Figure 5Trajectories 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Trajectories 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.


Figure 6 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) 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.


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

Figure 1A. 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.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.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.


Figure 2A . 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 samplesD . PEG pellets were subjected to OptiPrep flotation analysis . GPRC5C was detected only in the apical sEV samplesE . 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 samplesF . MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD63 antibody .

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

Figure 2A. 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 samplesD. PEG pellets were subjected to OptiPrep flotation analysis. GPRC5C was detected only in the apical sEV samplesE. 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 samplesF. MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody.


Figure 3A . 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 . 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 .

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

Figure 3A. 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.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.


Figure 4A . 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 . 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 . D . Quantification of the NTA data obtained in five independent experiments .

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

Figure 4A. 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.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.D. Quantification of the NTA data obtained in five independent experiments.


Figure 1Analyses 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 . 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 . 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 .

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

Figure 1Analyses 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.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.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.


Figure 2Fragments 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 . 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 . 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 .

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

Figure 2Fragments 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.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.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.


Figure 3Microscopy - 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 bindingMicroscopy - 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 . by immunoblotting using the indicated antibodies . Dimerisation assay in 293T cells transiently transfected with ATG16L1 - GFP and the indicated S - tagged ATG16L1 constructs and

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

Figure 3Microscopy-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 bindingMicroscopy-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.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


Figure 4ATG16L1 - / - 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 . 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 .

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

Figure 4ATG16L1-/- 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.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.


Figure 5ATG16L1 - / - 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 . 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 . 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 .

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

Figure 5ATG16L1-/- 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.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.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.


Figure 6Coomassie gel of recombinant ATG16L172 - 307 wild type and mutants . * indicates bacterial proteinLiposome 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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Figure 6Coomassie gel of recombinant ATG16L172-307 wild type and mutants. * indicates bacterial proteinLiposome 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.


Figure 7ATG16L1 - / - 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 punctaCells 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 . 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 .

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

Figure 7ATG16L1-/- 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 punctaCells 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.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.


Figure 1 ( 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 ) . ( 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 . ( 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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Figure 1(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).(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).


Figure 2 ( 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 . ( 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 .

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

Figure 2(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.(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.


Figure 3 ( 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 . ( 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 ) . ( e ) QRT - PCR analysis of Myc expression in tumours from Vil Apc and Vil Apc Huwe1hom mice .

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Figure 3(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.(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).(e) QRT-PCR analysis of Myc expression in tumours from Vil Apc and Vil Apc Huwe1hom mice.


Figure 4 ( 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 ) . ( 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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Figure 4(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).(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).


Figure 5 ( 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 . ( 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 .

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

Figure 5(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.(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.


Figure 6 ( 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 ) . ( 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 .

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

Figure 6(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).(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.


Figure 7 ( 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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Figure 7(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).


Figure 8 ( 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 ) . ( 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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Figure 8(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).(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).


Figure 1A ) 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 . 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 . E ) Cells treated as in D were stained for endogenous TRAPPC12 ( red ) and analysed by confocal microscopy . Scale bars = 10 μmF ) 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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Figure 1A) 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.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.E) Cells treated as in D were stained for endogenous TRAPPC12 (red) and analysed by confocal microscopy. Scale bars = 10 μmF) 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.


Figure 2A ) 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 . 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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Figure 2A) 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.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.


Figure 3B ) 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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Figure 3B) 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.


Figure 4A ) 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 ERCB ) 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 ) . 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 .

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Figure 4A) 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 ERCB) 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).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.


Figure 5A ) 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 . 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 ) .

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Figure 5A) 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.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).


Figure 6B ) 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 - testD ) 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 .

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Figure 6B) 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-testD) 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.


Figure 7A ) 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 . 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 .

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Figure 7A) 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.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.


Figure 8A ) 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 μmC ) 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 .

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Figure 8A) 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 μmC) 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.


Figure 9A ) 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 .

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Figure 9A) 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.


Figure 1 ( 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 sampleTypical 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 DLT358ParBF 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

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

Figure 1(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 sampleTypical 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 DLT358ParBF 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


Figure 2 ( A ) Normalized and rescaled ParBF binding profiles at different ParBF / parSF ratio . ChIP - seq density on the right side of parSF inserted at xylE were measured in DLT2075 induced ( 16 , 28 ) or not ( 0 . 4 ) with IPTG ( 100 and 500 µM ) , or carrying HCN plasmids pZC302 ( 0 . 04 ) or pJYB57 ( 0 . 016 ) , normalized as in Fig . 1C , E with the amplitudes of the curves rescaled by the indicated factors ( 1 . 2 , 10 or 50 ) to overlap with the curves of highest amplitude . The ParB / parS ratio is calculated relative to the one of F plasmid as determined from Western blot analyses ( Appendix Fig . S2B ) . Monte Carlo simulations and analytical formula are plotted with the same parameters as in Fig . 1E . Note ( i ) that the dips at ~ 9 - kbp are not visible for the low levels of available ParB since the signal is close to the basal level and ( ii ) that the ChIP - seq data at 100 µM IPTG induction ( 16 ) are the same as in Fig . 1E . Inset ; Same as in the main to display the density without rescaling . ( B ) ParBF are dispersed in the cell upon titration by HCN plasmids . ParBF - mVenus expressed from pJYB294 were imaged as in Fig . 1D in DLT3577 ( left ) and DLT3576 ( right ) carrying pZC302 and pJYB57 , respectively . The number of extra parSF per cell , indicated on top of each raw , are estimated from the copy number per cell of HCN plasmids carrying 10 specific binding sites . White bars : 1 µm . ( C ) The size of ParBF clusters is independent of the intracellular ParBF concentration . We considered two possible evolutions of the cluster size upon variations of ParB amount in the framework of " Nucleation & caging " with corresponding schematics drawn on the right . For direct comparison with ( A ) , all curves are displayed with a rescaling of the amplitude corresponding to the WT expression level . Top : constant ParB concentration ; supposing that clusters are compact , the cluster radius σ would depend on the number m of ParB like $ \ \ sigma = m ^ { \ frac { 1 } { 3 } } $ . Predictions profiles , plotted at different ratio of ParB / parS , vary within the range of the experimental levels tested . Bottom : constant cluster size ; ParB concentrations vary but the range of exploration remains the same resulting in

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

Figure 2(A) Normalized and rescaled ParBF binding profiles at different ParBF/parSF ratio. ChIP-seq density on the right side of parSF inserted at xylE were measured in DLT2075 induced (16, 28) or not (0.4) with IPTG (100 and 500 µM), or carrying HCN plasmids pZC302 (0.04) or pJYB57 (0.016), normalized as in Fig. 1C, E with the amplitudes of the curves rescaled by the indicated factors (1.2, 10 or 50) to overlap with the curves of highest amplitude. The ParB/parS ratio is calculated relative to the one of F plasmid as determined from Western blot analyses (Appendix Fig. S2B). Monte Carlo simulations and analytical formula are plotted with the same parameters as in Fig. 1E. Note (i) that the dips at ~9-kbp are not visible for the low levels of available ParB since the signal is close to the basal level and (ii) that the ChIP-seq data at 100 µM IPTG induction (16) are the same as in Fig. 1E. Inset; Same as in the main to display the density without rescaling. (B) ParBF are dispersed in the cell upon titration by HCN plasmids. ParBF-mVenus expressed from pJYB294 were imaged as in Fig. 1D in DLT3577 (left) and DLT3576 (right) carrying pZC302 and pJYB57, respectively. The number of extra parSF per cell, indicated on top of each raw, are estimated from the copy number per cell of HCN plasmids carrying 10 specific binding sites. White bars: 1 µm. (C) The size of ParBF clusters is independent of the intracellular ParBF concentration. We considered two possible evolutions of the cluster size upon variations of ParB amount in the framework of "Nucleation & caging" with corresponding schematics drawn on the right. For direct comparison with (A), all curves are displayed with a rescaling of the amplitude corresponding to the WT expression level. Top: constant ParB concentration; supposing that clusters are compact, the cluster radius σ would depend on the number m of ParB like$\ \sigma = m^{\frac{1}{3}}$. Predictions profiles, plotted at different ratio of ParB/parS, vary within the range of the experimental levels tested. Bottom: constant cluster size; ParB concentrations vary but the range of exploration remains the same resulting in overlappin


Figure 3 ( A ) The formation of secondary ParBF - DNA complexes requires the box II motif . EMSA were performed with a 144 - bp 32P - labelled DNA fragments ( C144 ) carrying a single 16 - bp parS binding motif . Reaction mixtures containing 100 µg . ml - 1 sonicated salmon sperm DNA were incubated in the absence ( - ) or the presence of increasing concentrations ( grey triangle ; 10 , 30 , 100 , 300 , and 1000 nM ) of ParBF or ParBF - 3R * . Positions of free and bound probes are indicated on the left . B1 represents complexes involving the specific interaction on the 16 - bp binding site , while B " 2 and B " 3 complexes represent secondary complexes involving the parSF site with one or two additional nsDNA - binding interactions , respectively ( Sanchez et al , ( B ) ParBF cluster formation requires the box II motif . Epifluorescence microscopy of ParBF - 3R * - mVenus from DLT3566 is displayed as in Fig . 1D . White bars : 1 µm ( C ) ParBF in vivo DNA binding in the vicinity of parSF sites requires the box II motif . ChIP - seq was performed on DLT3726 carrying parSF in the xylE chromosomal locus and expressing ParBF - 3R * variant . ParBF - 3R * DNA binding profile displayed the number of nucleotide reads as a function of the E . coli genomic coordinates . The peak at parSF covered approximately 950 - bp , which corresponds to the 402 - bp between the 1st and 10th specific binding sites and ~ 280 - bp on each sides ( representing the average size of the DNA library ; see Appendix Fig . S3E ) . No ParBF - 3R * enrichment was found on parSF - flanking DNA and elsewhere on the chromosome . Inset , zoom in on the right side of parSF over 5 - Kbp with the ParB density , normalized to 1 at the first bp after the last parS binding repeat , plotted as a function of the distance from parSF . Note that a highly similar DNA binding pattern is obtained with ParBF - 3R * - mVenus ( strain DLT3566 ; Appendix Fig . S3C )

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

Figure 3(A) The formation of secondary ParBF-DNA complexes requires the box II motif. EMSA were performed with a 144-bp 32P-labelled DNA fragments (C144) carrying a single 16-bp parS binding motif. Reaction mixtures containing 100 µg.ml-1 sonicated salmon sperm DNA were incubated in the absence (-) or the presence of increasing concentrations (grey triangle; 10, 30, 100, 300, and 1000 nM) of ParBF or ParBF-3R*. Positions of free and bound probes are indicated on the left. B1 represents complexes involving the specific interaction on the 16-bp binding site, while B"2 and B"3 complexes represent secondary complexes involving the parSF site with one or two additional nsDNA-binding interactions, respectively (Sanchez et al,(B) ParBF cluster formation requires the box II motif. Epifluorescence microscopy of ParBF-3R*-mVenus from DLT3566 is displayed as in Fig. 1D. White bars: 1 µm(C) ParBF in vivo DNA binding in the vicinity of parSF sites requires the box II motif. ChIP-seq was performed on DLT3726 carrying parSF in the xylE chromosomal locus and expressing ParBF-3R* variant. ParBF-3R* DNA binding profile displayed the number of nucleotide reads as a function of the E. coli genomic coordinates. The peak at parSF covered approximately 950-bp, which corresponds to the 402-bp between the 1st and 10th specific binding sites and ~280-bp on each sides (representing the average size of the DNA library; see Appendix Fig. S3E). No ParBF-3R* enrichment was found on parSF-flanking DNA and elsewhere on the chromosome. Inset, zoom in on the right side of parSF over 5-Kbp with the ParB density, normalized to 1 at the first bp after the last parS binding repeat, plotted as a function of the distance from parSF. Note that a highly similar DNA binding pattern is obtained with ParBF -3R*-mVenus (strain DLT3566; Appendix Fig. S3C)


Figure 4 ( A ) Schematic representation of the genomic locus of the chromosome 1 of V . cholerae with the three parS sites , named parS1 - 3 . The rRNA operon ( blue rectangle ) spans the genomic coordinates 53823 to 59123 ( B ) ChIP - seq performed on strain N16961 is displayed as the number of nucleotide reads in function of the genomic coordinates . Correspondence to the parS1 - 3 location represented in ( A ) is indicated by grey dotted lines . The number of reads in the Input sample ( grey line ) is normalized to the total number of reads in the IP sample . ( C ) We modeled the ChIP - Seq data as in Fig . 1C - E by means of MC simulations with a Freely jointed chain of size N = 2000 monomers of size a = 16 - bp . Data are normalized after background subtraction to the read value at parS1 ( genomic coordinate 62438 ) . The best fit was achieved with σ = 25nm and an amplitude κ = 0 . 15 leading to Nt ~ 50 ParB on the chromosome . In the MC simulation , we accounted for the finite width of the distribution around parS sites by including the average fragment size of the DNA

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

Figure 4(A) Schematic representation of the genomic locus of the chromosome 1 of V. cholerae with the three parS sites, named parS1-3. The rRNA operon (blue rectangle) spans the genomic coordinates 53823 to 59123 (B) ChIP-seq performed on strain N16961 is displayed as the number of nucleotide reads in function of the genomic coordinates. Correspondence to the parS1-3 location represented in (A) is indicated by grey dotted lines. The number of reads in the Input sample (grey line) is normalized to the total number of reads in the IP sample. (C) We modeled the ChIP-Seq data as in Fig.1C-E by means of MC simulations with a Freely jointed chain of size N=2000 monomers of size a=16-bp. Data are normalized after background subtraction to the read value at parS1 (genomic coordinate 62438). The best fit was achieved with σ=25nm and an amplitude κ=0.15 leading to Nt~50 ParB on the chromosome. In the MC simulation, we accounted for the finite width of the distribution around parS sites by including the average fragment size of the DNA librar