; green, tNHTT-150Q-EGFP; red, tested molecule).(f) RNAi of HSC70, Lamp2a or both alleviated the inhibitory effect of RHQ on the formation of inclusions containing tNHTT-150Q-EGFP inclusions.(g) Western blot analysis of soluble polyQ protein in 150Q Neuro2a cells transfected with tested constructs and shRNA for HSC70 and/or Lamp2a. Full-length western blots are presented in Supplementary Figure 13.(h) Silencing of HSC70, Lamp2a or both alleviated the inhibitory effect of RHQ on levels of soluble tNHTT-150Q-EGFP. Bars in d and f represent mean values ± s.e.m. from three independent experiments. Bars in h represent relative mean values ± s.e.m. from three independent experiments. For h, values for the R construct were arbitrarily set to 1.


figf5 ( a ) Filter trap assay analysis of HTT - polyQaggregation in nine R6 / 2 mouse brains injected with three different combinations of rAAV . ( b ) Quantification of the SDS - insoluble material detected by filter trap assay ( * , P = 0 . 037 ; * * , P = 0 . 0083 ; * * * , P = 0 . 0015 ) . ( c ) Western blot analysis of HTT aggregation ( gel top , EM48 antibody ) , soluble HTT ( 1C2 antibody ) , and higher molecular polyQ complexes by agarose gel electrophoresis for resolving aggregates ( AGERA ) . Full - length western blots are presented in Supplementary Figure 13 . ( d ) Quantification of the gel top - detected aggregation ( * , P = 0 . 093 ; * * , P = 0 . 0027 ; * * * , P = 0 . 0045 ) . ( e ) Quantification of soluble HTT - polyQ ( * , P = 0 . 034 ; * * , P = 0 . 00003 ; * * * , P = 0 . 0001 ) . ( f ) Brain sections stained with anti - RFP , EM48 and anti - ubiquitin antibodies ( bar , 40 μm ) . ( g ) Numbers of ubiquitin - positive inclusions in the transduced areas ( * , P = 0 . 0026 ; * * , P = 5 . 535 × 10 − 8 ; * * * , P = 2 . 009 × 10 − 8 ) . Bars in b , d , e and g represent the relative mean values ± s . e . m . from three brains injected with different rAAV in each striatum . R in R / Q and R / HQ , and Q in Q / HQ brains represent the control value of 1 .

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figf5(a) Filter trap assay analysis of HTT-polyQaggregation in nine R6/2 mouse brains injected with three different combinations of rAAV. (b) Quantification of the SDS-insoluble material detected by filter trap assay (*, P = 0.037; **, P = 0.0083; ***, P = 0.0015).(c) Western blot analysis of HTT aggregation (gel top, EM48 antibody), soluble HTT (1C2 antibody), and higher molecular polyQ complexes by agarose gel electrophoresis for resolving aggregates (AGERA). Full-length western blots are presented in Supplementary Figure 13. (d) Quantification of the gel top-detected aggregation (*, P = 0.093; **, P = 0.0027; ***, P = 0.0045). (e) Quantification of soluble HTT-polyQ (*, P = 0.034; **, P = 0.00003; ***, P = 0.0001).(f) Brain sections stained with anti-RFP, EM48 and anti-ubiquitin antibodies (bar, 40 μm). (g) Numbers of ubiquitin-positive inclusions in the transduced areas (*, P = 0.0026; **, P = 5.535 × 10−8; ***, P = 2.009 × 10−8). Bars in b, d, e and g represent the relative mean values ± s.e.m. from three brains injected with different rAAV in each striatum. R in R/Q and R/HQ, and Q in Q/HQ brains represent the control value of 1.


figf6 ( a ) Relative body weight changes in mice after bilateral injection with rAAV - R , rAAV - Q and rAAV - HQ at 6 - 14 weeks of age ( n = 11 for all three groups until 12 weeks ; at 14 weeks , n = 7 for R / R , n = 10 for Q / Q , n = 11 for HQ / HQ ) ( * , P = 0 . 0401 ; * * , P = 0 . 0081 ; * * * , P = 0 . 0182 ; * * * * , P = 0 . 0225 ) . ( b ) Clasping scores . n = 11 for all three groups ( scale : 0 , no clasping ; 1 , clasping of the forelimbs only ; 2 , clasping of both fore and hind limbs once or twice ; 3 , clasping of both fore and hind limbs more than 3 times or more than 5 s ) . ( c ) Rotarod performance ; n = 11 for all three groups ( * , P = 0 . 0055 ; * * , P = 0 . 002 ; * * * , P = 0 . 0455 ; * * * * , P = 0 . 0242 ) . ( d ) Survival curves for mice after bilateral injection with rAAV - R , rAAV - Q , and rAAV - HQ ; n = 11 for all groups . Median survival ( days ) : R / R - 108 ; Q / Q - 123 ; HQ / HQ - 140 . Mean survival ( days ) : R / R - 105 . 5 ± 3 . 9 ; Q / Q - 122 . 3 ± 4 . 8 ; HQ / HQ - 136 . 2 ± 4 . 4 . Log - rank test , P 0 . 0001 ; Wilcoxon test , P 0 . 0001 .

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figf6(a) Relative body weight changes in mice after bilateral injection with rAAV-R, rAAV-Q and rAAV-HQ at 6-14 weeks of age (n = 11 for all three groups until 12 weeks; at 14 weeks, n = 7 for R/R, n = 10 for Q/Q, n = 11 for HQ/HQ) (*, P = 0.0401; **, P = 0.0081; ***, P = 0.0182; ****, P = 0.0225).(b) Clasping scores. n = 11 for all three groups (scale: 0, no clasping; 1, clasping of the forelimbs only; 2, clasping of both fore and hind limbs once or twice; 3, clasping of both fore and hind limbs more than 3 times or more than 5 s).(c) Rotarod performance; n = 11 for all three groups (*, P = 0.0055; **, P = 0.002; ***, P = 0.0455; ****, P = 0.0242).(d) Survival curves for mice after bilateral injection with rAAV-R, rAAV-Q, and rAAV-HQ; n = 11 for all groups. Median survival (days): R/R-108; Q/Q-123; HQ/HQ-140. Mean survival (days): R/R-105.5 ± 3.9; Q/Q-122.3 ± 4.8; HQ/HQ-136.2 ± 4.4.Log-rank test, P 0.0001; Wilcoxon test, P 0.0001.


Figure 1F Minimal inhibitory concentrations of DHA treated wild - type , heterozygous ( YME1 / yme1 ) and homozygous ( yme1 / yme1 ) mutant strains . Plates were imaged after 72h .

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Figure 1F Minimal inhibitory concentrations of DHA treated wild-type, heterozygous (YME1/yme1) and homozygous (yme1/yme1) mutant strains. Plates were imaged after 72h.


Figure 2D Competitive growth assay of DHA resistant single cell clones . Wild - type ( mCherry + _ Cre _ Puro ) and knockout ( GFP + _ Puro ) sister clones were derived from mutant ( retrovirus - intron RE , darker shading , Tol2 transposon - intron T2 , lighter shading ) resistant colonies , treated with DHA , analyzed with flow cytometry and Sanger sequenced for insertion site mapping .

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Figure 2D Competitive growth assay of DHA resistant single cell clones. Wild-type (mCherry+_Cre_Puro) and knockout (GFP+_Puro) sister clones were derived from mutant (retrovirus - intron RE, darker shading, Tol2 transposon - intron T2, lighter shading) resistant colonies, treated with DHA, analyzed with flow cytometry and Sanger sequenced for insertion site mapping.


Figure 3A , B Cell survival of dihydroartemisinin treated ESCs in combination with ( A ) the Ppox inhibitor acifluorfen or upon ( B ) 5 - ALA administration . Alamar Blue staining was used to assess viability after 72h of treatment . C , D Cell survival of DHA treated primary human glioblastoma cells from cancer patients and in combination with ( C ) the Ppox inhibitor acifluorfen ( BTL90 cells ) or upon ( D ) 5 - ALA administration ( VBT12 cells ) . Viability was assessed using CellTiter - Glo after 72h . Experiments were performed in triplicate . E , F ( E ) ROS levels ( DHE staining , PE 582 / 15 nm - MFI ) and ( F ) cell survival of DHA ( 0 . 5μM ) , 5 - ALA ( 0 . 25mM ) or Ppox inhibitor ( 10μM ) treated human neuroblastoma cells ( SHSY5Y ) . Fluorescence of DHE ( ROS levels ) and cell numbers ( survival ) were assessed by flow cytometry and automated cell counting . The experiments were performed two times , in triplicate each .

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Figure 3A, B Cell survival of dihydroartemisinin treated ESCs in combination with (A) the Ppox inhibitor acifluorfen or upon (B) 5-ALA administration. Alamar Blue staining was used to assess viability after 72h of treatment.C, D Cell survival of DHA treated primary human glioblastoma cells from cancer patients and in combination with (C) the Ppox inhibitor acifluorfen (BTL90 cells) or upon (D) 5-ALA administration (VBT12 cells). Viability was assessed using CellTiter-Glo after 72h. Experiments were performed in triplicate.E, F (E) ROS levels (DHE staining, PE 582/15 nm - MFI) and (F) cell survival of DHA (0.5μM), 5-ALA (0.25mM) or Ppox inhibitor (10μM) treated human neuroblastoma cells (SHSY5Y). Fluorescence of DHE (ROS levels) and cell numbers (survival) were assessed by flow cytometry and automated cell counting. The experiments were performed two times, in triplicate each.


Figure 4C , D ( C ) Fluorescence and ( D ) brightfield images of DMSO ( control ) , 5 - ALA ( 0 . 0625mM ) , DHA ( 1μM ) , or 5 - ALA and DHA ( 0 . 0625mM and 1μM ) treated tumor organoids ( CNS - PNET - like , c - MYC overexpression ) . Organoids were imaged on day 1 , day 5 and day 7 . One experiment is shown , representative of four independent experiments . G ROS / DHE staining and flow cytometry analysis of dissociated tumor organoids ( PE , 582 / 15 nm ) . Mean fluorescence intensities ( MFI ) of DHE stained GFP + tumor cells and GFP - control cells are indicated . n = 2 , values are mean ± SD . H Quantification of γH2AX + cells in cerebral tumor organoids . 6 cryo - sections of 3 organoids each were stained for γH2AX , scanned and 25 regions of interest ( ROI 2 . 500μm2 ) were analyzed per condition . Raw data were normalized to organoid area ( per ROI ) and are shown as box plots ( 25th - 75th percentiles , median ) .

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Figure 4C, D (C) Fluorescence and (D) brightfield images of DMSO (control), 5-ALA (0.0625mM), DHA (1μM), or 5-ALA and DHA (0.0625mM and 1μM) treated tumor organoids (CNS-PNET-like, c-MYC overexpression). Organoids were imaged on day 1, day 5 and day 7. One experiment is shown, representative of four independent experiments.G ROS/ DHE staining and flow cytometry analysis of dissociated tumor organoids (PE, 582/15 nm). Mean fluorescence intensities (MFI) of DHE stained GFP+ tumor cells and GFP- control cells are indicated. n=2, values are mean ± SD.H Quantification of γH2AX+ cells in cerebral tumor organoids. 6 cryo-sections of 3 organoids each were stained for γH2AX, scanned and 25 regions of interest (ROI 2.500μm2) were analyzed per condition. Raw data were normalized to organoid area (per ROI) and are shown as box plots (25th-75th percentiles, median).


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

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


Figure 6A Representative brightfield images of 5 - ALA and DHA treated patient - derived glioblastoma spheroids ( VBT92 ) . Images were taken on day 3 of culture left untreated or exposed to the indicated treatments . Scale bar 500μm . B Re - plating of spheroids after 3 days of 5 - ALA and DHA treatment ( duplicates ) . Representative brightfield images are shown for glioblastoma VBT92 . C Quantification of the re - adhesion capacity of re - plated VBT92 spheroids as assessed by crystal violet staining and absorbance measurement . Data is shown as mean ± SD . Experiments were performed in quadruplicates . D Lipid peroxidation in VBT92 cells treated with 5 - ALA and DHA . Lipid peroxidation was measured using BODIPY ™ 581 / 591 C11 staining and green ( from red ) fluorescence shift was determined using flow cytometry . Experiments were performed in triplicate ; values are shown as mean ± SD .

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Figure 6A Representative brightfield images of 5-ALA and DHA treated patient-derived glioblastoma spheroids (VBT92). Images were taken on day 3 of culture left untreated or exposed to the indicated treatments. Scale bar 500μm.B Re-plating of spheroids after 3 days of 5-ALA and DHA treatment (duplicates). Representative brightfield images are shown for glioblastoma VBT92.C Quantification of the re-adhesion capacity of re-plated VBT92 spheroids as assessed by crystal violet staining and absorbance measurement. Data is shown as mean ± SD. Experiments were performed in quadruplicates.D Lipid peroxidation in VBT92 cells treated with 5-ALA and DHA. Lipid peroxidation was measured using BODIPY™ 581/591 C11 staining and green (from red) fluorescence shift was determined using flow cytometry. Experiments were performed in triplicate; values are shown as mean ± SD.


Figure 7B In vivo efficacy comparison of solvents ( control ) , 5 - ALA , artesunate ( ARS ) and combined treatment in an orthotopic glioblastoma model . 11 - 14 days after stereotactic intracerebral injection of 2 x 105 VBT529 cells , mice received the following treatments : Control ( solvents ) , 5 - ALA ( 80 mg / kg ) , ARS ( 40 mg / kg ) , or 5 - ALA plus ARS 5 times per week via intraperitoneal injection ( n ≥ 5 ) . C , D Quantification and representative images of the VBT529 flank tumor luminescence signal . 6 - 7 days after injection of 1 . 5 x 105 VBT529 cells into the flank , mice received the following treatments : Control ( solvents ) or 5 - ALA plus ARS 4 - 5 times per week for 5 weeks via intraperitoneal injection ( n = 6 ) . After 4 days of treatment , concentrations of 5 - ALA ( 100 mg / kg ) and ARS ( 50 mg / kg ) were increased to 120 mg / kg and 60 mg / kg , respectively . The 20 % dosage increase of both drugs was used as the body - weight , behaviour and overall health of the mice were not affected by both , the lower as well as higher dosages . E , F Quantification and representative images of VBT529 brain tumor luminescence signals . 6 - 7 days after stereotactic intracerebral injection of 1 . 5 x 105 VBT529 cells , mice received the treatments as described in ( C and D ) ( n = 6 ) . G Kaplan - Meier survival curves of mice injected with VBT529 glioblastoma cells into the flank and brain ( same mice as in C - F ; n = 6 ) . Mice were treated with solvents ( control ) or 5 - ALA plus ARS ( same mice as in C - F ) .

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Figure 7B In vivo efficacy comparison of solvents (control), 5-ALA, artesunate (ARS) and combined treatment in an orthotopic glioblastoma model. 11-14 days after stereotactic intracerebral injection of 2 x 105 VBT529 cells, mice received the following treatments: Control (solvents), 5-ALA (80 mg/kg), ARS (40 mg/kg), or 5-ALA plus ARS 5 times per week via intraperitoneal injection (n ≥ 5).C, D Quantification and representative images of the VBT529 flank tumor luminescence signal. 6-7 days after injection of 1.5 x 105 VBT529 cells into the flank, mice received the following treatments: Control (solvents) or 5-ALA plus ARS 4-5 times per week for 5 weeks via intraperitoneal injection (n = 6). After 4 days of treatment, concentrations of 5-ALA (100 mg/kg) and ARS (50 mg/kg) were increased to 120 mg/kg and 60 mg/kg, respectively. The 20% dosage increase of both drugs was used as the body-weight, behaviour and overall health of the mice were not affected by both, the lower as well as higher dosages. E, F Quantification and representative images of VBT529 brain tumor luminescence signals. 6-7 days after stereotactic intracerebral injection of 1.5 x 105 VBT529 cells, mice received the treatments as described in (C and D) (n = 6).G Kaplan-Meier survival curves of mice injected with VBT529 glioblastoma cells into the flank and brain (same mice as in C-F; n = 6). Mice were treated with solvents (control) or 5-ALA plus ARS (same mice as in C-F).


Figure 1A : Immunoblot analysis of wild type ( WT ) , FAM83D - / - knockout ( KO ) and FAM83DGFP / GFP knockin ( KI ) U2OS cell lines . Data information : All blots are representative of at least 3 independent experiments . D : AS or nocodazole - synchronised M KI cells were lysed and subjected to GFP TRAP immunoprecipitations ( IP ) . Extracts ( input ) and IP samples were analysed by immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . E : KI cells synchronised in mitosis with either nocodazole ( M Noc . ) or STLC ( M STLC ) were collected by shake - off , and drug - treated cells that remained adherent after shake - off ( AS Noc . ; AS STLC ) were lysed and subjected to GFP TRAP IP . AS cells and free - GFP - expressing 2G - PAI1 U2OS cells were included as controls . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . F : Propidium iodide staining analyses revealing cell cycle distribution profiles for the samplesG , H : AS or nocodazole - synchronised ( M ) WT U2OS cells were subjected to IP with IgG and either anti - FAM83D - coupled sepharose beads ( G ) , or anti - CK1α - coupled sepharose beads ( H ) . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . I : KI cells were synchronised in G2 with RO - 3306 , or arrested in mitosis ( M ) using STLC . STLC - treated shake - off cells were washed and re - plated , and cells lysed at the indicated time points after STLC wash - out . Cell lysates were subjected to GFP TRAP IP and input and IP extracts analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . J : Propidium iodide staining analyses revealing cell cycle distribution profiles for the samplesK : AS or STLC - synchronised ( M ) HeLa , A549 and HaCaT cells were subjected to IP with either IgG - or anti - FAM83D - coupled sepharose beads . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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Figure 1A: Immunoblot analysis of wild type (WT), FAM83D-/- knockout (KO) and FAM83DGFP/GFP knockin (KI) U2OS cell lines. Data information: All blots are representative of at least 3 independent experiments.D: AS or nocodazole-synchronised M KI cells were lysed and subjected to GFP TRAP immunoprecipitations (IP). Extracts (input) and IP samples were analysed by immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.E: KI cells synchronised in mitosis with either nocodazole (M Noc.) or STLC (M STLC) were collected by shake-off, and drug-treated cells that remained adherent after shake-off (AS Noc.; AS STLC) were lysed and subjected to GFP TRAP IP. AS cells and free-GFP-expressing 2G-PAI1 U2OS cells were included as controls. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.F: Propidium iodide staining analyses revealing cell cycle distribution profiles for the samplesG, H: AS or nocodazole-synchronised (M) WT U2OS cells were subjected to IP with IgG and either anti-FAM83D-coupled sepharose beads (G), or anti-CK1α-coupled sepharose beads (H). Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.I: KI cells were synchronised in G2 with RO-3306, or arrested in mitosis (M) using STLC. STLC-treated shake-off cells were washed and re-plated, and cells lysed at the indicated time points after STLC wash-out. Cell lysates were subjected to GFP TRAP IP and input and IP extracts analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.J: Propidium iodide staining analyses revealing cell cycle distribution profiles for the samplesK: AS or STLC-synchronised (M) HeLa, A549 and HaCaT cells were subjected to IP with either IgG- or anti-FAM83D-coupled sepharose beads. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


Figure 2A : STLC - synchronised mitotic ( M ) wild - type ( WT ) , FAM83D - / - knockout ( KO ) and FAM83DGFP / GFP knockin ( KI ) U2OS cells were subjected to anti - CK1α immunofluorescence and GFP fluorescence microscopy . DNA is stained with DAPI . Scale bars , 20 µm . B : STLC - synchronised mitotic ( M ) FAM83D - / - knockout ( KO ) , FAM83DGFP / GFP knockin ( KI ) and FAM83DGFP / GFP ( F283A ) knockin ( FA ) U2OS cells were subjected to anti - CK1α immunofluorescence and GFP fluorescence microscopy . DNA is stained with DAPI . Scale bars , 20 µC : Quantification of CK1α spindle localisation for the cells Cell images denote the measured regions used to calculate the ratios on the box plot . Box plot whiskers denote the minimum and maximum measured values . The middle line represents the median , and the box ranges depict the 25th / 75th percentiles . * * * , P < 0 . 0001 ; ANOVA . Analysis was performed on the indicated number of cells , n = 2 . D : The cell lines were STLC - synchronised and mitotic cells ( M ) isolated by shake off . Asynchronous ( AS ) cells were included as a control . Cells were lysed and subjected to GFP TRAP immunoprecipitation ( IP ) and subsequent immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . F : KI cells were infected with retroviruses encoding either VHL , aGFP . 16 , or VHL - aGFP . 16 . Uninfected cells were used as a control . Cells were lysed and subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . G : The cell lines were subjected to anti - CK1α immunofluorescence and GFP fluorescence microscopy . DNA is stained with DAPI . Scale bars , 20 µm .

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Figure 2A: STLC-synchronised mitotic (M) wild-type (WT), FAM83D-/- knockout (KO) and FAM83DGFP/GFP knockin (KI) U2OS cells were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µm.B: STLC-synchronised mitotic (M) FAM83D-/- knockout (KO), FAM83DGFP/GFP knockin (KI) and FAM83DGFP/GFP(F283A) knockin (FA) U2OS cells were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µC: Quantification of CK1α spindle localisation for the cells Cell images denote the measured regions used to calculate the ratios on the box plot. Box plot whiskers denote the minimum and maximum measured values. The middle line represents the median, and the box ranges depict the 25th/75th percentiles. ***, P<0.0001; ANOVA. Analysis was performed on the indicated number of cells, n=2.D: The cell lines were STLC-synchronised and mitotic cells (M) isolated by shake off. Asynchronous (AS) cells were included as a control. Cells were lysed and subjected to GFP TRAP immunoprecipitation (IP) and subsequent immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.F: KI cells were infected with retroviruses encoding either VHL, aGFP.16, or VHL-aGFP.16. Uninfected cells were used as a control. Cells were lysed and subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.G: The cell lines were subjected to anti-CK1α immunofluorescence and GFP fluorescence microscopy. DNA is stained with DAPI. Scale bars, 20 µm.


Figure 3B : FAM83D - / - ( KO ) , wild - type ( WT ) , and two independent clones from a CRISPR / Cas9 - mediated knockin rescue of FAM83D cDNA into FAM83D - / - cells ( c . 6 and c . 11 ) , were synchronized in mitosis ( M ) with STLC . Asynchronous ( AS ) cells were included as a control . Cells were lysed and subjected to immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . The cell lines were STLC - synchronized in mitosis , fixed , and stained with antibodies recognizing FAM83D ( C ) Representative images of mitotic cells are included . Scale bars , 20 µm . The cell lines were STLC - synchronized in mitosis , fixed , and stained with antibodies recognizing CK1 ⍺ ( D ) . Representative images of mitotic cells are included . Scale bars , 20 µm . E : Quantification of CK1 ⍺ spindle localization Box plot whiskers denote the minimum and maximum measured values . The middle line represents the median , and the box ranges depict the 25th / 75th percentiles . * * * , P < 0 . 0001 ; ANOVA . Analysis was performed on the indicated number of cells , n = 1 . F : The cell lines were STLC - synchronized in mitosis ( M ) or left AS , lysed and subjected to immunoprecipitation ( IP ) with anti - FAM83D - coupled sepharose beads , before IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . G : Asynchronous ( AS ) FAM83DGFP / GFP / mCherry / mCherryCSNK1A1 knockin U2OS cells were fixed and imaged . Representative images from the indicated cell cycle stages are included . Scale bars , 10 µm .

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Figure 3B: FAM83D-/- (KO), wild-type (WT), and two independent clones from a CRISPR/Cas9-mediated knockin rescue of FAM83D cDNA into FAM83D-/- cells (c. 6 and c. 11), were synchronized in mitosis (M) with STLC. Asynchronous (AS) cells were included as a control. Cells were lysed and subjected to immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.The cell lines were STLC-synchronized in mitosis, fixed, and stained with antibodies recognizing FAM83D (C) Representative images of mitotic cells are included. Scale bars, 20 µm.The cell lines were STLC-synchronized in mitosis, fixed, and stained with antibodies recognizing CK1⍺ (D). Representative images of mitotic cells are included. Scale bars, 20 µm.E: Quantification of CK1⍺ spindle localization Box plot whiskers denote the minimum and maximum measured values. The middle line represents the median, and the box ranges depict the 25th/75th percentiles. ***, P<0.0001; ANOVA. Analysis was performed on the indicated number of cells, n=1.F: The cell lines were STLC-synchronized in mitosis (M) or left AS, lysed and subjected to immunoprecipitation (IP) with anti-FAM83D-coupled sepharose beads, before IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.G: Asynchronous (AS) FAM83DGFP/GFP/ mCherry/mCherryCSNK1A1 knockin U2OS cells were fixed and imaged. Representative images from the indicated cell cycle stages are included. Scale bars, 10 µm.


Figure 4A : Nocodazole - synchronised mitotic FAM83DGFP / GFP knockin U2OS cells were lysed and subjected to GFP TRAP immunoprecipitation ( IP ) , followed by incubation ± λ - phosphatase . Asynchronous ( AS ) cells were used as a control . Whole cell extracts ( input ) and IP samples were subjected to SDS - PAGE and subsequent immunoblotting ( IB ) with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . STLC - synchronised mitotic wild - type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ( B ) AS cells were used as a control . Lysed extracts were subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . STLC - synchronised mitotic wild - type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± MG132 ( C ) AS cells were used as a control . Lysed extracts were subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . STLC - synchronised mitotic wild - type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± ProTAME ( D ) . AS cells were used as a control . Lysed extracts were subjected to IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . E : Wild - type U2OS cells were either left asynchronous ( AS ) , or arrested in mitosis with STLC and collected by shake - off ( M ) . AS and M cells were incubated in media containing combinations of RO - 3306 and MG132 as indicated , prior to lysis . MG132 was applied for 1 . 5 h , whereas RO - 3306 was applied for the last 1 h of incubation prior to lysis . Samples were lysed and extracts were subjected to SDS - PAGE , before IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . F : STLC - synchronised mitotic wild - type U2OS cells were subjected to qRT - PCR analysis using primers for FAM83D , HMMR , CSNK1A1 , and CCNB1 . AS cells were used as a control . Error bars , SEM ; * , P < 0 . 01 ; Student ' s T - test . n = 3H : STLC - synchronised mitotic wild - type ( WT ) and HMMR knockout ( KO ) mouse embryonic fibroblasts ( MEFs ) were lysed and subjected to IB with the indicated antibodies . AS cells were included as a control . Data information : All blots are representative of at least 3 independent experiments . The cells were STLC - synchronised in mitosis and subjected to immunofluorescence microscopy with an anti - HMMR antibody ( I ) DNA is stained with DAPI . Scale bars , 20 µm . The cells were STLC - synchronised in mitosis and subjected to immunofluorescence microscopy with an anti - CK1α antibody ( J ) . DNA is stained with DAPI . Scale bars , 20 µm .

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Figure 4A: Nocodazole-synchronised mitotic FAM83DGFP/GFP knockin U2OS cells were lysed and subjected to GFP TRAP immunoprecipitation (IP), followed by incubation ± λ-phosphatase. Asynchronous (AS) cells were used as a control. Whole cell extracts (input) and IP samples were subjected to SDS-PAGE and subsequent immunoblotting (IB) with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium (B) AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± MG132 (C) AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.STLC-synchronised mitotic wild-type U2OS cells were lysed at the indicated time points following STLC washout and release into medium ± ProTAME (D). AS cells were used as a control. Lysed extracts were subjected to IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.E: Wild-type U2OS cells were either left asynchronous (AS), or arrested in mitosis with STLC and collected by shake-off (M). AS and M cells were incubated in media containing combinations of RO-3306 and MG132 as indicated, prior to lysis. MG132 was applied for 1.5 h, whereas RO-3306 was applied for the last 1 h of incubation prior to lysis. Samples were lysed and extracts were subjected to SDS-PAGE, before IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.F: STLC-synchronised mitotic wild-type U2OS cells were subjected to qRT-PCR analysis using primers for FAM83D, HMMR, CSNK1A1, and CCNB1. AS cells were used as a control. Error bars, SEM; *, P<0.01; Student's T-test. n=3H: STLC-synchronised mitotic wild-type (WT) and HMMR knockout (KO) mouse embryonic fibroblasts (MEFs) were lysed and subjected to IB with the indicated antibodies. AS cells were included as a control. Data information: All blots are representative of at least 3 independent experiments.The cells were STLC-synchronised in mitosis and subjected to immunofluorescence microscopy with an anti-HMMR antibody (I) DNA is stained with DAPI. Scale bars, 20 µm.The cells were STLC-synchronised in mitosis and subjected to immunofluorescence microscopy with an anti-CK1α antibody (J). DNA is stained with DAPI. Scale bars, 20 µm.


Figure 5B : STLC - synchronised mitotic FAM83DGFP / GFP knockin ( KI ) , FAM83DGFP / GFP ( F283A ) ( FA ) , and FAM83DGFP / GFP ( F283A ) stably expressing aGFP . 16 - CK1α ( FA + aGFP . 16 - CK1α ) U2OS cells were subjected to GFP TRAP immunoprecipitation ( IP ) , followed by immunoblotting ( IB ) with the indicated antibodies . Asynchronous ( AS ) cells were used as controls . Data information : All blots are representative of at least 3 independent experiments . C : FA cells were infected with retroviruses encoding wild - type aGFP . 16 - CK1α ( WT ) , or aGFP . 16 - CK1α with one of two distinct CK1α kinase - inactive mutants ( D136N or K46D ) . Uninfected cells ( - ) were included as a control . Cells were lysed , subjected to anti - GFP IP and IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments . D : Immunofluorescence analysis for the cells described in ( B ) following synchronisation with STLC . Cells were stained using anti - CK1α antibody , and DNA is stained with DAPI . Scale bars , 20 µm . F : FAM83D - / - knockout ( KO ) , KI , and FA cells were synchronised in mitosis ( M ) using STLC . Lysed extracts were subjected to anti - GFP IPs , followed by ATP [ γ - 32P ] kinase assays , using an optimised CK1 substrate peptide ( CK1tide ) . AS cells were used as controls . Error bars , SEM ; P < 0 . 01 ; ANOVA . n = 3 . Input and IP samples were analysed by IB with the indicated antibodies . Data information : All blots are representative of at least 3 independent experiments .

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Figure 5B: STLC-synchronised mitotic FAM83DGFP/GFP knockin (KI), FAM83DGFP/GFP (F283A) (FA), and FAM83DGFP/GFP(F283A) stably expressing aGFP.16-CK1α (FA + aGFP.16-CK1α) U2OS cells were subjected to GFP TRAP immunoprecipitation (IP), followed by immunoblotting (IB) with the indicated antibodies. Asynchronous (AS) cells were used as controls. Data information: All blots are representative of at least 3 independent experiments.C: FA cells were infected with retroviruses encoding wild-type aGFP.16-CK1α (WT), or aGFP.16-CK1α with one of two distinct CK1α kinase-inactive mutants (D136N or K46D). Uninfected cells (-) were included as a control. Cells were lysed, subjected to anti-GFP IP and IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.D: Immunofluorescence analysis for the cells described in (B) following synchronisation with STLC. Cells were stained using anti-CK1α antibody, and DNA is stained with DAPI. Scale bars, 20 µm.F: FAM83D-/- knockout (KO), KI, and FA cells were synchronised in mitosis (M) using STLC. Lysed extracts were subjected to anti-GFP IPs, followed by ATP [γ-32P] kinase assays, using an optimised CK1 substrate peptide (CK1tide). AS cells were used as controls. Error bars, SEM; P<0.01; ANOVA. n=3. Input and IP samples were analysed by IB with the indicated antibodies. Data information: All blots are representative of at least 3 independent experiments.


Figure 6A . Representative images initiating at metaphase for mitotic U2OS cell lines stained with Hoechst , taken every 5 min as they progress through division . Mitotic stage was determined by chromosome condensation and is indicated by the coloured boxes . Scale bar , 20 μm . B . Graphical representation for the kinetics of transition from metaphase alignment ( yellow ) to anaphase ( green ) , and cytokinesis ( blue ) . 100 mitotic cells per genotype are plotted ; n = 2 . C . Length of time needed to transition from metaphase to anaphase . Mean of 100 mitotic cells per genotype are plotted ; n = 2 . Error bars , SEM . * * * , P < 0 . 0001 ; ANOVA . D . Representative bright field images for mitotic U2OS cells indicating the long axis during interphase ( white line ) preceding mitosis , and the division axis determined at anaphase ( yellow line ) . Scale bar , 20 μm . E . Percentage of oriented divisions for U2OS cells grown in subconfluent cultures . An oriented division axis was defined as being less than 30 ° removed from the long axis of the interphase cell . Mean of 50 cells per genotype are plotted , n = 2 . Error bars , SEM . * * * , P < 0 . 0001 ; ANOVA . F . Representative images of mitotic U2OS cells stained with Hoechst and grown on L - shaped micropatterns previously coated with fibronectin . The position of metaphase chromosomes is indicated ( yellow line ) as is the presence of cortical blebbing ( red arrows ) . Scale bar , 20 μm . G . Circular graphs , superimposed on L - shaped micropattern , show the distribution of cell division angles measured at anaphase . Angles for 100 U2OS cells are plotted per genotype , n = 2 . Metaphase position is indicated ( yellow line ) and the percentages of division angles ± 15 ° from the expected axis ( red line ) are indicated . H . Percentage of metaphase U2OS cells that align chromosomes outside of the expected axis ( angles ± 15 ° ) . Mean of 100 cells per genotype are plotted , n = 2 . Error bars , SEM . * * * , P < 0 . 0001 ; ANOVA . I . Representative images of RFP - actin localization in mitotic U2OS cells grown on fibronectin - coated , L - shaped micropatterns , which is superimposed . Arrowheads indicate polarized cortical actin . Heatmap shows the intensity of RFP - actin localization ( Image J z - projection standard deviation ) as the cell progresses from prophase to metaphase ( 12 - 24 min ) . Scale bar , 20 μm . J . Heatmap additive intensities of RFP - actin localization in two representative mitotic U2OS cells for each genotype grown on fibronectin - coated , L - shaped micropatterns . Arrowheads indicate polarized cortical actin .

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Figure 6A. Representative images initiating at metaphase for mitotic U2OS cell lines stained with Hoechst, taken every 5 min as they progress through division. Mitotic stage was determined by chromosome condensation and is indicated by the coloured boxes. Scale bar, 20 μm.B. Graphical representation for the kinetics of transition from metaphase alignment (yellow) to anaphase (green), and cytokinesis (blue). 100 mitotic cells per genotype are plotted; n=2.C. Length of time needed to transition from metaphase to anaphase. Mean of 100 mitotic cells per genotype are plotted; n=2. Error bars, SEM. ***, P< 0.0001; ANOVA.D. Representative bright field images for mitotic U2OS cells indicating the long axis during interphase (white line) preceding mitosis, and the division axis determined at anaphase (yellow line). Scale bar, 20 μm.E. Percentage of oriented divisions for U2OS cells grown in subconfluent cultures. An oriented division axis was defined as being less than 30° removed from the long axis of the interphase cell. Mean of 50 cells per genotype are plotted, n=2. Error bars, SEM. ***, P< 0.0001; ANOVA.F. Representative images of mitotic U2OS cells stained with Hoechst and grown on L-shaped micropatterns previously coated with fibronectin. The position of metaphase chromosomes is indicated (yellow line) as is the presence of cortical blebbing (red arrows). Scale bar, 20 μm.G. Circular graphs, superimposed on L-shaped micropattern, show the distribution of cell division angles measured at anaphase. Angles for 100 U2OS cells are plotted per genotype, n=2. Metaphase position is indicated (yellow line) and the percentages of division angles ± 15° from the expected axis (red line) are indicated.H. Percentage of metaphase U2OS cells that align chromosomes outside of the expected axis (angles ± 15°). Mean of 100 cells per genotype are plotted, n=2. Error bars, SEM. ***, P< 0.0001; ANOVA.I. Representative images of RFP-actin localization in mitotic U2OS cells grown on fibronectin-coated, L-shaped micropatterns, which is superimposed. Arrowheads indicate polarized cortical actin. Heatmap shows the intensity of RFP-actin localization (Image J z-projection standard deviation) as the cell progresses from prophase to metaphase (12- 24 min). Scale bar, 20 μm.J. Heatmap additive intensities of RFP-actin localization in two representative mitotic U2OS cells for each genotype grown on fibronectin-coated, L-shaped micropatterns. Arrowheads indicate polarized cortical actin.


Figure 4A ) ALS patients from the UPenn Biobank neuroimaging cohort with higher wPRS exhibited greater reduction of cortical thickness in the orbital prefrontal cortex , anterior cingulate cortex , premotor cortex , lateral temporal cortex , and hippocampus . The heatmap indicates the associated T - statistic for each voxel , with light blue representing the highest value .

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Figure 4A) ALS patients from the UPenn Biobank neuroimaging cohort with higher wPRS exhibited greater reduction of cortical thickness in the orbital prefrontal cortex, anterior cingulate cortex, premotor cortex, lateral temporal cortex, and hippocampus. The heatmap indicates the associated T-statistic for each voxel, with light blue representing the highest value.


Figure 2 ( C ) Change in the expression levels of 36 genes defining the host - targeted anti - viral signature . log2 fold change at 24 hours post SARS - CoV - 2 infection from A549 cells are shown . ( D ) Change in the expression levels of 17 genes defining anti - cytokine signature ; log2 fold change at 24 hours post high SARS - CoV - 2 infection from A549 - ACE2 cells are shown .

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Figure 2(C) Change in the expression levels of 36 genes defining the host-targeted anti-viral signature. log2 fold change at 24 hours post SARS-CoV-2 infection from A549 cells are shown.(D) Change in the expression levels of 17 genes defining anti-cytokine signature; log2 fold change at 24 hours post high SARS-CoV-2 infection from A549-ACE2 cells are shown.


Figure 5 ( A ) Representative fluorescence images of Mock , SARS - CoV - 2 infected ( Ctrl ) , and Salmeterol - treated wells analyzed with the Multiwavelength Cell Scoring application in MetaXpress . Grayscales of the images were adjusted to enable direct comparison of the relative levels of fluorescence among the treatments : Segmentation images show how cells were segmented and identified as spike positive . Purple , nuclei ; cyan , spike . Scale bar , 100 μm . ( B ) Violin plots of Vero - E6 cells labeled for Spike protein . The Multi - wavelength Cell Scoring algorithm in MetaXpress was used to determine the integrated fluorescent signal in individual cells as a measure of the amount of Spike protein within each cell . The plots show the population distribution of the integrated signal for all of the treatments . The Boxes in the plot show the interquartile range ( IQR ) with the top and bottom edges marking the 75th and 25th percentiles , respectively . The horizontal line in the box is the median value , and the whiskers are defined to be 1 . 5 IQR . The ordinate is a log scale . The effect of the treatment is assessed quantitatively by changes in the median signal level , and qualitatively by observing changes in the modes . The dashed line is 3 standard deviations above the mean signal in the Mock samples and is used as a cut - off to quantify the number of cells that are positive or negative for the Spike signal . The statistics table below the plots shows the number of cells counted in each treatment group , and the median of the population .

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Figure 5(A) Representative fluorescence images of Mock, SARS-CoV-2 infected (Ctrl), and Salmeterol-treated wells analyzed with the Multiwavelength Cell Scoring application in MetaXpress. Grayscales of the images were adjusted to enable direct comparison of the relative levels of fluorescence among the treatments: Segmentation images show how cells were segmented and identified as spike positive. Purple, nuclei; cyan, spike. Scale bar, 100 μm.(B) Violin plots of Vero-E6 cells labeled for Spike protein. The Multi-wavelength Cell Scoring algorithm in MetaXpress was used to determine the integrated fluorescent signal in individual cells as a measure of the amount of Spike protein within each cell. The plots show the population distribution of the integrated signal for all of the treatments. The Boxes in the plot show the interquartile range (IQR) with the top and bottom edges marking the 75th and 25th percentiles, respectively. The horizontal line in the box is the median value, and the whiskers are defined to be 1.5 IQR. The ordinate is a log scale. The effect of the treatment is assessed quantitatively by changes in the median signal level, and qualitatively by observing changes in the modes. The dashed line is 3 standard deviations above the mean signal in the Mock samples and is used as a cut-off to quantify the number of cells that are positive or negative for the Spike signal. The statistics table below the plots shows the number of cells counted in each treatment group, and the median of the population.


Figure 6HEK293 acceptor cells transfected with or without ACE2 and TMPRSS2 were seeded in 384 well plates , pretreated with 7 - point gradients of test compounds for 1 - 2 h , and co - cultured for 4 hours with HEK293 donor cells expressing SARS - CoV - 2 spike and GFP , or donor cells expressing GFP only ( no spike ) . Images of GFP - positive objects were acquired on a confocal high - content imager and analyzed for syncytia formation and integrated GFP area ( total GFP ) as a measure of cytotoxicity , using a CNT algorithm as described in the Materials and Methods . Data are the aggregate of 8 independent biological repeats ; where errors are shown they represent SD from matching concentrations in at least three experiments .

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Figure 6HEK293 acceptor cells transfected with or without ACE2 and TMPRSS2 were seeded in 384 well plates, pretreated with 7-point gradients of test compounds for 1-2 h, and co-cultured for 4 hours with HEK293 donor cells expressing SARS-CoV-2 spike and GFP, or donor cells expressing GFP only (no spike). Images of GFP-positive objects were acquired on a confocal high-content imager and analyzed for syncytia formation and integrated GFP area (total GFP) as a measure of cytotoxicity, using a CNT algorithm as described in the Materials and Methods. Data are the aggregate of 8 independent biological repeats; where errors are shown they represent SD from matching concentrations in at least three experiments.


Figure 1Peptide array dot blot analysis to identify LIR motifs in the indicated SNARE proteins . Amino acids for the identified LIRs from positive signals are marked on each SNARE . Amino acids are denoted as amino acid single letter codes in the blot . Co - immunoprecipitation ( Co - IP ) analysis of interactions between FLAG - tagged Stx16 / Vti1a / Stx6 and EGFP - tagged LC3B or GABARAP overexpressed in HEK293T cells . * indicates mouse IgG heavy chain of precipitated mouse - anti - FLAG antibody . GST pull - down analysis of interactions between radiolabeled Myc - Stx16 and GST - tagged mAtg8 proteins . GST pull - down analysis of interactions between wild type ( WT ) or LIR - mutant ( L219A / V222A ) Stx16 and GST - tagged LC3C or GABARAP . Lower panel shows percentages of WT or LIR - mutant Stx16 bound to GST - LC3C or GST - GABARAP . Data shown as means ± SEM of precipitated Stx16 , n = 3 . GST pull - down analysis of interactions between WT or different types of LIR - mutant Stx16 and GST - tagged LC3C or GABARAP . Lower panel shows percentages of WT or LIR - mutant Stx16 bound to GST - LC3C or GST - GABARAP . GST pull - down analysis of interactions between radiolabeled Myc - Stx16 and WT or LDS - mutant GABARAP ( GABARAP - Y49A and - Y49A / F104A ) . Lower panel shows percentages of Stx16 bound to WT or LDS - mutant GABARAP . Data shown as means ± SEM of precipitated Stx16 , n = 3 .

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Figure 1Peptide array dot blot analysis to identify LIR motifs in the indicated SNARE proteins. Amino acids for the identified LIRs from positive signals are marked on each SNARE. Amino acids are denoted as amino acid single letter codes in the blot.Co-immunoprecipitation (Co-IP) analysis of interactions between FLAG-tagged Stx16/Vti1a/Stx6 and EGFP-tagged LC3B or GABARAP overexpressed in HEK293T cells. * indicates mouse IgG heavy chain of precipitated mouse-anti-FLAG antibody.GST pull-down analysis of interactions between radiolabeled Myc-Stx16 and GST-tagged mAtg8 proteins.GST pull-down analysis of interactions between wild type (WT) or LIR-mutant (L219A/V222A) Stx16 and GST-tagged LC3C or GABARAP. Lower panel shows percentages of WT or LIR-mutant Stx16 bound to GST-LC3C or GST-GABARAP. Data shown as means ± SEM of precipitated Stx16, n = 3.GST pull-down analysis of interactions between WT or different types of LIR-mutant Stx16 and GST-tagged LC3C or GABARAP. Lower panel shows percentages of WT or LIR-mutant Stx16 bound to GST-LC3C or GST-GABARAP.GST pull-down analysis of interactions between radiolabeled Myc-Stx16 and WT or LDS-mutant GABARAP (GABARAP-Y49A and -Y49A/F104A). Lower panel shows percentages of Stx16 bound to WT or LDS-mutant GABARAP. Data shown as means ± SEM of precipitated Stx16, n = 3.


Figure 2WT or STX17 - knockout ( STX17KO ) HeLa cells were starved with or without the presence of bafilomycin A1 ( Baf A1 , 100 nM ) for 2 h , and cell lysates were subjected to Western blot analysis of LC3B and p62 . Quantifications of LC3B - II levels normalized to β - actin from cells treated as in ( A ) ; Data shown as means ± SEM of LC3B - II and β - actin ratios , n = 3 ; * , p < 0 . 05 ( one - way ANOVA ) . WT , STX16KO , STX17KO or STX16 / STX17 double KO ( STX16 / STX17DKO ) HeLa cells were starved with or without the presence of Baf A1 ( 100 nM ) for 2 h , and cell lysates were subjected to Western blot analysis of LC3B . Quantifications of LC3B - II levels normalized to β - actin from ( C ) ; Data shown as means ± SEM of LC3B - II and β - actin ratios , n = 3 ; † , not significant ; * * , p < 0 . 01 ( one - way ANOVA ) . WT or STX16 / STX17DKO HeLa cells were starved in EBSS for 2 h , and subjected to ultrastructural analysis of the autophagic vesicles ( AV ) with electron microscopy . AVi : initial autophagic vacuoles ; AVd : degradative autophagic vacuoles ; G : Golgi apparatus . Image acquisition and counting as in Methods . Scale bars , 1 μm and 0 . 5 µm ( top sections ) . Quantifications of autophagic vesicles in WT and STX16 / STX17DKO HeLa cells treated as ( E ) ; Data shown as means ± SEM of AV profiles relative to cytoplasmic area ; † , not significant ; * , p < 0 . 05 ( two - way ANOVA ) . AV profiles from 47 images of each samples were counted .

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Figure 2WT or STX17-knockout (STX17KO) HeLa cells were starved with or without the presence of bafilomycin A1 (Baf A1, 100 nM) for 2 h, and cell lysates were subjected to Western blot analysis of LC3B and p62.   Quantifications of LC3B-II levels normalized to β-actin from cells treated as in (A); Data shown as means ± SEM of LC3B-II and β-actin ratios, n = 3; *, p < 0.05 (one-way ANOVA).   WT, STX16KO, STX17KO or STX16/STX17 double KO (STX16/STX17DKO) HeLa cells were starved with or without the presence of Baf A1 (100 nM) for 2 h, and cell lysates were subjected to Western blot analysis of LC3B.   Quantifications of LC3B-II levels normalized to β-actin from (C); Data shown as means ± SEM of LC3B-II and β-actin ratios, n = 3; †, not significant; **, p < 0.01 (one-way ANOVA).   WT or STX16/STX17DKO HeLa cells were starved in EBSS for 2 h, and subjected to ultrastructural analysis of the autophagic vesicles (AV) with electron microscopy. AVi: initial autophagic vacuoles; AVd: degradative autophagic vacuoles; G: Golgi apparatus. Image acquisition and counting as in Methods. Scale bars, 1 μm and 0.5 µm (top sections).   Quantifications of autophagic vesicles in WT and STX16/STX17DKO HeLa cells treated as (E); Data shown as means ± SEM of AV profiles relative to cytoplasmic area; †, not significant; *, p < 0.05 (two-way ANOVA). AV profiles from 47 images of each samples were counted.


Figure 3WT or STX16 / STX17DKO HeLa - YFP - Parkin cells were treated with CCCP ( 10 µM ) or oligomycin A ( 5 µM ) and antimycin A ( 10 µM ) ( OA ) for 16 hours , and subjected to high - content microscopy ( HCM ) analysis of mitochondria clearance . Masks : blue , nuclei ; red , mitochondria stained with mitochondrial DNA ( mtDNA ) antibody . Scale bar : 20 µm . Quantifications of mtDNA by object count or total area per cell in WT or STX16 / STX17DKO HeLa - YFP - Parkin cells treated as in ( A ) ; Data shown as means ± SEM of mtDNA object count ( upper panel ) or object total area ( lower panel ) per cell ; minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) . WT or STX16 / STX17DKO Huh7 cells were treated with H2O2 ( 0 . 4 mM ) for indicated time points , and autophagic clearance of peroxisomes was measured by the protein levels of PMP70 , PEX14 and p62 . Quantifications of peroxisomal proteins PMP70 and PEX14 for WT or STX16 / STX17DKO Huh7 cells treated as in ( C ) ; Data shown as means ± SEM of PEX14 or PMP70 and β - actin ratios , n = 3 ; † , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ( two - way ANOVA ) . WT , STX16KO , STX17KO or STX16 / STX17DKO THP - 1 cells were infected with Mycobacterium tuberculosis ( Mtb ) , followed by starvation to induce autophagy . Autophagic clearance of infected Mtb was measured by Mtb colonies grown on Middlebrook 7H11 agar plates ; Data shown as means ± SEM of Mtb colonies , n = 4 ; * , p < 0 . 05 ; * * , p < 0 . 01 ( two - way ANOVA ) .

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Figure 3WT or STX16/STX17DKO HeLa-YFP-Parkin cells were treated with CCCP (10 µM) or oligomycin A (5 µM) and antimycin A (10 µM) (OA) for 16 hours, and subjected to high-content microscopy (HCM) analysis of mitochondria clearance. Masks: blue, nuclei; red, mitochondria stained with mitochondrial DNA (mtDNA) antibody. Scale bar: 20 µm.   Quantifications of mtDNA by object count or total area per cell in WT or STX16/STX17DKO HeLa-YFP-Parkin cells treated as in (A); Data shown as means ± SEM of mtDNA object count (upper panel) or object total area (lower panel) per cell; minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).   WT or STX16/STX17DKO Huh7 cells were treated with H2O2 (0.4 mM) for indicated time points, and autophagic clearance of peroxisomes was measured by the protein levels of PMP70, PEX14 and p62.   Quantifications of peroxisomal proteins PMP70 and PEX14 for WT or STX16/STX17DKO Huh7 cells treated as in (C); Data shown as means ± SEM of PEX14 or PMP70 and β-actin ratios, n = 3; †, not significant; *, p < 0.05; **, p < 0.01 (two-way ANOVA).   WT, STX16KO, STX17KO or STX16/STX17DKO THP-1 cells were infected with Mycobacterium tuberculosis (Mtb), followed by starvation to induce autophagy. Autophagic clearance of infected Mtb was measured by Mtb colonies grown on Middlebrook 7H11 agar plates; Data shown as means ± SEM of Mtb colonies, n = 4; *, p < 0.05; **, p < 0.01 (two-way ANOVA).


Figure 4Validation of STX16 / STX17 DKO by Western blot analysis in HEK293 cells stably expressing RPS3 - Keima ( left panel ) and HCT116 cells stably expressing RPL28 - Keima ( right panel ) . WT or STX16 / STX17DKO HEK293 RPS3 - Keima cells were cultured in full medium or starved for 8 hours , and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm . Masks : white , cells identified based on nuclei ; red , Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm , respectively ; lower panel shows Keima puncta without masks . Scale bar : 20 µm . Quantifications of autolysosomal Keima puncta in WT or STX16 / STX17DKO HEK293 RPS3 - Keima cells treated as in ( C ) . Data shown as means ± SEM of Keima puncta per cell , minimum 1000 cells were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) . WT or STX16 / STX17DKO HCT116 RPL28 - Keima cells were cultured in full medium or starved for 8 hours , and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm . Masks : white , cells identified based on nuclei ; red , Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm , respectively ; lower panel shows Keima puncta without masks . Scale bar : 20 µm . Quantifications of autolysosomal Keima puncta in WT or STX16 / STX17DKO HCT116 RPL28 - Keima cells treated as in ( E ) . Data shown as means ± SEM of Keima puncta per cell , minimum 1000 cells were counted each well from at least 12 wells , 3 independent experiments ; † , not significant ; * * , p < 0 . 01 ( two - way ANOVA ) .

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

Figure 4Validation of STX16/STX17 DKO by Western blot analysis in HEK293 cells stably expressing RPS3-Keima (left panel) and HCT116 cells stably expressing RPL28-Keima (right panel).WT or STX16/STX17DKO HEK293 RPS3-Keima cells were cultured in full medium or starved for 8 hours, and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm. Masks: white, cells identified based on nuclei; red, Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm, respectively; lower panel shows Keima puncta without masks. Scale bar: 20 µm.   Quantifications of autolysosomal Keima puncta in WT or STX16 /STX17DKO HEK293 RPS3-Keima cells treated as in (C). Data shown as means ± SEM of Keima puncta per cell, minimum 1000 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).  WT or STX16/STX17DKO HCT116 RPL28-Keima cells were cultured in full medium or starved for 8 hours, and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm. Masks: white, cells identified based on nuclei; red, Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm, respectively; lower panel shows Keima puncta without masks. Scale bar: 20 µm.   Quantifications of autolysosomal Keima puncta in WT or STX16/STX17DKO HCT116 RPL28-Keima cells treated as in (E). Data shown as means ± SEM of Keima puncta per cell, minimum 1000 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; **, p < 0.01 (two-way ANOVA).


Figure 5 ( A ) WT or STX16KO HeLa cells were starved in EBSS for 2 h and subjected to HCM analysis of LAMP2 and LC3 puncta . Masks : white , cells identified based on nuclei ; red , LC3 puncta ; green , LAMP2 puncta ; yellow , overlap of LC3 and LAMP2 . Scale bar : 20 µm . ( B - D ) Quantifications of LC3 puncta per cell ( B ) , LC3 and LAMP2 overlap area per cell ( C ) , and LAMP2 puncta per cell ( D ) in WT or STX16KO cells treated Data shown as means ± SEM of puncta or overlap area per cell , minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ( two - way ANOVA ) . ( E ) Western blot analysis of LAMP1 and LAMP2 protein levels in WT and separate clones of STX16KO HeLa , Huh7 , and U2OS cells . ( F ) Western blot analysis of LAMP2 protein levels in WT and separate clones of STX6KO or VTI1AKO HeLa cells . ( G ) Time course of mTOR inactivation during EBSS starvation in WT vs . STX16KO HeLa cells analyzed by Western blotting for mTOR substrates ULK1 and 4E - BP1 . ( H ) WT or STX16KO HeLa cells were starved in EBSS for 1 h and subjected to HCM analysis of mTOR puncta . Masks : white , cells identified based on nuclei ; red , mTOR puncta . Scale bar : 20 µm . ( I ) Quantifications of mTOR puncta in WT or STX16KO cells treated as in ( H ) . Data shown as means ± SEM of mTOR puncta per cell , minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) .

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 O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT or STX16KO HeLa cells were starved in EBSS for 2 h and subjected to HCM analysis of LAMP2 and LC3 puncta. Masks: white, cells identified based on nuclei; red, LC3 puncta; green, LAMP2 puncta; yellow, overlap of LC3 and LAMP2. Scale bar: 20 µm.(B-D) Quantifications of LC3 puncta per cell (B), LC3 and LAMP2 overlap area per cell (C), and LAMP2 puncta per cell (D) in WT or STX16KO cells treated Data shown as means ± SEM of puncta or overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (two-way ANOVA).(E) Western blot analysis of LAMP1 and LAMP2 protein levels in WT and separate clones of STX16KO HeLa, Huh7, and U2OS cells.(F) Western blot analysis of LAMP2 protein levels in WT and separate clones of STX6KO or VTI1AKO HeLa cells.(G) Time course of mTOR inactivation during EBSS starvation in WT vs. STX16KO HeLa cells analyzed by Western blotting for mTOR substrates ULK1 and 4E-BP1.(H) WT or STX16KO HeLa cells were starved in EBSS for 1 h and subjected to HCM analysis of mTOR puncta. Masks: white, cells identified based on nuclei; red, mTOR puncta. Scale bar: 20 µm. (I) Quantifications of mTOR puncta in WT or STX16KO cells treated as in (H). Data shown as means ± SEM of mTOR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).


Figure 6WT or STX16KO HeLa cells were starved in EBSS for 1 h , followed by starvation with the presence of LysoTracker Red DND - 99 ( LTR ) ( 100 nM ) for additional 30 min , and subjected to HCM analysis of LTR . Scale bar : 20 µm . Quantifications of LTR count per cell or total area per cell in WT and STX16KO HeLa cells treated as in ( A ) . Data shown as means ± SEM of LTR puncta per cell or object total area per cell , minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; † , not significant ( two - way ANOVA ) . WT or STX16KO HeLa cells were starved in EBSS for 1 h , followed by starvation with the presence of LTR ( 100 nM ) for additional 30 min , and subjected to HCM analysis of the colocalization between LTR and TGN46 . Masks : white , cells identified based on nuclei ; red , LTR puncta ; green , TGN46 puncta ; yellow , overlap between LTR and TGN46 . Scale bar : 20 µm . Quantifications of overlaps between LysoTracker Red and TGN46 in WT and STX16KO cells treated as in ( C ) . Data shown as means ± SEM of LTR and TGN46 overlap area per cell , minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) . WT or STX16 / STX17DKO HeLa cells were starved in EBSS for 2 h , and subjected to ultrastructural analysis of the Golgi apparatus with electron microscopy . VF of Golgi : volume fraction of Golgi relative to total cytoplasmic area . Scale bar , 2 μm . Red arrows indicate Golgi apparatus .

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Figure 6WT or STX16KO HeLa cells were starved in EBSS for 1 h, followed by starvation with the presence of LysoTracker Red DND-99 (LTR) (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm.   Quantifications of LTR count per cell or total area per cell in WT and STX16KO HeLa cells treated as in (A). Data shown as means ± SEM of LTR puncta per cell or object total area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant (two-way ANOVA).   WT or STX16KO HeLa cells were starved in EBSS for 1 h, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of the colocalization between LTR and TGN46. Masks: white, cells identified based on nuclei; red, LTR puncta; green, TGN46 puncta; yellow, overlap between LTR and TGN46. Scale bar: 20 µm.   Quantifications of overlaps between LysoTracker Red and TGN46 in WT and STX16KO cells treated as in (C). Data shown as means ± SEM of LTR and TGN46 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).   WT or STX16/STX17DKO HeLa cells were starved in EBSS for 2 h, and subjected to ultrastructural analysis of the Golgi apparatus with electron microscopy. VF of Golgi: volume fraction of Golgi relative to total cytoplasmic area. Scale bar, 2 μm. Red arrows indicate Golgi apparatus.


Figure 7WT , LC3TKO , GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 90 min and subjected to HCM analysis of overlaps between LAMP2 and Stx16 . Masks : white , cells identified based on nuclei ; green , LAMP2 puncta ; red , Stx16 puncta ; yellow , overlap of LAMP2 and Stx16 . Scale bar : 20 µm . Quantifications of overlaps between LAMP2 and Stx16 in WT , LC3TKO , GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LAMP2 and Stx16 overlap area per cell , minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ( one - way ANOVA ) . Quantifications of overlaps between LBPA and Stx16 in WT , LC3TKO , GABARAPTKO or HexaKO HeLa cells grown in full medium or treated Data shown as means ± SEM of LBPA and Stx16 overlap area per cell , minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ( one - way ANOVA ) . WT , LC3TKO , GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 1 hour , followed by starvation with the presence of LTR ( 100 nM ) for additional 30 min , and subjected to HCM analysis of LTR . Scale bar : 20 µm . Quantifications of LTR puncta in WT , LC3TKO , GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LTR puncta per cell , minimum 500 cells were counted each well from at least 12 wells , 3 independent experiments ; † , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ( one - way ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 90 min and subjected to HCM analysis of overlaps between LAMP2 and Stx16. Masks: white, cells identified based on nuclei; green, LAMP2 puncta; red, Stx16 puncta; yellow, overlap of LAMP2 and Stx16. Scale bar: 20 µm.Quantifications of overlaps between LAMP2 and Stx16 in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LAMP2 and Stx16 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (one-way ANOVA).Quantifications of overlaps between LBPA and Stx16 in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells grown in full medium or treated Data shown as means ± SEM of LBPA and Stx16 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (one-way ANOVA).WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 1 hour, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm.Quantifications of LTR puncta in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LTR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; *, p < 0.05; **, p < 0.01 (one-way ANOVA).


Figure 1ELISA assay of fecal IgA1 ( A ) , IgA2 ( B ) , total IgA ( C ) , levels from non - IBD ( n = 7 ) , CD ( n = 18 ) , and UC ( n = 12 ) patients ' stool . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney test . P - values are as follows : ( A ) * * : p = 0 , 0098 ; ( B ) * : p = 0 , 0406 ; ( C ) Non - IBD v . UC * : p = 0 , 0479 , CD vs . UC * : p = 0 , 0166 ; ELISA assay of secretory component ( D ) levels from non - IBD ( n = 7 ) , CD ( n = 18 ) , and UC ( n = 12 ) patients ' stool . Data were further separated according to disease activity for IgA1 ( E ) and IgA2 ( F ) . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney test . P - values are as follows : ( F ) * : p = 0 , 0167In vitro assay of purified IgA1 ( G ) and IgA2 ( H ) reverse - transcytosis abilities on an inverted model of FAE from Caco2 and Raji cells co - culture . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney test . P - values are as follows : ( G ) * : p = 0 , 0441 ; ( H ) * : p = 0 , 423 ; For some patients , antibody purification did not yield a high enough concentration , so samples had to be excluded . N are thus as follows : IgA1 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 4 ; IgA2 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 5 . All patient samples ( biological replicates ) have been tested in technical duplicates meaning nx2 . ( I ) ELISA assay of IgA1 - Dectin - 1 binding , at a rate of one receptor per 10 IgA ; ( J ) ELISA assay of IgA2 - Dectin - 1 binding , at a rate of one receptor per 10 IgA . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney test . P - values are as follows : ( J ) Non - IBD vs . UC * : p = 0 , 0199 , CD vs . UC * : p = 0 , 0243 ; For some patients , antibody purification did not yield a high enough concentration , so samples had to be excluded . N are thus as follows : IgA1 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 4 ; IgA2 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 5 . All patient samples ( biological replicates ) have been tested in technical duplicates meaning nx2 . Percent of IgA1 ( K , L ) and IgA2 ( M , N ) reverse - transcytosis for weak ( K , M ) and strong ( L , N ) Dectin - 1 binding . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney test . P - values are as follows : ( K ) * : p = 0 , 0221 ; ( L ) * : p = 0 , 0486 . For some patients , antibody purification did not yield a high enough concentration , so samples had to be excluded . N are thus as follows : IgA1 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 4 ; IgA2 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 5 . All patient samples ( biological replicates ) have been tested in technical duplicates meaning nx2 .

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

Figure 1ELISA assay of fecal IgA1 (A), IgA2 (B), total IgA (C), levels from non-IBD (n= 7), CD (n=18), and UC (n=12) patients' stool. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (A)** : p= 0,0098; (B) *: p=0,0406; (C) Non-IBD v. UC *: p=0,0479, CD vs. UC *: p=0,0166;ELISA assay of secretory component (D) levels from non-IBD (n= 7), CD (n=18), and UC (n=12) patients' stool.Data were further separated according to disease activity for IgA1 (E) and IgA2 (F). Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (F) *: p=0,0167In vitro assay of purified IgA1 (G) and IgA2 (H) reverse-transcytosis abilities on an inverted model of FAE from Caco2 and Raji cells co-culture. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (G) *: p=0,0441; (H) *: p=0,423 ; For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.(I) ELISA assay of IgA1-Dectin-1 binding, at a rate of one receptor per 10 IgA; (J) ELISA assay of IgA2-Dectin-1 binding, at a rate of one receptor per 10 IgA. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (J) Non-IBD vs. UC *: p=0,0199 , CD vs. UC *: p= 0,0243; For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.Percent of IgA1 (K, L) and IgA2 (M, N) reverse-transcytosis for weak (K, M) and strong (L, N) Dectin-1 binding. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (K) *: p=0,0221 ; (L) *: p=0,0486 . For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.


Figure 3Heatmaps displaying standardized log2 - transformed signal intensities per glycan for IgA1 ( A ) and IgA2 ( B ) in non - IBD , CD and UC groups , plotted by hierarchical clustering of Euclidean distance .

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Figure 3Heatmaps displaying standardized log2-transformed signal intensities per glycan for IgA1 (A) and IgA2 (B) in non-IBD, CD and UC groups, plotted by hierarchical clustering of Euclidean distance.


Figure 4Flow cytometry analysis of total IgA1 - bound ( A ) and IgA2 - bound ( B ) fecal microbiota . ( C ) Flow cytometry analysis of stool IgA1 + IgA2 - bacteria ( grey dots ) , IgA1 - IgA2 + bacteria ( black dots ) and IgA1 + IgA2 + bacteria ( empty dots ) . ( A - C ) Data were analysed with a Kruskall - Wallis multiple comparison test . In ( C ) * : p = 0 , 0226 . Non - IBD : n = 7 , CD : n = 18 , and UC : n = 12 .

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Figure 4Flow cytometry analysis of total IgA1-bound (A) and IgA2-bound (B) fecal microbiota. (C) Flow cytometry analysis of stool IgA1+ IgA2- bacteria (grey dots), IgA1- IgA2+ bacteria (black dots) and IgA1+ IgA2+ bacteria (empty dots). (A-C) Data were analysed with a Kruskall-Wallis multiple comparison test. In (C) *: p= 0,0226. Non-IBD: n= 7, CD: n=18, and UC: n=12.