A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( I ) hepatic phosphorylation of AKT ( p - AKTSer473 ) were analyzed . Compounds were administered by daily subcutaneous injections . To assess insulin - stimulated p - AKTSer473 , insulin ( n = 5 ) or saline ( n = 3 ) was injected 10 min prior to liver sampling . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (I) hepatic phosphorylation of AKT (p-AKTSer473) were analyzed. Compounds were administered by daily subcutaneous injections. To assess insulin-stimulated p-AKTSer473, insulin (n = 5) or saline (n = 3) was injected 10 min prior to liver sampling. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( A ) Plasma lipoprotein fractions . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (A) Plasma lipoprotein fractions. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( B ) liver cholesterol levels . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (B) liver cholesterol levels. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( C ) expression of genes implicated in hepatic cholesterol and lipoprotein uptake . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (C) expression of genes implicated in hepatic cholesterol and lipoprotein uptake. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( D ) expression of genes implicated in cholesterol and bile acid metabolism and excretion were analyzed . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (D) expression of genes implicated in cholesterol and bile acid metabolism and excretion were analyzed. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( A ) body weight . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (A) body weight. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( B ) cumulative food intake . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (B) cumulative food intake. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( C , D ) body composition . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (C, D) body composition. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( E , F ) glucose tolerance . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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

A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (E, F) glucose tolerance. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


( A ) Bar chart showing the relative abundance ( signal intensity ) of the phosphorylated tau species identified using an exploratory IP - MS approach using HT7 antibody on TBS - soluble control and Alzheimer ' s disease ( AD ) pools . Blue dashed line indicates the two phosphorylated tau peptides that underwent targeted IP - MS : mono - phosphorylated p - tau231 and double phosphorylated p - tau ( 231 + 235 ) .

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

(A) Bar chart showing the relative abundance (signal intensity) of the phosphorylated tau species identified using an exploratory IP-MS approach using HT7 antibody on TBS-soluble control and Alzheimer's disease (AD) pools. Blue dashed line indicates the two phosphorylated tau peptides that underwent targeted IP-MS: mono-phosphorylated p-tau231 and double phosphorylated p-tau(231+235).


( A ) Box - and - whiskers plot showing the levels of CSF p - tau235 in neurological controls ( n = 21 ) and biologically defined Alzheimer ' s disease ( AD ) cases ( n = 19 ) .

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(A) Box-and-whiskers plot showing the levels of CSF p-tau235 in neurological controls (n=21) and biologically defined Alzheimer's disease (AD) cases (n=19).


( A ) Box - and - whiskers plot showing CSF p - tau235 concentrations in the different groups : amyloid - negative cognitively unimpaired ( CU - ) participants ( n = 50 ) , participants across the Alzheimer ' s disease ( AD ) continuum ( amyloid - positive cognitively unimpaired ( CU + , n = 32 ) ; amyloid - positive mild cognitive impairment ( MCI + , n = 20 ) ; AD ( n = 20 ) and non - AD cases ( n = 19 ) . CSF p - tau235 is highly specific for AD pathology . Cut - off value for CSF p - tau235 positivity is displayed with black dashed line ( 19 . 92 pg / mL ) .

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

(A) Box-and-whiskers plot showing CSF p-tau235 concentrations in the different groups: amyloid-negative cognitively unimpaired (CU-) participants (n=50), participants across the Alzheimer's disease (AD) continuum (amyloid-positive cognitively unimpaired (CU+, n=32); amyloid-positive mild cognitive impairment (MCI+, n=20); AD (n=20) and non-AD cases (n=19). CSF p-tau235 is highly specific for AD pathology. Cut-off value for CSF p-tau235 positivity is displayed with black dashed line (19.92 pg/mL).


( A ) Box - and - whiskers plot showing CSF p - tau235 concentrations in A - T - participants and across preclinical Alzheimer ' s disease ( AD , dichotomised as A + T - and A + T + ) . CSF p - tau235 was increased already in A + T - , which represents the early cases within preclinical AD . A prominent increase was observed from A + T - to A + T + cases , the latter representing late preclinical AD cases . Cut - off value for CSF p - tau235 positivity is displayed with black dashed line ( 19 . 92 pg / mL ) .

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(A) Box-and-whiskers plot showing CSF p-tau235 concentrations in A-T- participants and across preclinical Alzheimer's disease (AD, dichotomised as A+T- and A+T+). CSF p-tau235 was increased already in A+T-, which represents the early cases within preclinical AD. A prominent increase was observed from A+T- to A+T+ cases, the latter representing late preclinical AD cases. Cut-off value for CSF p-tau235 positivity is displayed with black dashed line (19.92 pg/mL).


Trajectories of the different tau biomarkers in preclinical AD using a local weighted regression method ( Loess curve ) . Changes on biomarkers levels in CSF are represented as z - scores using Aβ PET in centiloid scale ( CL ) as a proxy of disease progression . Abnormal biomarker levels were determined as two standard deviations ( SD ) above the mean . Incipient Aβ pathology positivity was determined as Aβ PET higher than CL 12 ( Mila - Aloma et al . , 2020 ) . All samples were run in singlicates .

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

Trajectories of the different tau biomarkers in preclinical AD using a local weighted regression method (Loess curve). Changes on biomarkers levels in CSF are represented as z-scores using Aβ PET in centiloid scale (CL) as a proxy of disease progression. Abnormal biomarker levels were determined as two standard deviations (SD) above the mean. Incipient Aβ pathology positivity was determined as Aβ PET higher than CL 12 (Mila-Aloma et al., 2020). All samples were run in singlicates.


( A ) Stacked bar chart depicting the percentages of [ 231 + / 235 + ] , [ 231 + / 235 - ] , [ 231 - / 235 - ] and [ 231 - / 235 + ] cases in each preclinical group . Table below indicates the exact percentages of participants in each group ( exact number of participants in parenthesis ) . Discordant cases with the sequential phosphorylation hypothesis [ 231 - / + 235 ] shown in grey .

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(A) Stacked bar chart depicting the percentages of [231+/235+], [231+/235-], [231-/235-] and [231-/235+] cases in each preclinical group. Table below indicates the exact percentages of participants in each group (exact number of participants in parenthesis). Discordant cases with the sequential phosphorylation hypothesis [231-/+235] shown in grey.


( B ) Correlation between CSF p - tau235 and p - tau231 in the whole ALFA + cohort ( Spearman ' s rank correlation : rS = 0 . 80 , P ˂ 0 . 0001 ) . Assay cut - offs were determined as the mean + 2 SD of the A - T - group ( defining p - tau231 and p - tau235 positivity or negativity in each participant ) . Cut - off values are indicated in red ( 19 . 92 and 9 . 59 pg / mL for CSF p - tau235 and p - tau231 respectively ) and displayed with black dashed lines , resulting in four quadrants , each of them representing the four different positive or negatively status for each biomarker . Discordant cases with the sequential phosphorylation hypothesis ( [ 231 - / + 235 ] ) showed on the lower right quadrant ( 2 . 35 % of the total , 9 participants out 383 ) . All samples were run in singlicates .

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

(B) Correlation between CSF p-tau235 and p-tau231 in the whole ALFA+ cohort (Spearman's rank correlation: rS = 0.80, P˂0.0001). Assay cut-offs were determined as the mean + 2 SD of the A-T- group (defining p-tau231 and p-tau235 positivity or negativity in each participant). Cut-off values are indicated in red (19.92 and 9.59 pg/mL for CSF p-tau235 and p-tau231 respectively) and displayed with black dashed lines, resulting in four quadrants, each of them representing the four different positive or negatively status for each biomarker. Discordant cases with the sequential phosphorylation hypothesis ([231-/+235]) showed on the lower right quadrant (2.35% of the total, 9 participants out 383). All samples were run in singlicates.


A . MDCK cells were cultured on cell culture inserts for 4 days . On the last day , the culture medium was replaced with EV - depleted medium . EVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation . Cell lysates and EV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated . Note that the PEG pellets did not contain mitochondrial protein TOMM20 , suggesting that the PEG pellets were not contaminated by intracellular organelles .

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

A. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. EVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and EV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. Note that the PEG pellets did not contain mitochondrial protein TOMM20, suggesting that the PEG pellets were not contaminated by intracellular organelles.


B . PEG pellets were subjected to OptiPrep flotation analysis .

B. PEG pellets were subjected to OptiPrep flotation analysis.


C . MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD63 antibody .

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C. MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody.


D . sEVs were eluted from the beads with a glycine buffer and analyzed by nanoparticle tracking analysis ( NTA ) . Representative NTA traces were shown . E . Quantification of the NTA data obtained in five independent experiments .

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D. sEVs were eluted from the beads with a glycine buffer and analyzed by nanoparticle tracking analysis (NTA). Representative NTA traces were shown. E. Quantification of the NTA data obtained in five independent experiments.


F . MDCK cells were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody .

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F. MDCK cells were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody.


G . sEVs were eluted from the beads with a glycine buffer and analyzed by NTA . Representative NTA traces were shown . H . Quantification of the NTA data obtained in five independent experiments .

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

G. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown. H. Quantification of the NTA data obtained in five independent experiments.


A . MDCK cells were cultured on cell culture inserts for 4 days . On the last day , the culture medium was replaced with serum - free medium . sEVs released from the apical and basolateral sides of MDCK cells were purified by ultracentrifugation . Apical and basolateral sEVs ( P100 ) were analyzed by silver staining .

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

A. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with serum-free medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by ultracentrifugation. Apical and basolateral sEVs (P100) were analyzed by silver staining.


C . MDCK cells were cultured on cell culture inserts for 4 days . On the last day , the culture medium was replaced with EV - depleted medium . sEVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation . Cell lysates and sEV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated . GPRC5C was detected only in the apical sEV

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

C. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and sEV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. GPRC5C was detected only in the apical sEV samples


D . PEG pellets were subjected to OptiPrep flotation analysis . GPRC5C was detected only in the apical sEV

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D. PEG pellets were subjected to OptiPrep flotation analysis. GPRC5C was detected only in the apical sEV samples


E . MDCK cells were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . GPRC5C was detected only in the apical sEV

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

E. MDCK cells were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. GPRC5C was detected only in the apical sEV samples


F . MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD63 antibody .

F. MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody.


A . MDCK cells were transfected with siControl or the siRNAs indicated , and the cells were transferred to cell culture inserts and cultured for 4 days . On the last day , the culture medium was replaced with EV - depleted medium . sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated .

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A. MDCK cells were transfected with siControl or the siRNAs indicated, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated.


B . The intensity of the bands was measured in three independent experiments .

B. The intensity of the bands was measured in three independent experiments.


C . sEVs were eluted from the beads with a glycine buffer and analyzed by NTA . Representative NTA traces were shown .

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C. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown.


D . Quantification of the NTA data obtained in five independent experiments .

D. Quantification of the NTA data obtained in five independent experiments.


A . MDCK cells were transfected with siControl or siALIX , and the cells were transferred to cell culture inserts and cultured for 4 days . On the last day , the culture medium was replaced with EV - depleted medium with or without 10 nM GW4869 . sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated .

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A. MDCK cells were transfected with siControl or siALIX, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium with or without 10 nM GW4869. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated.


Analyses of protein expression in lysates of various cell lines by western blotting against the indicated antibodies .

Analyses of protein expression in lysates of various cell lines by western blotting against the indicated antibodies.


Fluorescence analyses of GFP - ATG5 or ATG16L1 - GFP stably expressed in ATG5 - / - or ATG16L1 - / - . Cells were amino acid starved for 2 hr prior to fixation and imaging of the GFP fluorescence . Scale bar : 10 μm .

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Fluorescence analyses of GFP-ATG5 or ATG16L1-GFP stably expressed in ATG5-/- or ATG16L1-/-. Cells were amino acid starved for 2 hr prior to fixation and imaging of the GFP fluorescence. Scale bar: 10 μm.


Assessment of ATG16L1 levels in the cytosolic ( C ) and membrane ( M ) fractions in lysates of the indicated cell lines using western blot analyses and antibodies against ATG16L1 . ATG16L1 - / - and ATG5 - / - stably expressed ATG16L1 - GFP while ATG3 - / - stably expressed Flag - S - ATG16L1 . Antibodies against α - tubulin and integrin β1 were used as controls for fractionation . Exogenous ( exo ) ATG16L1 was detected using antibodies against ATG16L1 .

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Assessment of ATG16L1 levels in the cytosolic (C) and membrane (M) fractions in lysates of the indicated cell lines using western blot analyses and antibodies against ATG16L1. ATG16L1-/- and ATG5-/- stably expressed ATG16L1-GFP while ATG3-/- stably expressed Flag-S-ATG16L1. Antibodies against α-tubulin and integrin β1 were used as controls for fractionation. Exogenous (exo) ATG16L1 was detected using antibodies against ATG16L1.


Lack of WIPI2 expression is confirmed by western blot analyses in wild type MEFs ( WIPI2 + / + ) and WIPI2 - / - cells .

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Lack of WIPI2 expression is confirmed by western blot analyses in wild type MEFs (WIPI2+/+) and WIPI2-/- cells.


Immunofluorescence analyses of ATG16L1 - / - and WIPI2 - / - stably expressing Flag - S - ATG16L1 . Cells were amino acid starved ( AA starve ) for 2 hr in the presence or absence of 3 ' MA ( and additional pretreatment for 30 min ) followed by fixation and immuno - staining using antibodies against Flag tag to detect ATG16L1 . Scale bar : 9 μm .

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Immunofluorescence analyses of ATG16L1-/- and WIPI2-/- stably expressing Flag-S-ATG16L1. Cells were amino acid starved (AA starve) for 2 hr in the presence or absence of 3'MA (and additional pretreatment for 30 min) followed by fixation and immuno-staining using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm.


Fragments depicted in ( A ) were expressed in U2OS cells and amino acid starved for 5 hr followed by fixation and immunostaining against S tag to detect S - ATG16L1 . Scale bar : 10 μm .

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Fragments depicted in (A) were expressed in U2OS cells and amino acid starved for 5 hr followed by fixation and immunostaining against S tag to detect S-ATG16L1. Scale bar: 10 μm.


U2OS cells expressing Flag - ATG16L1 ∆ 2 or Flag - ATG16L1 ∆ 2 ∆ 182 - 205 were treated as in ( B ) and stained using antibodies against Flag tag ( to detect ATG16L1 , green ) and FIP200 ( red ) . Scale bar : 10 μm .

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U2OS cells expressing Flag-ATG16L1∆2 or Flag-ATG16L1∆2∆182-205 were treated as in (B) and stained using antibodies against Flag tag (to detect ATG16L1, green) and FIP200 (red). Scale bar: 10 μm.


Protein - protein interaction assay in 293T cells transiently transfected with the indicated Flag - S - tagged ATG16L1 constructs . S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies .

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Protein-protein interaction assay in 293T cells transiently transfected with the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.


Homodimerisation assay in 293T cells transiently transfected with ATG16L1 - GFP and the indicated Flag - S - tagged ATG16L1 constructs . S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies .

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Homodimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated Flag-S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.


Flag - ATG16L1WT or Flag - ATG16L1 ∆ 182 - 205 were stably expressed in ATG5 - / - cells and analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1 . Scale bar : 9 μm .

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Flag-ATG16L1WT or Flag-ATG16L1∆182-205 were stably expressed in ATG5-/- cells and analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm.


Microscopy - based protein - liposome binding assay . ATG16L1 - GFP immobilised on beads incubated in the presence of rhodamine - labelled liposome preparations containing the indicated phosphoinositides . Scale bar : 50 μm .

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Microscopy-based protein-liposome binding assay. ATG16L1-GFP immobilised on beads incubated in the presence of rhodamine-labelled liposome preparations containing the indicated phosphoinositides. Scale bar: 50 μm.


Quantification of relative liposome binding


Microscopy - based protein - liposome binding assay as in ( B ) . ATG16L1WT - and ATG16L1LD - GFP immobilised on beads were incubated with rhodamine - labelled , PI3P - positive liposome preparations . Scale bar : 50 μm . Right panel shows quantification of liposome binding relative to ATG16L1WT from 3 independent experiments including SEM values .

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Microscopy-based protein-liposome binding assay as in (B). ATG16L1WT- and ATG16L1LD-GFP immobilised on beads were incubated with rhodamine-labelled, PI3P-positive liposome preparations. Scale bar: 50 μm. Right panel shows quantification of liposome binding relative to ATG16L1WT from 3 independent experiments including SEM values.


Protein - protein interaction assay in 293T cells transiently transfected with the indicated S - tagged ATG16L1 constructs . S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies .

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Protein-protein interaction assay in 293T cells transiently transfected with the indicated S-tagged ATG16L1 constructs. S tag pull down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies.


by immunoblotting using the indicated antibodies . Dimerisation assay in 293T cells transiently transfected with ATG16L1 - GFP and the indicated S - tagged ATG16L1 constructs and

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by immunoblotting using the indicated antibodies. Dimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated S-tagged ATG16L1 constructs and analysed


ATG16L1 - / - stably expressing the indicated Flag - S - ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag ( green ) and ATG5 ( red ) . Scale bar : 9 μm . Right panels show quantifications of average number of Flag - and ATG5 - positive dots per cell .

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ATG16L1-/- stably expressing the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag (green) and ATG5 (red). Scale bar: 9 μm. Right panels show quantifications of average number of Flag- and ATG5-positive dots per cell.


ATG16L1 - / - stably expressing the indicated Flag - S - ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against S tag ( to detect ATG16L1 , red ) and WIPI2 ( green ) . Scale bar : 9 μm . Lower panel shows quantification of average number of WIPI2 positive dots per cell .

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ATG16L1-/- stably expressing the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against S tag (to detect ATG16L1, red) and WIPI2 (green). Scale bar: 9 μm. Lower panel shows quantification of average number of WIPI2 positive dots per cell.


Average intensities of individual Flag - ATG16L1 dots in ( A ) . Underlying grey circles represent individual data points . Dots were quantified ( n = 1225 for ATG16L1WT ; n = 334 for ATG16L1LD ) .

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Average intensities of individual Flag-ATG16L1 dots in (A). Underlying grey circles represent individual data points. Dots were quantified (n=1225 for ATG16L1WT; n=334 for ATG16L1LD).


WIPI2 - / - cells reconstituted with the indicated Flag - S - ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag ( green ) and FIP200 ( red ) . Scale bar : 9 μm . Lower panel shows quantification of average number of Flag positive dots per puncta - positive cell .

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WIPI2-/- cells reconstituted with the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 hr prior to immunofluorescence analyses using antibodies against Flag tag (green) and FIP200 (red). Scale bar: 9 μm. Lower panel shows quantification of average number of Flag positive dots per puncta-positive cell.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . Cells were amino acid starved ( AA starved ) to induce mTORC1 - dependent autophagy in the presence or absence of BafA1 for 2 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Cells were amino acid starved (AA starved) to induce mTORC1-dependent autophagy in the presence or absence of BafA1 for 2 hr prior to lysis.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . Mitophagy was induced by CCCP ( 10 μM ) treatment for 6 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Mitophagy was induced by CCCP (10 μM) treatment for 6 hr prior to lysis.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . Glucose starvation ( Glu starved ) for 20 hr was used to induce mTORC1 - independent autophagy . BafA1 was added to all conditions 2 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Glucose starvation (Glu starved) for 20 hr was used to induce mTORC1-independent autophagy. BafA1 was added to all conditions 2 hr prior to lysis.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . LC3 - associated phagocytosis ( LAP ) - like LC3 lipidation was induced by treating cells with CCCP ( 100 μM ) for 2 hr prior to lysis . Right panel shows quantification of LC3 - II / LC3 - I ratio .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. LC3-associated phagocytosis (LAP)-like LC3 lipidation was induced by treating cells with CCCP (100 μM) for 2 hr prior to lysis. Right panel shows quantification of LC3-II/LC3-I ratio.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LD - expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies . LAP - like LC3 lipidation induced by monensin or ammonium chloride ( NH4Cl ) for 2 hr prior to lysis .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LD-expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. LAP-like LC3 lipidation induced by monensin or ammonium chloride (NH4Cl) for 2 hr prior to lysis.


Coomassie gel of recombinant ATG16L172 - 307 wild type and mutants . * indicates bacterial

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Coomassie gel of recombinant ATG16L172-307 wild type and mutants. * indicates bacterial protein


Liposome co - sedimentation assay using recombinant ATG16L1 protein and sonicated liposomes . Supernatant ( S ) and pellet ( P ) were analysed by immunoblotting against T7 tag to detect ATG16L1 . Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments ( right panel ) .

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Liposome co-sedimentation assay using recombinant ATG16L1 protein and sonicated liposomes. Supernatant (S) and pellet (P) were analysed by immunoblotting against T7 tag to detect ATG16L1. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel).


Liposome co - sedimentation assay , as in ( C ) , using ATG16L172 - 307LE and sonicated liposomes that contain or lack PI3P . Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments ( right panel ) with error bars depicting SEM values .

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Liposome co-sedimentation assay, as in (C), using ATG16L172-307LE and sonicated liposomes that contain or lack PI3P. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel) with error bars depicting SEM values.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LE - expression constructs and autophagy assessed during amino acid starvation ( AA starve ) followed by immunoblotting or immunofluorescence analyses . Immunofluorescence analyses of amino acid starved cells ( 2 hr ) using antibodies against WIPI2 ( red ) or ATG16L1 ( green ) . Scale bar : 9 μm . Cells as in ( A ) with the addition of 3 ' MA ( and pretreatment with the drug for 30 min ) . Scale bar : 9 μm .

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ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LE-expression constructs and autophagy assessed during amino acid starvation (AA starve) followed by immunoblotting or immunofluorescence analyses. Immunofluorescence analyses of amino acid starved cells (2 hr) using antibodies against WIPI2 (red) or ATG16L1 (green). Scale bar: 9 μm. Cells as in (A) with the addition of 3'MA (and pretreatment with the drug for 30 min). Scale bar: 9 μm.


Quantification of percentage of cells positive for ATG16L1 puncta


Cells were left untreated , amino acid starved for 2 hr , or amino acid starved for 2 hr followed by refeeding with full growth media for 1 hr . Puncta formation was assessed by immunofluorescence staining using antibodies against S tag to detect ATG16L1 . Scale bar : 10 μm .

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Cells were left untreated, amino acid starved for 2 hr, or amino acid starved for 2 hr followed by refeeding with full growth media for 1 hr. Puncta formation was assessed by immunofluorescence staining using antibodies against S tag to detect ATG16L1. Scale bar: 10 μm.


Immunoblot analyses of amino acid starved cells in the presence of BafA1 for 2 hr . Cell lysates were analysed using the indicated antibodies .

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Immunoblot analyses of amino acid starved cells in the presence of BafA1 for 2 hr. Cell lysates were analysed using the indicated antibodies.


WIPI2 - / - cells reconstituted with the indicated ATG16L1 constructs were amino acid starved for 2 hr . BafA1 treatment was included in all conditions for 2 hr prior to lysis . Cell lysates were analysed by immunoblotting using the indicated antibodies . Quantification of LC3 - II / LC3 - I ratio of three independent experiments is shown on the right panel .

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WIPI2-/- cells reconstituted with the indicated ATG16L1 constructs were amino acid starved for 2 hr. BafA1 treatment was included in all conditions for 2 hr prior to lysis. Cell lysates were analysed by immunoblotting using the indicated antibodies. Quantification of LC3-II/LC3-I ratio of three independent experiments is shown on the right panel.


Ferroptosis assay in ATG16L1 - / - cells stably expressing ATG16L1WT , ATG16L1LD or ATG16L1LE . Cells were cultured in amino acid free media ( AA starve ) in the presence of 10 % FBS or 10 % dialysed FBS ( diFBS ) for 24 hr . Quantification of percentage of PI - positive cells from at least three independent experiments is shown .

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Ferroptosis assay in ATG16L1-/- cells stably expressing ATG16L1WT, ATG16L1LD or ATG16L1LE. Cells were cultured in amino acid free media (AA starve) in the presence of 10% FBS or 10% dialysed FBS (diFBS) for 24 hr. Quantification of percentage of PI-positive cells from at least three independent experiments is shown.


( a ) Kaplan - Meier survival plot of cohorts of induced Vil Apc , Vil Apc Huwe1het and Vil Apc Huwe1hom mice . Deletion of Huwe1 led to a significant reduction in survival of these animals ( Vil Apc vs Vil Apc Huwe1het / Vil Apc Huwe1hom , Log Rank , p < 0 . 001 , n ≥ 10 ) .

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(a) Kaplan-Meier survival plot of cohorts of induced Vil Apc, Vil Apc Huwe1het and Vil Apc Huwe1hom mice. Deletion of Huwe1 led to a significant reduction in survival of these animals (Vil Apc vs Vil Apc Huwe1het / Vil Apc Huwe1hom, Log Rank, p < 0.001, n ≥ 10).


( b ) Wholemount isolation of small intestines from Vil Apc and Vil Apc Huwe1hom mice culled at clinical endpoint . Note the huge numbers of tiny macroscopic adenomas visible in the Vil Apc Huwe1hom intestine ( red arrows ) .

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(b) Wholemount isolation of small intestines from Vil Apc and Vil Apc Huwe1hom mice culled at clinical endpoint. Note the huge numbers of tiny macroscopic adenomas visible in the Vil Apc Huwe1hom intestine (red arrows).


( c ) H & amp ; amp ; E staining of intestinal cross sections from Vil Apc and Vil Apc Huwe1hom mice demonstrating the increased adenoma burden following Huwe1 deletion . Black arrows indicate individual adenomas . Scale bars = 200 µm .

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(c) H&amp;E staining of intestinal cross sections from Vil Apc and Vil Apc Huwe1hom mice demonstrating the increased adenoma burden following Huwe1 deletion. Black arrows indicate individual adenomas. Scale bars = 200 µm.


( d ) Quantification of total tumour numbers per gut in sacrificed Vil Apc , Vil Apc Huwe1het and Vil Apc Huwe1hom mice . Deletion of Huwe1 led to a significant increase in the number of tumours per gut ( Mann Whitney , n ≥ 5 ) .

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(d) Quantification of total tumour numbers per gut in sacrificed Vil Apc, Vil Apc Huwe1het and Vil Apc Huwe1hom mice. Deletion of Huwe1 led to a significant increase in the number of tumours per gut (Mann Whitney, n ≥ 5).


( a ) H & amp ; amp ; E staining of control and Huwe1 deleted intestinal epithelium . Shortened villi in Huwe1 deficient tissue are indicated . Scale bars = 100 µm . ( b ) BrdU IHC of control and Huwe1 deleted intestinal epithelium . Scale bars = 100 µm . ( c ) PAS staining identifying Goblet cells . No gross changes were observed . Scale bars = 100 µm . ( d ) Lysozyme staining ( Paneth cell marker ) of control and Huwe1 deleted small intestine . Note the occurrence of lysozyme positive cells away from the crypt base ( black arrows ) . Scale bars = 100 µm .

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(a) H&amp;E staining of control and Huwe1 deleted intestinal epithelium. Shortened villi in Huwe1 deficient tissue are indicated. Scale bars = 100 µm. (b) BrdU IHC of control and Huwe1 deleted intestinal epithelium. Scale bars = 100 µm. (c) PAS staining identifying Goblet cells. No gross changes were observed. Scale bars = 100 µm. (d) Lysozyme staining (Paneth cell marker) of control and Huwe1 deleted small intestine. Note the occurrence of lysozyme positive cells away from the crypt base (black arrows). Scale bars = 100 µm.


( e ) Dual Periodic acid - Schiff / Alcian blue staining to identify Paneth cell secretory vesicles ( light blue / pink - marked with black arrows ) and goblet cells ( dark blue / purple - marked with red arrows ) . Note Paneth cell secretory vesicles are restricted to crypt base in both control and Huwe1 deficient small intestines ( inset - black arrows ) . Scale bars = 100µm .

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(e) Dual Periodic acid-Schiff / Alcian blue staining to identify Paneth cell secretory vesicles (light blue / pink - marked with black arrows) and goblet cells (dark blue / purple - marked with red arrows). Note Paneth cell secretory vesicles are restricted to crypt base in both control and Huwe1 deficient small intestines (inset - black arrows). Scale bars = 100µm.


( f ) MMP7 staining of control and Huwe1 deleted small intestine . Note MMP7 staining is restricted to crypt base in Huwe1 deficient intestines . Scale bars = 100µm .

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(f) MMP7 staining of control and Huwe1 deleted small intestine. Note MMP7 staining is restricted to crypt base in Huwe1 deficient intestines. Scale bars = 100µm.


( a ) HUWE1 and MYC Western blot analysis of epithelial cell extractions from control and Huwe1 deficient intestines . Levels of MYC protein are significantly increased in normal intestines lacking HUWE1 ( Mann Whitney , p = 0 . 04 , n = 3 ) .

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(a) HUWE1 and MYC Western blot analysis of epithelial cell extractions from control and Huwe1 deficient intestines. Levels of MYC protein are significantly increased in normal intestines lacking HUWE1 (Mann Whitney, p = 0.04, n = 3).


( b ) QRT - PCR analysis of Myc expression in control and Huwe1 deficient intestines .

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(b) QRT-PCR analysis of Myc expression in control and Huwe1 deficient intestines.


( c ) MYC IHC in tumours from Vil Apc and Vil Apc Huwe1hom mice . Scale bars = 50 µm .

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(c) MYC IHC in tumours from Vil Apc and Vil Apc Huwe1hom mice. Scale bars = 50 µm.


( d ) MYC Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours . Levels of MYC protein are significantly increased in tumours lacking HUWE1 ( Mann Whitney , p = 0 . 04 , n = 3 ) .

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(d) MYC Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours. Levels of MYC protein are significantly increased in tumours lacking HUWE1 (Mann Whitney, p = 0.04, n = 3).


( e ) QRT - PCR analysis of Myc expression in tumours from Vil Apc and Vil Apc Huwe1hom mice .

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(e) QRT-PCR analysis of Myc expression in tumours from Vil Apc and Vil Apc Huwe1hom mice.


( a ) Kaplan - Meier survival plot of cohorts of induced Vil Apc Pten , Vil Apc Pten Huwe1 , Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice . Heterozygous deletion of Myc led to a specific and significant increase in survival of Huwe1 deleted animals ( Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc , Log Rank , p < 0 . 001 , n ≥ 16 ) .

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(a) Kaplan-Meier survival plot of cohorts of induced Vil Apc Pten, Vil Apc Pten Huwe1, Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice. Heterozygous deletion of Myc led to a specific and significant increase in survival of Huwe1 deleted animals (Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc, Log Rank, p < 0.001, n ≥ 16).


( b ) Quantification of total tumour numbers per gut in sacrificed Vil Apc Pten , Vil Apc Pten Huwe1 , Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice . Heterozygous deletion of Myc did not reduce the number of tumours in Huwe1 deleted mice ( Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc . Mann Whitney , n ≥ 10 ) .

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(b) Quantification of total tumour numbers per gut in sacrificed Vil Apc Pten, Vil Apc Pten Huwe1, Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice. Heterozygous deletion of Myc did not reduce the number of tumours in Huwe1 deleted mice (Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc. Mann Whitney, n ≥ 10).


( c ) Kaplan - Meier survival plot of cohorts of induced AhCre - ERT Apc Pten and AhCre - ERT Apc Pten MycT58A mice . Overexpression of proteolytically stabilised MYC led to a significant reduction in survival ( Log Rank , p < 0 . 001 , n = 14 vs 21 ) .

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(c) Kaplan-Meier survival plot of cohorts of induced AhCre-ERT Apc Pten and AhCre-ERT Apc Pten MycT58A mice. Overexpression of proteolytically stabilised MYC led to a significant reduction in survival (Log Rank, p < 0.001, n = 14 vs 21).


( d ) Quantification of total tumour numbers per gut in sacrificed Apc Pten and Apc Pten MycT58A mice . Overexpression of proteolytically stabilised MYC did not increase the number of tumours in Apc Pten mice ( Mann Whitney , n = 9 vs 19 ) .

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(d) Quantification of total tumour numbers per gut in sacrificed Apc Pten and Apc Pten MycT58A mice. Overexpression of proteolytically stabilised MYC did not increase the number of tumours in Apc Pten mice (Mann Whitney, n = 9 vs 19).


( a ) H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells . Band intensity relative to βactin displayed under each lane ( Mann - Whitney , p = 0 . 04 , n = 3 vs 3 ) .

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(a) H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells. Band intensity relative to βactin displayed under each lane (Mann-Whitney, p = 0.04, n = 3 vs 3).


( b ) γ - H2AX IHC showing increased positivity in Huwe1 deleted intestinal cells . Scale bars = 50 µm .

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(b) γ-H2AX IHC showing increased positivity in Huwe1 deleted intestinal cells. Scale bars = 50 µm.


( c ) γ - H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells ( Mann - Whitney , p = 0 . 04 , n = 3 vs 3 ) .

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(c) γ-H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells (Mann-Whitney, p = 0.04, n = 3 vs 3).


( d ) γ - H2AX IHC of Vil Apc and Vil Apc Huwe1hom tumours , note increased positivity in Huwe1 deficient tumours . Scale bars = 50 µm .

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(d) γ-H2AX IHC of Vil Apc and Vil Apc Huwe1hom tumours, note increased positivity in Huwe1 deficient tumours. Scale bars = 50 µm.


( a ) Caspase 3 IHC of tumours from Lgr5 Apc and Lgr5 Apc Huwe1 mice either untreated or 6h post treatment with 7 . 5mg / kg cisplatin . Arrows identify caspase 3 positive cells . Scale bars = 50 µm . ( b ) Quantification of cisplatin treatment showing a significant increase in apoptosis in Huwe1 deficient tumour cells ( Mann - Whitney , n = 3 vs 5 ) .

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(a) Caspase 3 IHC of tumours from Lgr5 Apc and Lgr5 Apc Huwe1 mice either untreated or 6h post treatment with 7.5mg/kg cisplatin. Arrows identify caspase 3 positive cells. Scale bars = 50 µm. (b) Quantification of cisplatin treatment showing a significant increase in apoptosis in Huwe1 deficient tumour cells (Mann-Whitney, n = 3 vs 5).


( c ) Comparison of somatic mutation rate ( mutations / Mb ) between human tumours carrying HUWE1 mutations or not . Note the significant increase in mutational burden in HUWE1 mutated tumours ( Mann - Whitney , p = 0 . 0002 , n ≥ 15 ) .

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(c) Comparison of somatic mutation rate (mutations / Mb) between human tumours carrying HUWE1 mutations or not. Note the significant increase in mutational burden in HUWE1 mutated tumours (Mann-Whitney, p = 0.0002, n ≥ 15).


( d ) Comparison of somatic mutation rate ( mutations / Mb ) between human tumours carrying HUWE1 mutations or not grouped according to MLH1 status . Note that the increased mutation rate is found primarily in tumours where MLH1 is not silenced indicating increased mutation rate ( and HUWE1 mutation itself ) is not due to silencing of MLH1 ( Mann - Whitney , p = 0 . 0007 , n ≥ 11 ) .

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(d) Comparison of somatic mutation rate (mutations / Mb) between human tumours carrying HUWE1 mutations or not grouped according to MLH1 status. Note that the increased mutation rate is found primarily in tumours where MLH1 is not silenced indicating increased mutation rate (and HUWE1 mutation itself) is not due to silencing of MLH1 (Mann-Whitney, p = 0.0007, n ≥ 11).


( e ) Survival analysis of colorectal cancer patients treated with adjuvant chemotherapy divided by HUWE1 expression levels . Note the lowest HUWE1 expressing quartile of patients respond significantly better to chemotherapy . ( f ) Survival analysis of colorectal cancer patients not treated with adjuvant chemotherapy divided by HUWE1 expression levels . Note the lowest HUWE1 expressing quartile of patients do not survive significantly longer than those expressing higher levels of HUWE1 if not treated with chemotherapy .

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(e) Survival analysis of colorectal cancer patients treated with adjuvant chemotherapy divided by HUWE1 expression levels. Note the lowest HUWE1 expressing quartile of patients respond significantly better to chemotherapy. (f) Survival analysis of colorectal cancer patients not treated with adjuvant chemotherapy divided by HUWE1 expression levels. Note the lowest HUWE1 expressing quartile of patients do not survive significantly longer than those expressing higher levels of HUWE1 if not treated with chemotherapy.


( b ) Kaplan - Meier survival plot of cohorts of induced Lgr5 Apc and Lgr5 Apc Huwe1 mice . Deletion of Huwe1 led to a significant reduction in survival of these animals ( Log Rank , p < 0 . 001 , n ≥ 15 ) .

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(b) Kaplan-Meier survival plot of cohorts of induced Lgr5 Apc and Lgr5 Apc Huwe1 mice. Deletion of Huwe1 led to a significant reduction in survival of these animals (Log Rank, p < 0.001, n ≥ 15).


( c ) Lysozyme IHC demonstrating increased lysozyme positive cell numbers in tumours from Lgr5 Apc Huwe1 mice . Scale bars = 200 µm ( low magnification ) , 100 µm ( high magnification inset ) . ( d ) OLFM4 IHC demonstrating expanded stem cell population in Lgr5 Apc Huwe1 tumours . Scale bars = 200 µm ( low magnification ) , 100 µm ( high magnification inset ) .

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(c) Lysozyme IHC demonstrating increased lysozyme positive cell numbers in tumours from Lgr5 Apc Huwe1 mice. Scale bars = 200 µm (low magnification), 100 µm (high magnification inset). (d) OLFM4 IHC demonstrating expanded stem cell population in Lgr5 Apc Huwe1 tumours. Scale bars = 200 µm (low magnification), 100 µm (high magnification inset).


( a ) MCL1 Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours . Levels of MCL1 protein are significantly increased in tumours lacking HUWE1 ( Mann Whitney , p = 0 . 04 , n = 3 ) .

(a) MCL1 Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours. Levels of MCL1 protein are significantly increased in tumours lacking HUWE1 (Mann Whitney, p = 0.04, n = 3).


( b ) Kaplan - Meier survival plot of cohorts of induced Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1 mice . Deletion of one copy of Mcl1 led to a significant increase in survival of these animals ( Log Rank , p = 0 . 0052 , n = 9 vs 10 ) .

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(b) Kaplan-Meier survival plot of cohorts of induced Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1 mice. Deletion of one copy of Mcl1 led to a significant increase in survival of these animals (Log Rank, p = 0.0052, n = 9 vs 10).


( c ) Ratio of adenoma / indolent lesions observed in Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1het mice . Note the decreased ration of adenoma / indolent lesions observed upon Mcl1 deletion ( Mann - Whitney , p = 0 . 0281 , n = 8 v 8 ) .

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(c) Ratio of adenoma / indolent lesions observed in Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1het mice. Note the decreased ration of adenoma / indolent lesions observed upon Mcl1 deletion (Mann-Whitney, p=0.0281, n = 8 v 8).


A ) GST pulldown used to analyse TBC1D14 interactors . Recombinant GST or GST - TBC1D14 was incubated with or without HEK 293A lysate . Bound proteins were eluted from the beads using Laemmli sample buffer and subjected to SDS - PAGE , with visible bands being excised from the gel and analysed by mass spectrometry .

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A) GST pulldown used to analyse TBC1D14 interactors. Recombinant GST or GST-TBC1D14 was incubated with or without HEK 293A lysate. Bound proteins were eluted from the beads using Laemmli sample buffer and subjected to SDS-PAGE, with visible bands being excised from the gel and analysed by mass spectrometry.


B ) Lysates of HEK293A cells expressing either GFP or GFP - TBC1D14 were subjected to immunoprecipitation ( IP ) with GFP - Trap , and then immunoblotted for GFP , TRAPPC12 and TRAPPC4 .

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B) Lysates of HEK293A cells expressing either GFP or GFP-TBC1D14 were subjected to immunoprecipitation (IP) with GFP-Trap, and then immunoblotted for GFP, TRAPPC12 and TRAPPC4.


C ) Lysates of cells expressing GFP or GFP - TRAPPC3 were subjected to IP with GFP - Trap , and then immunoblotted for TRAPPC4 , GFP and TBC1D14 .

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C) Lysates of cells expressing GFP or GFP-TRAPPC3 were subjected to IP with GFP-Trap, and then immunoblotted for TRAPPC4, GFP and TBC1D14.


D ) Cells expressing GFP - TBC1D14 , generating a tubulated endosomal compartment , were fed Alexa647 labelled Transferrin ( white , blue in merge ) for 15 minutes in full medium , fixed , stained for endogenous TRAPPC4 ( red ) and analysed by confocal microscopy . White arrowheads indicate transfected cells , yellow arrowheads depict regions of colocalisation in inset . Scale bars = 10 μm .

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D) Cells expressing GFP-TBC1D14, generating a tubulated endosomal compartment, were fed Alexa647 labelled Transferrin (white, blue in merge) for 15 minutes in full medium, fixed, stained for endogenous TRAPPC4 (red) and analysed by confocal microscopy. White arrowheads indicate transfected cells, yellow arrowheads depict regions of colocalisation in inset. Scale bars = 10 μm.


E ) Cells treated as in D were stained for endogenous TRAPPC12 ( red ) and analysed by confocal microscopy . Scale bars = 10

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E) Cells treated as in D were stained for endogenous TRAPPC12 (red) and analysed by confocal microscopy. Scale bars = 10 μm


F ) Cells in complete medium were fixed , stained for endogenous TBC1D14 ( green ) , RAB1B ( red ) and TRAPPC4 ( white , blue in merge ) and analysed by confocal microscopy . Inset shows protein localisation in juxtanuclear area . Yellow arrowheads show regions of colocalisation Scale bars = 10 μm . Data shown are representative from 3 independent experiments .

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F) Cells in complete medium were fixed, stained for endogenous TBC1D14 (green), RAB1B (red) and TRAPPC4 (white, blue in merge) and analysed by confocal microscopy. Inset shows protein localisation in juxtanuclear area. Yellow arrowheads show regions of colocalisation Scale bars = 10 μm. Data shown are representative from 3 independent experiments.


A ) HEK293A cells transfected with GFP ( upper panels ) or GFP - TBC1D14 ( lower panels ) ( green ) were labelled with anti - RAB11A ( red ) and anti - RAB1B ( white , blue in merge ) antibodies . Arrows depict transfected cells . Inset : GFP - TBC1D14 transfected cell showing triple co - localisation between GFP - TBC1D14 , RAB11A and RAB1B ( white arrows ) and double co - localisation between GFP - TBC1D14 and RAB11A ( yellow arrows ) .

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A) HEK293A cells transfected with GFP (upper panels) or GFP-TBC1D14 (lower panels) (green) were labelled with anti-RAB11A (red) and anti-RAB1B (white, blue in merge) antibodies. Arrows depict transfected cells. Inset: GFP-TBC1D14 transfected cell showing triple co-localisation between GFP-TBC1D14, RAB11A and RAB1B (white arrows) and double co-localisation between GFP-TBC1D14 and RAB11A (yellow arrows).


B ) Co - localisation of GFP - TBC1D14 , TRAPPC4 ( red ) and RAB11A ( white , blue in merge ) . White arrowheads indicate regions of triple co - localisation .

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B) Co-localisation of GFP-TBC1D14, TRAPPC4 (red) and RAB11A (white, blue in merge). White arrowheads indicate regions of triple co-localisation.


C ) Co - localisation of GFP - TBC1D14 , TRAPPC4 ( red ) and RAB1B ( white , blue in merge ) . White arrowheads indicate regions of triple co - localisation , yellow arrowheads regions of double co - localisation between GFP - TBC1D14 and TRAPPC4 . All scale bars = 20 μm .

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C) Co-localisation of GFP-TBC1D14, TRAPPC4 (red) and RAB1B (white, blue in merge). White arrowheads indicate regions of triple co-localisation, yellow arrowheads regions of double co-localisation between GFP-TBC1D14 and TRAPPC4. All scale bars = 20 μm.


B ) HEK 293A cell lysates ( from approximately 8 × 106 cells per construct ) expressing the indicated GFP fusions were subjected to IP and immunoblotting for GFP , TRAPPC12 and TRAPPC4 . Data are representative of 3 independent experiments .

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B) HEK 293A cell lysates (from approximately 8 × 106 cells per construct) expressing the indicated GFP fusions were subjected to IP and immunoblotting for GFP, TRAPPC12 and TRAPPC4. Data are representative of 3 independent experiments.


A ) Cells expressing the indicated GFP - fusions ( green ) were loaded with Alexa647 - Transferrin ( red ; arrowheads indicate transfected cells ) for 15 minutes , fixed and the morphology of the transferrin positive recycling endosomes analysed by confocal microscopy . Scale bars = 10 μm . Arrows in inset indicate the juxtanuclear

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A) Cells expressing the indicated GFP-fusions (green) were loaded with Alexa647-Transferrin (red; arrowheads indicate transfected cells) for 15 minutes, fixed and the morphology of the transferrin positive recycling endosomes analysed by confocal microscopy. Scale bars = 10 μm. Arrows in inset indicate the juxtanuclear ERC


B ) Cells transfected as in ( A ) were loaded with Alexa - 647 transferrin for 15 minutes and the fluorescent transferrin chased out for the indicated time periods . At the end of the timecourse the cells were fixed and the transferrin content of GFP positive cells analysed by flow cytometry . Results are expressed as a percentage of Tfn fluorescence at t = 0 and are the mean of 3 independent experiments , ± s . e . m .

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B) Cells transfected as in (A) were loaded with Alexa-647 transferrin for 15 minutes and the fluorescent transferrin chased out for the indicated time periods. At the end of the timecourse the cells were fixed and the transferrin content of GFP positive cells analysed by flow cytometry. Results are expressed as a percentage of Tfn fluorescence at t=0 and are the mean of 3 independent experiments, ± s.e.m.


C ) Cells transfected as in ( A ) were stained for the cis - Golgi marker GM130 . Arrowheads depict transfected cells . Scale bars = 10 μm . Bar chart is quantification of fragmented Golgi complex ( > 100 cells per condition from three independent experiments , ± s . e . m . * * p < 0 . 01 , one way ANOVA with Sidak ' s post - test ) .

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C) Cells transfected as in (A) were stained for the cis-Golgi marker GM130. Arrowheads depict transfected cells. Scale bars = 10 μm. Bar chart is quantification of fragmented Golgi complex (> 100 cells per condition from three independent experiments, ± s.e.m. ** p < 0.01, one way ANOVA with Sidak's post-test).


D ) HeLa C1 cells transfected with CFP or CFP - TBR were treated with D / D solubiliser ( Clontech ) or D / D solubiliser plus 2 . 5 μg / ml Brefeldin A ( BFA ) for the indicated time periods , trypsinised , fixed and their GFP content analysed by flow cytometry . Results are expressed as a percentage of GFP levels at t = 0 , and the mean of 3 independent experiments ± s . e . m .

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

D) HeLa C1 cells transfected with CFP or CFP-TBR were treated with D/D solubiliser (Clontech) or D/D solubiliser plus 2.5 μg/ml Brefeldin A (BFA) for the indicated time periods, trypsinised, fixed and their GFP content analysed by flow cytometry. Results are expressed as a percentage of GFP levels at t=0, and the mean of 3 independent experiments ± s.e.m.


A ) HEK293A cells expressing GFP , GFP - TBC1D14 224 - 669 or GFP - TBR were treated in duplicate with EBSS , EBSS plus 100 nM BafA1 or not for two hours , lysed and subjected to immunoblotting for LC3B , tubulin and GFP . The amount of LC3B / tubulin for each condition from three independent experiments is shown on the bar graph , ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , one way ANOVA with Sidak ' s multiple comparison test .

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

A) HEK293A cells expressing GFP, GFP-TBC1D14 224-669 or GFP-TBR were treated in duplicate with EBSS, EBSS plus 100 nM BafA1 or not for two hours, lysed and subjected to immunoblotting for LC3B, tubulin and GFP. The amount of LC3B/tubulin for each condition from three independent experiments is shown on the bar graph, ±s.e.m. * p<0.05, ** p<0.01, one way ANOVA with Sidak's multiple comparison test.


B ) HEK293A GFP - LC3B ( 2GL9 ) cells were transfected with mCherry or mCherry - TBR , treated with EBSS for two hours , fixed and analysed by confocal microscopy .

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

B) HEK293A GFP-LC3B (2GL9) cells were transfected with mCherry or mCherry-TBR, treated with EBSS for two hours, fixed and analysed by confocal microscopy.


C ) HEK293A cells were treated as in ( B ) , fixed , stained for WIPI2 and analysed by confocal microscopy .

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

C) HEK293A cells were treated as in (B), fixed, stained for WIPI2 and analysed by confocal microscopy.


D ) HEK293A cells stably expressing GFP - DFCP1 were treated as in ( B ) , fixed and analysed by confocal microscopy . Scale bars = 20 μm . For quantification of ( B ) , ( C ) and ( D ) , 10 fields of view containing transfected cells were imaged for each of three experiments , and the number of GFP - LC3B , WIPI2 or GFP - DFCP1 puncta per cell enumerated using Imaris software ( Bitplane ) .

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

D) HEK293A cells stably expressing GFP-DFCP1 were treated as in (B), fixed and analysed by confocal microscopy. Scale bars = 20 μm. For quantification of (B), (C) and (D), 10 fields of view containing transfected cells were imaged for each of three experiments, and the number of GFP-LC3B, WIPI2 or GFP-DFCP1 puncta per cell enumerated using Imaris software (Bitplane).


B ) Samples prepared according to ( A ) ( 8 × 107 HEK293A cells per transfection ) were lysed in TNTE , split equally and denatured with 1 % SDS ( D ) or not ( ND ) and subjected to 1 hour streptavidin resin pulldown at room temperature . The bound proteins were eluted in 2x Laemmli sample buffer plus 3 mM Biotin and immunoblotted for the indicated proteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Samples prepared according to (A) (8 × 107 HEK293A cells per transfection) were lysed in TNTE, split equally and denatured with 1% SDS (D) or not (ND) and subjected to 1 hour streptavidin resin pulldown at room temperature. The bound proteins were eluted in 2x Laemmli sample buffer plus 3 mM Biotin and immunoblotted for the indicated proteins.


C ) siRNA transfected control cells ( RISC Free ) or TRAPPC8 knockdown ( TRAPPC8 ) ( 1x10cm plate per condition ) were lysed in TNTE and used in pulldown assays with immobilised recombinant GST or GST - TBR . Bound proteins were subjected to immunoblot analysis using TRAPPC8 , TRAPPC4 and tubulin antibodies . Ponceau staining was used to verify loading of the GST fusion proteins ( GST - TBR indicated with arrowhead ) . The bar chart expresses the relative amount of TRAPPC4 isolated from siTRAPPC8 compared to siRISC Free control cells from three independent experiments , error bars are ± s . e . m . * * p < 0 . 01 , unpaired T -

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

C) siRNA transfected control cells (RISC Free) or TRAPPC8 knockdown (TRAPPC8) (1x10cm plate per condition) were lysed in TNTE and used in pulldown assays with immobilised recombinant GST or GST-TBR. Bound proteins were subjected to immunoblot analysis using TRAPPC8, TRAPPC4 and tubulin antibodies. Ponceau staining was used to verify loading of the GST fusion proteins (GST-TBR indicated with arrowhead). The bar chart expresses the relative amount of TRAPPC4 isolated from siTRAPPC8 compared to siRISC Free control cells from three independent experiments, error bars are ± s.e.m. ** p<0.01, unpaired T-test


D ) HEK293T cells stably depleted of TRAPPC8 ( shC8 ) or not ( shCtrl ) were transfected with constructs encoding GFP or GFP - TRAPPC3 and subjected to GFP Trap IP . Precipitated proteins were immunoblotted for GFP , TRAPPC4 and TRAPPC8 . Bar chart : the amount of TRAPPC4 isolated was normalised to the amount of GFP - TRAPPC3 precipitated from three independent experiments and plotted ± s . e . m . , ns = non - significant , unpaired T - test .

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

D) HEK293T cells stably depleted of TRAPPC8 (shC8) or not (shCtrl) were transfected with constructs encoding GFP or GFP-TRAPPC3 and subjected to GFP Trap IP. Precipitated proteins were immunoblotted for GFP, TRAPPC4 and TRAPPC8. Bar chart: the amount of TRAPPC4 isolated was normalised to the amount of GFP-TRAPPC3 precipitated from three independent experiments and plotted ± s.e.m., ns = non-significant, unpaired T-test.


A ) HEK293A cells treated with transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 and RAB11A and B ( knockdown confirmed by immunoblot analysis ) were fed with Alexa - 647 Transferrin for 15 minutes in full medium , and the fluorescent transferrin chased out for the indicated time periods . At the end of the time course , cells were trypsinised , fixed and the Alexa - 647 fluorescence analysed by flow cytometry . Results are plotted as percentage of transferrin fluorescence at t = 0 for the RISC free control cells , expressed as the mean of three independent experiments ± s . e . m .

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

A) HEK293A cells treated with transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 and RAB11A and B (knockdown confirmed by immunoblot analysis) were fed with Alexa-647 Transferrin for 15 minutes in full medium, and the fluorescent transferrin chased out for the indicated time periods. At the end of the time course, cells were trypsinised, fixed and the Alexa-647 fluorescence analysed by flow cytometry. Results are plotted as percentage of transferrin fluorescence at t=0 for the RISC free control cells, expressed as the mean of three independent experiments ± s.e.m.


B ) Cells transiently transfected with RISC Free control siRNA , or siRNA duplexes directed against TRAPPC8 or RAB1A and B were stained for the cis - Golgi markers GM130 or RAB1B and analysed by confocal microscopy . Scale bars = 20 μm in main panels , 10 μm in inset panels . Bar graph : > 100 cells per siRNA were scored for fragmented or normal juxtanuclear Golgi stacks from three independent experiments . Error bars ± s . e . m . , * p < 0 . 05 , * * p < 0 . 01 , one way ANOVA with Sidak ' s multiple comparison test .

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

B) Cells transiently transfected with RISC Free control siRNA, or siRNA duplexes directed against TRAPPC8 or RAB1A and B were stained for the cis-Golgi markers GM130 or RAB1B and analysed by confocal microscopy. Scale bars = 20 μm in main panels, 10 μm in inset panels. Bar graph: > 100 cells per siRNA were scored for fragmented or normal juxtanuclear Golgi stacks from three independent experiments. Error bars ± s.e.m., * p<0.05, ** p<0.01, one way ANOVA with Sidak's multiple comparison test.


C ) HeLa C1 cells transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 or RAB1A and B ( knockdown confirmed by immunoblot analysis ) , were treated with D / D solubiliser for the indicated time periods , trypsinised , fixed and their GFP content analysed by flow cytometry . The line graph shows the GFP fluorescence as a percentage of t = 0 , ± s . e . m . from three independent experiments .

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

C) HeLa C1 cells transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 or RAB1A and B (knockdown confirmed by immunoblot analysis), were treated with D/D solubiliser for the indicated time periods, trypsinised, fixed and their GFP content analysed by flow cytometry. The line graph shows the GFP fluorescence as a percentage of t=0, ± s.e.m. from three independent experiments.


A ) ATG9 positive membranes were immunoisolated from approximately 2 × 107 fed ( F ) or starved ( S ) HEK293A cells using a monoclonal hamster anti - ATG9 ( ATG9 ) or control hamster IgM and immunoblotted for ATG9 using a rabbit polyclonal antibody , TRAPPC8 , TRAPPC4 and actin .

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

A) ATG9 positive membranes were immunoisolated from approximately 2 × 107 fed (F) or starved (S) HEK293A cells using a monoclonal hamster anti-ATG9 (ATG9) or control hamster IgM and immunoblotted for ATG9 using a rabbit polyclonal antibody, TRAPPC8, TRAPPC4 and actin.


B ) HEK293A cells transfected with GFP or GFP - TBR ( green ) were stained for ATG9 using hamster anti - ATG9 ( red ) and analysed by confocal microscopy . Scale bar = 20

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

B) HEK293A cells transfected with GFP or GFP-TBR (green) were stained for ATG9 using hamster anti-ATG9 (red) and analysed by confocal microscopy. Scale bar = 20 μm


C ) HEK293A cells transiently transfected with RISC Free siRNA or siRNA against TRAPPC8 were stained for RAB1B ( green ) and ATG9 using hamster anti - ATG9 ( red ) and imaged using confocal microscopy .

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

C) HEK293A cells transiently transfected with RISC Free siRNA or siRNA against TRAPPC8 were stained for RAB1B (green) and ATG9 using hamster anti-ATG9 (red) and imaged using confocal microscopy.


D ) Stills at t = 30 minutes from time lapse imaging of 9B9 cells ( stably expressing GFP - LC3 and mRFP - ATG9 ) expressing either CFP or CFP - TBR ( blue ) . Scale bars = 20 μm . Inset panels show mRFP - ATG9 and GFP - LC3 contact events at the indicated time points . Bar chart shows number of times a GFP - LC3 spot was within 1 μm of an mRFP - ATG9 spot ( a contact event ) , expressed as total mRFP - ATG9 contacts per GFP - LC3 spot , n = 18 cells per condition pooled from 4 independent experiments .

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

D) Stills at t = 30 minutes from time lapse imaging of 9B9 cells (stably expressing GFP-LC3 and mRFP-ATG9) expressing either CFP or CFP-TBR (blue). Scale bars = 20 μm. Inset panels show mRFP-ATG9 and GFP-LC3 contact events at the indicated time points. Bar chart shows number of times a GFP-LC3 spot was within 1 μm of an mRFP-ATG9 spot (a contact event), expressed as total mRFP-ATG9 contacts per GFP-LC3 spot, n = 18 cells per condition pooled from 4 independent experiments.


A ) Cells depleted of ULK1 ( siULK1 ) or not ( RISC Free ) were transfected with GFP or GFP - TBR ( green ) , starved ( EBSS ) or not ( - EBSS ) and stained for ATG9 using hamster anti - ATG9 ( red ) . Arrows indicate transfected cells . Western blot indicates ULK1 knockdown . Scale bars = 20 μm .

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

A) Cells depleted of ULK1 (siULK1) or not (RISC Free) were transfected with GFP or GFP-TBR (green), starved (EBSS) or not (-EBSS) and stained for ATG9 using hamster anti-ATG9 (red). Arrows indicate transfected cells. Western blot indicates ULK1 knockdown. Scale bars = 20 μm.


B ) Cells depleted of ULK1 ( siULK1 ) or not ( RISC Free ) were transfected with myc - RAB1B or myc - RAB1B ( S22N ) ( green ) , starved ( EBSS ) or not ( - EBSS ) and stained for ATG9 using hamster anti - ATG9 ( red ) Arrows indicate transfected cells . Western blot indicates ULK1 knockdown . Scale bars = 20 μm .

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

B) Cells depleted of ULK1 (siULK1) or not (RISC Free) were transfected with myc-RAB1B or myc-RAB1B (S22N) (green), starved (EBSS) or not (-EBSS) and stained for ATG9 using hamster anti-ATG9 (red) Arrows indicate transfected cells. Western blot indicates ULK1 knockdown. Scale bars = 20 μm.


( B ) ParB clusters on F plasmid in vivo . Typical E . coli cells ( DLT3594 ) displays foci of ParBF - mVenus protein ( top ) expressed from the endogenous genetic locus of the F plasmid ( F1 - 10B - mVenus ) . The nucleoid is labelled with Hu - mCherry ( central ) . The overlay ( bottom ) combines the two fluorescent channels . Over 99 % of cells harbor ParBF foci . White bars : 1

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

(B) ParB clusters on F plasmid in vivo. Typical E. coli cells (DLT3594) displays foci of ParBF-mVenus protein (top) expressed from the endogenous genetic locus of the F plasmid (F1-10B-mVenus). The nucleoid is labelled with Hu-mCherry (central). The overlay (bottom) combines the two fluorescent channels. Over 99 % of cells harbor ParBF foci. White bars: 1 µm


( C ) ParBF binding outside parSF on the F plasmid is compatible with a power - law decay . High resolution ChIP - seq performed on DLT3586 carrying the F plasmid ( F1 - 10B ) . The ParB density , normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction , is displayed over 14 - Kbp on the right side of parSF . Monte Carlo simulations and analytic formula are represented in red and dotted black lines , respectively . MC simulations were performed with a Freely - Jointed Chain of linear length L = 15 - Kbp and a cluster radius σ = 75nm . The two other parameters , the Kuhn length a = 10 - bp and the total number of proteins on the F plasmid Nt = 360 ( related to the normalization constant of the protein concentration κ = 0 . 41 ) were fitted from the ChIP - seq data ( see text and Fig . EV2 ) . As a benchmark for simulations , the analytics are obtained from Eq . ( 1 ) with the same parameters . Inset ; The ParBF binding profile ( black line ) is represented as the number of nucleotide reads over 80 - Kbp centered at parS . The number of reads in the Input sample ( grey line ) is normalized to the total number of reads in the IP

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

(C) ParBF binding outside parSF on the F plasmid is compatible with a power-law decay. High resolution ChIP-seq performed on DLT3586 carrying the F plasmid (F1-10B). The ParB density, normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction, is displayed over 14-Kbp on the right side of parSF. Monte Carlo simulations and analytic formula are represented in red and dotted black lines, respectively. MC simulations were performed with a Freely-Jointed Chain of linear length L=15-Kbp and a cluster radius σ=75nm. The two other parameters, the Kuhn length a=10-bp and the total number of proteins on the F plasmid Nt=360 (related to the normalization constant of the protein concentration κ=0.41) were fitted from the ChIP-seq data (see text and Fig. EV2). As a benchmark for simulations, the analytics are obtained from Eq.(1) with the same parameters. Inset; The ParBF binding profile (black line) is represented as the number of nucleotide reads over 80-Kbp centered at parS. The number of reads in the Input sample (grey line) is normalized to the total number of reads in the IP sample


Typical E . coli cell displays foc expressed from the endogenous genetic locus of the F plasmid ( F1 - 10B - mVenus ) . The nucleoid is labelled with Hu - mCherry ( central ) . The overlay ( bottom ) combines the two fluorescent channels parSF inserted at the xylE locus on E . coli chromosome from

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

Typical E. coli cell displays foc expressed from the endogenous genetic locus of the F plasmid (F1-10B-mVenus). The nucleoid is labelled with Hu-mCherry (central). The overlay (bottom) combines the two fluorescent channels parSF inserted at the xylE locus on E. coli chromosome from DLT358


ParBF binding outside parSF on the F plasmid is compatible with a power - law decay . High resolution ChIP - seq performed on DLT3586 carrying the F plasmid ( F1 - 10B ) . The ParB density , normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction , is displayed over 14 - Kbp on the right side of parSF . Monte Carlo simulations and analytic formula are represented in red and dotted black lines , respectively . MC simulations were performed with a Freely - Jointed Chain of linear length L = 15 - Kbp and a cluster radius σ = 75nm . The two other parameters , the Kuhn length a = 10 - bp and the total number of proteins on the F plasmid Nt = 360 ( related to the normalization constant of the protein concentration κ = 0 . 41 ) were fitted from the ChIP - seq dat parSF inserted at the xylE locus on E . coli chromosome fro

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

ParBF binding outside parSF on the F plasmid is compatible with a power-law decay. High resolution ChIP-seq performed on DLT3586 carrying the F plasmid (F1-10B). The ParB density, normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction, is displayed over 14-Kbp on the right side of parSF. Monte Carlo simulations and analytic formula are represented in red and dotted black lines, respectively. MC simulations were performed with a Freely-Jointed Chain of linear length L=15-Kbp and a cluster radius σ=75nm. The two other parameters, the Kuhn length a=10-bp and the total number of proteins on the F plasmid Nt=360 (related to the normalization constant of the protein concentration κ=0.41) were fitted from the ChIP-seq dat parSF inserted at the xylE locus on E. coli chromosome fro DLT207