( C ) Western blot analysis of EVs isolated by UC from HCT116 cells cultured in glutamine - replete and - depleted medium for 24 h , following transduction with a Rab11a or control non - targeting ( NT ) shRNA knockdown construct over previous two days . Bar chart shows change in putative exosome proteins in EVs secreted from Rab11a knockdown cells relative to NT - treated cells in glutamine - depleted conditions , following initial normalisation to cell lysate protein . ( C ' ) Growth curves are for HCT116 recipient cells pre - treated with EVs isolated as in ( C ) or with vehicle ( PBS ) . * * * * P colour denotes significant increase relative to EVs from glutamine - replete cells ( black ) and Rab11a knockdown cells ( red ) .

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(C) Western blot analysis of EVs isolated by UC from HCT116 cells cultured in glutamine-replete and -depleted medium for 24 h, following transduction with a Rab11a or control non-targeting (NT) shRNA knockdown construct over previous two days. Bar chart shows change in putative exosome proteins in EVs secreted from Rab11a knockdown cells relative to NT-treated cells in glutamine-depleted conditions, following initial normalisation to cell lysate protein. (C') Growth curves are for HCT116 recipient cells pre-treated with EVs isolated as in (C) or with vehicle (PBS). ****P colour denotes significant increase relative to EVs from glutamine-replete cells (black) and Rab11a knockdown cells (red).


( D ) Left hand group of four images show cells grown in glutamine - replete ( 2 . 00 mM ) and glutamine - depleted ( 0 . 15 mM ) conditions for 24 h , then incubated with an anti - CD63 antibody ( yellow ) for 30 min at 4oC , washed with PBS , chased at 37oC for 30 min , then fixed , immunostained and imaged . Right hand group of four images show cells grown in glutamine - replete and glutamine - depleted conditions for 24 h , incubated with Tfn - Alexa Fluor ® 488 ( yellow ) for 30 min at 4oC , shifted to 37oC for 30 min , then washed , immediately fixed and imaged .

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(D) Left hand group of four images show cells grown in glutamine-replete (2.00 mM) and glutamine-depleted (0.15 mM) conditions for 24 h, then incubated with an anti-CD63 antibody (yellow) for 30 min at 4oC, washed with PBS, chased at 37oC for 30 min, then fixed, immunostained and imaged. Right hand group of four images show cells grown in glutamine-replete and glutamine-depleted conditions for 24 h, incubated with Tfn-Alexa Fluor® 488 (yellow) for 30 min at 4oC, shifted to 37oC for 30 min, then washed, immediately fixed and imaged.


( E ) Western blot showing levels of anti - CD63 heavy chain and biotin - conjugated Tfn in HCT116 cells cultured for 24 h in glutamine - replete or - depleted conditions , incubated for 30 min in medium containing these molecules at 4oC , then chased at 37oC for 15 min ( the chase step was not performed for Tfn in D , to reduce loss due to rapid recycling of Tfn ) .

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(E) Western blot showing levels of anti-CD63 heavy chain and biotin-conjugated Tfn in HCT116 cells cultured for 24 h in glutamine-replete or -depleted conditions, incubated for 30 min in medium containing these molecules at 4oC, then chased at 37oC for 15 min (the chase step was not performed for Tfn in D, to reduce loss due to rapid recycling of Tfn).


( F ) Western blot analysis of EVs isolated by UC from HCT116 cells in glutamine - replete medium , transduced with a Rab7 or non - targeting ( NT ) control shRNA knockdown construct . Bar charts show changes in putative exosome proteins in EVs isolated by ultracentrifugation , following normalisation to cell lysate protein . ( F ' ) Growth curves are for HCT116 recipient cells pre - treated with EVs isolated as in ( F ) or with vehicle ( PBS ) .

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(F) Western blot analysis of EVs isolated by UC from HCT116 cells in glutamine-replete medium, transduced with a Rab7 or non-targeting (NT) control shRNA knockdown construct. Bar charts show changes in putative exosome proteins in EVs isolated by ultracentrifugation, following normalisation to cell lysate protein. (F') Growth curves are for HCT116 recipient cells pre-treated with EVs isolated as in (F) or with vehicle (PBS).


( A ) Western blot analysis of ERK phosphorylation ( p - ERK ) in recipient serum - deprived HCT116 cells pre - treated with EVs isolated by SEC from glutamine - replete or - depleted HCT116 cells or vehicle ( PBS ) . Bar chart shows ratio of p - ERK to ERK levels from triplicate 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 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

(A) Western blot analysis of ERK phosphorylation (p-ERK) in recipient serum-deprived HCT116 cells pre-treated with EVs isolated by SEC from glutamine-replete or -depleted HCT116 cells or vehicle (PBS). Bar chart shows ratio of p-ERK to ERK levels from triplicate independent experiments.


( B ) Bar chart shows HCT116 recipient cell growth over 120 h , after pre - treatment with EV preparations isolated as in ( A ) or with PBS , and then incubation for the first 24 h of culture in the presence or absence of the ERK inhibitor SCH772984 ( 1 . 00 µM ) .

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(B) Bar chart shows HCT116 recipient cell growth over 120 h, after pre-treatment with EV preparations isolated as in (A) or with PBS, and then incubation for the first 24 h of culture in the presence or absence of the ERK inhibitor SCH772984 (1.00 µM).


( C ) Western blot analysis showing levels of the EGFR ligand , Amphiregulin ( AREG ) in EVs isolated by SEC of medium from HCT116 cells cultured in glutamine - replete ( 2 . 00 mM ) or - depleted ( 0 . 15 mM ) conditions for 24 h . Gel loading was normalised to cell lysate protein levels . AREG ' s molecular weight ( approximately 26 - 28 kDa ) suggests it is in its membrane - associated form ( see Appendix Fig S8D ) . In the bar chart , the levels of putative exosome proteins were normalised to cell lysate protein levels . Significantly decreased levels are in blue and increased levels are in red . ( C ' ) Growth curves in low ( 1 % ) serum are for HCT116 recipient cells pre - treated with the EV preparations [ isolated as in ( C ) ] , which had themselves been pre - treated with and without anti - AREG neutralising antibody or with a control IgG . Solid red line shows growth - promoting effect of EVs isolated under glutamine depletion , which is blocked by anti - AREG antibody ( red dashed line ) . * * * P colour denotes significant increase relative to EVs from glutamine - replete cells ( black ) and anti - AREG - treated cells ( red ) . ( C ' ' ) Western blot analysis of ERK phosphorylation ( p - ERK ) in recipient serum - deprived HCT116 cells pre - treated with EVs isolated by SEC from glutamine - replete ( 2 . 00 mM ) or - depleted ( 0 . 15 mM ) HCT116 cells or vehicle ( PBS ) , which were pre - treated with anti - AREG ( AREGab ) or a control immunoglobulin ( Ig ) . Note increase in ERK phosphorylation using EVs from glutamine - depleted cells , which is blunted by the addition of the anti - AREG antibody .

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

(C) Western blot analysis showing levels of the EGFR ligand, Amphiregulin (AREG) in EVs isolated by SEC of medium from HCT116 cells cultured in glutamine-replete (2.00 mM) or -depleted (0.15 mM) conditions for 24 h. Gel loading was normalised to cell lysate protein levels. AREG's molecular weight (approximately 26-28 kDa) suggests it is in its membrane-associated form (see Appendix Fig S8D). In the bar chart, the levels of putative exosome proteins were normalised to cell lysate protein levels. Significantly decreased levels are in blue and increased levels are in red.(C') Growth curves in low (1%) serum are for HCT116 recipient cells pre-treated with the EV preparations [isolated as in (C)], which had themselves been pre-treated with and without anti-AREG neutralising antibody or with a control IgG. Solid red line shows growth-promoting effect of EVs isolated under glutamine depletion, which is blocked by anti-AREG antibody (red dashed line). ***P colour denotes significant increase relative to EVs from glutamine-replete cells (black) and anti-AREG-treated cells (red). (C'') Western blot analysis of ERK phosphorylation (p-ERK) in recipient serum-deprived HCT116 cells pre-treated with EVs isolated by SEC from glutamine-replete (2.00 mM) or -depleted (0.15 mM) HCT116 cells or vehicle (PBS), which were pre-treated with anti-AREG (AREGab) or a control immunoglobulin (Ig). Note increase in ERK phosphorylation using EVs from glutamine-depleted cells, which is blunted by the addition of the anti-AREG antibody.


HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three - day intervals with vehicle ( PBS ) , or EVs isolated by UC from glutamine - replete or glutamine - depleted HCT116 cells . Tumours were excised 24 h after last of four injections for analysis . Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System . ( A ) Sections immunostained for CD31 , which labels endothelial cells and blood vessels , with blood vessel number ( upper ) and total area ( lower ) represented in bar charts .

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

HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three-day intervals with vehicle (PBS), or EVs isolated by UC from glutamine-replete or glutamine-depleted HCT116 cells. Tumours were excised 24 h after last of four injections for analysis. Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System. (A) Sections immunostained for CD31, which labels endothelial cells and blood vessels, with blood vessel number (upper) and total area (lower) represented in bar charts.


HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three - day intervals with vehicle ( PBS ) , or EVs isolated by UC from glutamine - replete or glutamine - depleted HCT116 cells . Tumours were excised 24 h after last of four injections for analysis . Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System . ( B ) Sections immunostained for Ki67 , which stains proliferative cells . Proportion of tumour cells with Ki69 staining is represented in bar chart .

O O O O O O O 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 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

HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three-day intervals with vehicle (PBS), or EVs isolated by UC from glutamine-replete or glutamine-depleted HCT116 cells. Tumours were excised 24 h after last of four injections for analysis. Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System. (B) Sections immunostained for Ki67, which stains proliferative cells. Proportion of tumour cells with Ki69 staining is represented in bar chart.


HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three - day intervals with vehicle ( PBS ) , or EVs isolated by UC from glutamine - replete or glutamine - depleted HCT116 cells . Tumours were excised 24 h after last of four injections for analysis . Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System . ( C ) Sections stained with haematoxylin and eosin , which highlights necrotic regions ( pale staining ) . Proportion of tumour area that is necrotic is represented in bar chart .

O O O O O O O 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 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 O O O O

HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three-day intervals with vehicle (PBS), or EVs isolated by UC from glutamine-replete or glutamine-depleted HCT116 cells. Tumours were excised 24 h after last of four injections for analysis. Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System. (C) Sections stained with haematoxylin and eosin, which highlights necrotic regions (pale staining). Proportion of tumour area that is necrotic is represented in bar chart.


HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three - day intervals with vehicle ( PBS ) , or EVs isolated by UC from glutamine - replete or glutamine - depleted HCT116 cells . Tumours were excised 24 h after last of four injections for analysis . Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System . ( D ) Sections immunostained for CA9 , which is expressed in hypoxic regions . Proportion of tumour cells with CA9 staining is represented in bar chart .

O O O O O O O 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 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

HCT116 flank tumours produced by subcutaneous injection were established for 20 days before injection at three-day intervals with vehicle (PBS), or EVs isolated by UC from glutamine-replete or glutamine-depleted HCT116 cells. Tumours were excised 24 h after last of four injections for analysis. Panels show representative immunostained histological sections of tumour tissue quantified using the Visiopharm Integrator System. (D) Sections immunostained for CA9, which is expressed in hypoxic regions. Proportion of tumour cells with CA9 staining is represented in bar chart.


( D ) Western blot analysis of a strain expressing TurboID - HA - Emc6 , Bpl1 - AID * - 9myc and OsTIR1 which was grown overnight in SD media with or without auxin . The cells were then diluted into fresh SD media and grown to mid logarithmic phase ( ~ 4hours ) with or without auxin ( respectively ) . When required , biotin was added either 30min before harvesting , or for the entire 4hours . H3 ( histone H3 ) is used as a loading control .

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(D) Western blot analysis of a strain expressing TurboID-HA-Emc6, Bpl1-AID*-9myc and OsTIR1 which was grown overnight in SD media with or without auxin. The cells were then diluted into fresh SD media and grown to mid logarithmic phase (~4hours) with or without auxin (respectively). When required, biotin was added either 30min before harvesting, or for the entire 4hours. H3 (histone H3) is used as a loading control.


( B ) Volcano plots for each of the four distinct LC - MS / MS experiments highlighted in Figure 2A showing - log ( p - value ) against log2 ( fold - change ) for all proteins identified in the appropriate Emc6 / Sbh1 samples . Proteins enriched in the Emc6 samples are colour / pattern - coded as follows : dotted outline for HA - IP of BioID2 - HA - Emc6 ; blue fill for streptavidin - AP of BioID2 - HA - Emc6 ; black , solid outline for streptavidin - AP of TurboID - HA - Emc6 ; and yellow fill for streptavidin - AP of TurboID - HA - Emc6 / ABOLISH . Grey dots represent proteins that passed the p - value and fold - change criteria , but were only identified by one peptide . Only EMC complex components or proteins with EMC client features are named .

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(B) Volcano plots for each of the four distinct LC-MS/MS experiments highlighted in Figure 2A showing -log(p-value) against log2(fold-change) for all proteins identified in the appropriate Emc6/Sbh1 samples. Proteins enriched in the Emc6 samples are colour/pattern-coded as follows: dotted outline for HA-IP of BioID2-HA-Emc6; blue fill for streptavidin-AP of BioID2-HA-Emc6; black, solid outline for streptavidin-AP of TurboID-HA-Emc6; and yellow fill for streptavidin-AP of TurboID-HA-Emc6/ABOLISH. Grey dots represent proteins that passed the p-value and fold-change criteria, but were only identified by one peptide. Only EMC complex components or proteins with EMC client features are named.


( A ) Fluorescence microscopy images of control or Δemc3 strains containing either Alg1 N ' tagged with GFP , Gnp1 C ' tagged with GFP , Pdr12 N ' tagged with GFP or Pho84 C ' tagged with GFP . For the Gnp1 - GFP strain , the quantified signal depletion was ~ 10 % ( p < 0 . 0001 ) . Topological schematics feature beneath each respective image , with predicted TMDs highlighted in blue ( Weill et al , 2019 ) . All images are from two independent biological repeats and are digitally coloured using the ' fire ' lookup table ( LUT ) on Fiji . The same contrast settings were used for each image pair .

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(A) Fluorescence microscopy images of control or Δemc3 strains containing either Alg1 N' tagged with GFP, Gnp1 C' tagged with GFP, Pdr12 N' tagged with GFP or Pho84 C' tagged with GFP. For the Gnp1-GFP strain, the quantified signal depletion was ~10% (p < 0.0001). Topological schematics feature beneath each respective image, with predicted TMDs highlighted in blue (Weill et al, 2019). All images are from two independent biological repeats and are digitally coloured using the 'fire' lookup table (LUT) on Fiji. The same contrast settings were used for each image pair.


( C ) Streptavidin blots of diploid strains expressing either AviTag - Fks1 , - Gnp1 , - Pdr5 , - Pdr12 or - Stv1 ( as negative control ) together with BirA - Emc6 , grown in the conditions described in Figure 1E . For each strain , blots showing both the ' lower ' ( L ) and ' higher ' ( H ) contrast settings are shown side - by - side . The expected molecular weight in kDa for each tested protein including AviTag is written in parentheses after the protein name .

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(C) Streptavidin blots of diploid strains expressing either AviTag-Fks1, -Gnp1, -Pdr5, -Pdr12 or -Stv1 (as negative control) together with BirA-Emc6, grown in the conditions described in Figure 1E. For each strain, blots showing both the 'lower' (L) and 'higher' (H) contrast settings are shown side-by-side. The expected molecular weight in kDa for each tested protein including AviTag is written in parentheses after the protein name.


A . Protein lysates from the melanocytic - like melanoma cells 501mel , MM011 , MM074 and MM117 or the mesenchymal - like melanoma cells MM029 , MM047 and MM099 were immuno - blotted for proteins as indicated . Molecular mass of the proteins is indicated ( kDa ) .

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A. Protein lysates from the melanocytic-like melanoma cells 501mel, MM011, MM074 and MM117 or the mesenchymal-like melanoma cells MM029, MM047 and MM099 were immuno-blotted for proteins as indicated. Molecular mass of the proteins is indicated (kDa).


B . Melanoma cells were treated with increasing concentrations of THZ1 as indicated for 72h . Mean growth is shown relative to vehicle ( DMSO ) - treated cells . IC50 for each cell line is indicated . Melanocytic - type ( MITF - High , proliferative ) melanoma cells are shown in red , while mesenchymal - like ( MITF - low , invasive ) melanoma cells are shown in blue . Data information : data are presented as mean values + Standard Deviation ( SD ) for three replicates ( n = 3 ) .

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B. Melanoma cells were treated with increasing concentrations of THZ1 as indicated for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. IC50 for each cell line is indicated. Melanocytic-type (MITF-High, proliferative) melanoma cells are shown in red, while mesenchymal-like (MITF-low, invasive) melanoma cells are shown in blue. Data information: data are presented as mean values + Standard Deviation (SD) for three replicates (n=3).


D . Protein lysates from MM074 , MM074BRAFi - R or MM074CDK7i - R were immuno - blotted for indicated proteins . Molecular sizes of the proteins are indicated ( kDa ) . The numbers below the gel lanes represent relative protein level , which was determined from the band intensity using ImageJ software normalized to each relative Vinculin control .

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D. Protein lysates from MM074, MM074BRAFi-R or MM074CDK7i-R were immuno-blotted for indicated proteins. Molecular sizes of the proteins are indicated (kDa). The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software normalized to each relative Vinculin control.


B . Protein lysates from the indicated cells were immuno - blotted for the indicated proteins . Molecular sizes of the proteins are indicated in kDa . The numbers below the gel lanes represent relative protein level , which was determined from the band intensity using ImageJ software normalized relative to each relative ACTIN control .

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B. Protein lysates from the indicated cells were immuno-blotted for the indicated proteins. Molecular sizes of the proteins are indicated in kDa. The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software normalized relative to each relative ACTIN control.


E - H . qRT - PCR analysis showing average TBP - normalized expression of GATA6 ( E ) , AMIGO2 ( F ) , SERPINE1 ( G ) and ABCG2 ( H ) in the indicated cells treated with either siCTL or siGATA6 for 72h . Data information : data are presented as mean values + SD for six replicates ( n = 6 ) . The p - value ( Student ' s t - test ) is indicated , * * < 0 . 01 , * * * < 0 . 005 and ns = non -

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E-H. qRT-PCR analysis showing average TBP-normalized expression of GATA6 (E), AMIGO2 (F), SERPINE1 (G) and ABCG2 (H) in the indicated cells treated with either siCTL or siGATA6 for 72h. Data information: data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, **<0.01, ***<0.005 and ns=non-significant


J . MM029 ( left ) and MM099 ( right ) were pre - treated with either siCTL or siGATA6 for 48h and treated with increasing concentrations of THZ1 for 72h . Mean growth is shown relative to vehicle ( DMSO ) - treated cells . Data information : data are presented as mean values + SD for six replicates ( n = 6 ) . The p - value ( Student ' s t - test ) is indicated , * * < 0 . 01 , * * * < 0 . 005 and ns = non -

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

J. MM029 (left) and MM099 (right) were pre-treated with either siCTL or siGATA6 for 48h and treated with increasing concentrations of THZ1 for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. Data information: data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, **<0.01, ***<0.005 and ns=non-significant


C - D . MM099 ( c ) and MM029 ( d ) were pre - treated with either siCTL or siABCG2 as indicated and treated with increasing concentrations of THZ1 for 72h . Mean growth is shown relative to vehicle ( DMSO ) - treated cells . E - F . MM099 ( e ) and MM029 ( f ) were pre - treated with either siCTL or siABCG2 as indicated and treated with increasing doses of Vemurafenib for 72h . Mean growth is shown relative to vehicle ( DMSO ) - treated cells . D Data information : data are presented as mean values + SD for three replicates ( n = 3 ) . IC50 for each cell line is indicated .

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

C-D. MM099 (c) and MM029 (d) were pre-treated with either siCTL or siABCG2 as indicated and treated with increasing concentrations of THZ1 for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. E-F. MM099 (e) and MM029 (f) were pre-treated with either siCTL or siABCG2 as indicated and treated with increasing doses of Vemurafenib for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. D Data information: data are presented as mean values + SD for three replicates (n=3). IC50 for each cell line is indicated.


C - E . qRT - PCR analysis showing average TBP - normalized fold expression of MITF ( C ) , SOX10 ( D ) and GATA6 ( E ) in 501mel treated either with DMSO / THZ1 ( 50nM ) ( upper ) or DMSO / JQ1 ( 10μM ) ( lower ) for 24h . Data information : data are presented as mean values + SD for six replicates ( n = 6 ) . The p - value ( Student ' s t - test ) is indicated , * < 0 . 05 .

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

C-E. qRT-PCR analysis showing average TBP-normalized fold expression of MITF (C), SOX10 (D) and GATA6 (E) in 501mel treated either with DMSO/THZ1 (50nM) (upper) or DMSO/JQ1 (10μM) (lower) for 24h. Data information: data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, *<0.05.


A . qRT - PCR analysis showing average TBP - normalized fold expression of SOX10 , MITF and GATA6 in 501mel treated with either siCTL or siSOX10 for 48h . Data information : , data are presented as mean values + SD for six replicates ( n = 6 ) . The p - value ( Student ' s t - test ) is indicated , * < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. qRT-PCR analysis showing average TBP-normalized fold expression of SOX10, MITF and GATA6 in 501mel treated with either siCTL or siSOX10 for 48h. Data information: , data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, *<0.05.


D . MM099MITF - SOX10 - PAX3 expressing inducible MITF - SOX10 - PAX3 genes was treated or not with Doxycycline ( 1μg / ml ) for 24h and protein lysates were immuno - blotted for the indicated protein . The numbers below the gel lanes represent relative protein level , which was determined from the band intensity using ImageJ software , and normalized relative to VINCULIN .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. MM099MITF-SOX10-PAX3 expressing inducible MITF-SOX10-PAX3 genes was treated or not with Doxycycline (1μg/ml) for 24h and protein lysates were immuno-blotted for the indicated protein. The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software, and normalized relative to VINCULIN.


E . qRT - PCR analysis showing average TBP - normalized fold expression of GATA6 , ABCG2 , AMIGO2 or SERPINE1 in MM099MITF - SOX10 - PAX3 treated or not with Doxycycline ( 1μg / ml ) for 24h . Data information : data are presented as mean values + SD for three biological triplicates . The p - value ( Student ' s t - test ) is indicated , * < 0 . 05 , * * < 0 . 01 ; * * * < 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. qRT-PCR analysis showing average TBP-normalized fold expression of GATA6, ABCG2, AMIGO2 or SERPINE1 in MM099MITF-SOX10-PAX3 treated or not with Doxycycline (1μg/ml) for 24h. Data information: data are presented as mean values + SD for three biological triplicates. The p-value (Student's t-test) is indicated, *<0.05, **<0.01; ***<0.001.


A . Tumor sections were immuno - labelled ( IHC ) with anti - GATA6 ( red ) and anti - SOX10 ( green ) antibodies and images were captured by confocal microscopy at the indicated magnification . We analysed six tumor sections of metastases and observed significant GATA6 expression in only one of them . Scale bar 250 μM for 40x , 100μM for 100x .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Tumor sections were immuno-labelled (IHC) with anti-GATA6 (red) and anti-SOX10 (green) antibodies and images were captured by confocal microscopy at the indicated magnification. We analysed six tumor sections of metastases and observed significant GATA6 expression in only one of them. Scale bar 250 μM for 40x, 100μM for 100x.


D - E . Graphs showing the average expression of GATA6 ( d ) and MITF ( e ) ( RPKM ) for each phenotype cluster in T0 ( drug naïve ) ( blue ) , phases 1 - 2 ( MDR ) ( green and yellow ) and phase 3 ( drug - resistance ) ( red ) . Data information : data are presented as mean values + SEM ( n = 6 , 574 cells from 5 PDX ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E. Graphs showing the average expression of GATA6 (d) and MITF (e) (RPKM) for each phenotype cluster in T0 (drug naïve) (blue), phases 1-2 (MDR) (green and yellow) and phase 3 (drug-resistance) (red). Data information: data are presented as mean values + SEM (n=6,574 cells from 5 PDX).


( A ) Confocal images of representative eye cryosections immunostained with anti - Lamp1 antibody from 2 - month - old miR - 211 - / - and control mice sacrificed 3h after light on , at 10 AM ( diurnal condition ) . Enlarged boxes highlight Lamp1 - positive structures ( white arrowheads ) in the RPE . Autofluorescence from lipofuscin granules are visible in all cryosections of miR - 211 - / - compared to control littermates . Nuclei were counterstained with DAPI ( blue ) . At least n = 6 mice per group . Scale bar 10 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer nuclear layer .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images of representative eye cryosections immunostained with anti-Lamp1 antibody from 2-month-old miR-211-/- and control mice sacrificed 3h after light on, at 10 AM (diurnal condition). Enlarged boxes highlight Lamp1-positive structures (white arrowheads) in the RPE. Autofluorescence from lipofuscin granules are visible in all cryosections of miR-211-/- compared to control littermates. Nuclei were counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 10 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer.


( B ) RPE were isolated at 10 AM ( diurnal condition ) from 2 - month - old WT and miR - 211 - / - mice . Representative Western blot analysis of the Lamp1 , Cln5 , Trpml1 , Sqstm1 / p62 , Cathepsin D ( CtsD ) and LC3 proteins from WT and miR - 211 - / - mice . Note a decrease of both proCtsD and its maturation CtsD heavy chain ( hc ) . The plots show the quantification of the indicated proteins normalized to the Gapdh loading control . Bar graphs represent mean values ± s . e . m . of independent experiments ( n = 6 mice ) Mann and Whitney test ( miR - 211 - / - vs WT ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 . Data information : ( RPE ) Retinal Pigment

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RPE were isolated at 10 AM (diurnal condition) from 2-month-old WT and miR-211-/- mice. Representative Western blot analysis of the Lamp1, Cln5, Trpml1, Sqstm1/p62, Cathepsin D (CtsD) and LC3 proteins from WT and miR-211-/- mice. Note a decrease of both proCtsD and its maturation CtsD heavy chain (hc). The plots show the quantification of the indicated proteins normalized to the Gapdh loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=6 mice) Mann and Whitney test (miR-211-/- vs WT), *p ≤ 0.05, **p ≤ 0.01. Data information: (RPE) Retinal Pigment Epithelium


( C ) Cathepsin B ( CtsB ) activity in RPE lysates from 2 - month - old miR - 211 - / - and control mice sacrificed 3h after light on at 10 AM ( diurnal condition ) . CtsB was reduced in miR - 211 - / - compared to control mice . Bar graphs represent percentage of CtsB activity ± s . e . m . of independent experiments ( n = 3 mice ) Mann and Whitney test ( miR - 211 - / - vs WT ) , * p ≤ 0 . 05 . Data information : ( RPE ) Retinal Pigment

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cathepsin B (CtsB) activity in RPE lysates from 2-month-old miR-211-/- and control mice sacrificed 3h after light on at 10 AM (diurnal condition). CtsB was reduced in miR-211-/- compared to control mice. Bar graphs represent percentage of CtsB activity ± s.e.m. of independent experiments (n=3 mice) Mann and Whitney test (miR-211-/- vs WT), *p ≤ 0.05. Data information: (RPE) Retinal Pigment Epithelium


( D ) Representative images of conventional TEM analysis of RPE of both 2 - month - old and 3 - month - old WT and miR - 211 - / - mice . miR - 211 - / - mice show accumulation of phagolysosomes ( red arrows ) in the RPE . Scale bar 2 μm . Enlarged boxes highlight phagolysosome - like structures containing poorly processed POS . Scale bar 1 . 5 μm . Representative images of conventional TEM analysis of RPE shown at higher magnification also highlighted accumulation of lipofuscin - like granules ( blue arrows ) in 3 - month - old miR - 211 - / - compared to control WT mice . Melanosomes are indicated by white asterisks . Scale bar 500 nm ( n = 3 mice ) . ( E ) Graphs showing number of lipofuscin - like granules ( n / 100 μm2 ) from the RPE of 3 - month - old miR - 211 - / - mice as in ( D ) . Bar graphs represent mean values ± s . e . m . Mann and Whitney test ( miR - 211 - / - vs WT ) * * * p ≤ 0 . 005 ( n = 6 mice ) . Data information : ( RPE ) Retinal Pigment

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of conventional TEM analysis of RPE of both 2-month-old and 3-month-old WT and miR-211-/- mice. miR-211-/- mice show accumulation of phagolysosomes (red arrows) in the RPE. Scale bar 2 μm. Enlarged boxes highlight phagolysosome-like structures containing poorly processed POS. Scale bar 1.5 μm. Representative images of conventional TEM analysis of RPE shown at higher magnification also highlighted accumulation of lipofuscin-like granules (blue arrows) in 3-month-old miR-211-/- compared to control WT mice. Melanosomes are indicated by white asterisks. Scale bar 500 nm (n=3 mice). (E) Graphs showing number of lipofuscin-like granules (n/100 μm2) from the RPE of 3-month-old miR-211-/- mice as in (D). Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- vs WT) ***p ≤ 0.005 (n=6 mice). Data information: (RPE) Retinal Pigment Epithelium


( A ) Representative confocal images of eye cryosections immunostained with anti - Ezrin ( green ) and anti - ZO - 1 ( red ) antibodies from 2 - month - old WT mice sacrificed 3h after light on at 10 AM ( diurnal condition - LIGHT ) and 3h after light off at 10 PM ( nocturnal condition - DARK ) , nuclei were counterstained with DAPI ( blue ) . Enlarged boxes highlight Ezrin staining in both basal infoldings ( BI ) and apical extension ( AE ) of the RPE . At least n = 6 mice per group . Scale bar 10 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer nuclear layer ; ( BI ) RPE basal infoldings ( AE ) RPE apical extensions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative confocal images of eye cryosections immunostained with anti-Ezrin (green) and anti-ZO-1 (red) antibodies from 2-month-old WT mice sacrificed 3h after light on at 10 AM (diurnal condition - LIGHT) and 3h after light off at 10 PM (nocturnal condition - DARK), nuclei were counterstained with DAPI (blue). Enlarged boxes highlight Ezrin staining in both basal infoldings (BI) and apical extension (AE) of the RPE. At least n = 6 mice per group. Scale bar 10 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer; (BI) RPE basal infoldings (AE) RPE apical extensions.


( B ) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2 - month - old WT mice during dark / light and light / dark transitions . Mice were sacrificed at determined time points as indicated on the X axis . Scheme at the top describes light / dark adaptation regime . Values normalized to Hprt , represent means ± s . e . m . Mann and Whitney test * p ≤ 0 . 05 ( n = 3 mice for each time point ) . Values from 12h dark - adapted mice ( T0 ) were set to one .

(B) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2-month-old WT mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated on the X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m. Mann and Whitney test *p ≤ 0.05 (n=3 mice for each time point). Values from 12h dark-adapted mice (T0) were set to one.


( C ) Confocal images of representative eye cryosections immunostained with anti - Ezrin ( green ) and anti - ZO - 1 ( red ) antibodies from 2 - month - old miR - 211 - / - and WT control mice sacrificed 3h after light on at 10 AM ( diurnal condition ) . Enlarged boxes highlight Ezrin staining in both basal infoldings ( BI ) and apical extension ( AE ) of the RPE . At least n = 6 mice per group . Scale bar 10 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer nuclear layer ; ( BI ) RPE basal infoldings ( AE ) RPE apical extensions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images of representative eye cryosections immunostained with anti-Ezrin (green) and anti-ZO-1 (red) antibodies from 2-month-old miR-211-/- and WT control mice sacrificed 3h after light on at 10 AM (diurnal condition). Enlarged boxes highlight Ezrin staining in both basal infoldings (BI) and apical extension (AE) of the RPE. At least n = 6 mice per group. Scale bar 10 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer; (BI) RPE basal infoldings (AE) RPE apical extensions.


( D ) qRT - PCR assay for Ezrin from RPE 2 - month - old WT and miR - 211 - / - mice sacrificed 3h after light on at 10 AM . The graph shows the expression level of Ezrin normalized to Hprt . Bar graphs represent mean values ± s . e . m . Mann and Whitney test ( miR - 211 - / - vs WT ) , * * * p ≤ 0 . 005 ( n = 6 mice ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qRT-PCR assay for Ezrin from RPE 2-month-old WT and miR-211-/- mice sacrificed 3h after light on at 10 AM. The graph shows the expression level of Ezrin normalized to Hprt. Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- vs WT), ***p ≤ 0.005 (n=6 mice).


( E ) RPE were isolated at 10 AM ( diurnal condition ) from 2 - month - old WT and miR - 211 - / - mice . Representative image at low exposure of Western blot analysis of the Ezrin protein from WT and miR - 211 - / - mice . The graph shows the quantification of Ezrin normalized to the Gapdh loading control . Bar graphs represent mean values ± s . e . m . of independent experiments ( n = 5 mice ) Mann and Whitney test ( miR - 211 - / - vs WT ) , * * p ≤ 0 . 01 .

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

(E) RPE were isolated at 10 AM (diurnal condition) from 2-month-old WT and miR-211-/- mice. Representative image at low exposure of Western blot analysis of the Ezrin protein from WT and miR-211-/- mice. The graph shows the quantification of Ezrin normalized to the Gapdh loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=5 mice) Mann and Whitney test (miR-211-/- vs WT), **p ≤ 0.01.


( F ) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2 - month - old miR - 211 - / - mice during dark / light and light / dark transitions . Mice were sacrificed at determined time points as indicated in X axis . Scheme at the top describes light / dark adaptation regime . Values normalized to Hprt , represent means ± s . e . m . ( n = 3 mice for each time point ) . Values from 12h dark adapted mice ( T0 ) were set to one . ( G ) Graph representing the kinetic expression pattern of Trpm1 levels in the RPE from 2 - month - old WT mice during dark / light and light / dark transitions . Mice were sacrificed at determined time points as indicated in X axis . Scheme at the top describes light / dark adaptation regime . Values normalized to Hprt , represent means ± s . e . m ( n = 3 mice for each time point ) . Values from 12h dark adapted mice ( T0 ) were set to one .

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

(F) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2-month-old miR-211-/- mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated in X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m. (n=3 mice for each time point). Values from 12h dark adapted mice (T0) were set to one. (G) Graph representing the kinetic expression pattern of Trpm1 levels in the RPE from 2-month-old WT mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated in X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m (n=3 mice for each time point). Values from 12h dark adapted mice (T0) were set to one.


( H ) qRT - PCR assay for Mtmr10 and Klf13 from RPE 2 - month - old WT and miR - 211 - / - mice sacrificed 3h after on at 10 AM . The graph shows the expression level of these genes normalized to the Hprt . Bar graphs represent mean values ± s . e . m . ( n = 3 mice )

(H) qRT-PCR assay for Mtmr10 and Klf13 from RPE 2-month-old WT and miR-211-/- mice sacrificed 3h after on at 10 AM. The graph shows the expression level of these genes normalized to the Hprt. Bar graphs represent mean values ± s.e.m. (n=3 mice)


( A ) Representative images from ARPE - 19 cells treated with DMSO and NSC668394 . All cells were fixed and stained with anti - LC3 ( green ) and anti - LAMP1 ( red ) antibodies and nuclei were counterstained with DAPI ( blue ) . Scale bar 5 μm . ( B ) The plot shows the quantification of the numbers of LC3 / LAMP1 - positive vesicles per cell ( n ≥ 100 ) . Values represent means ± s . e . m from 3 independent experiments . Student ' s t - test ( NSC668394 vs DMSO ) * * * p ≤ 0 . 005 .

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

(A) Representative images from ARPE-19 cells treated with DMSO and NSC668394. All cells were fixed and stained with anti-LC3 (green) and anti-LAMP1 (red) antibodies and nuclei were counterstained with DAPI (blue). Scale bar 5 μm. (B) The plot shows the quantification of the numbers of LC3/LAMP1-positive vesicles per cell (n ≥ 100). Values represent means ± s.e.m from 3 independent experiments. Student's t-test (NSC668394 vs DMSO) ***p ≤ 0.005.


( C ) Representative Western blots of LAMP1 , EZRIN , EZRIN - pT567 , CLN5 , TRPML1 , SQSTM1 / p62 , CTSD and LC3 proteins from DMSO or NSC668394 treated cells . Note an increase of both proCTSD and its maturation CTSD heavy chain ( hc ) . The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control . Bar graphs represent mean values ± s . e . m . of at least n = 6 independent experiments . Mann and Whitney test ( NSC668394 vs DMSO ) * p ≤ 0 . 05 , * * * p ≤ 0 . 005 .

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

(C) Representative Western blots of LAMP1, EZRIN, EZRIN-pT567, CLN5, TRPML1, SQSTM1/p62, CTSD and LC3 proteins from DMSO or NSC668394 treated cells. Note an increase of both proCTSD and its maturation CTSD heavy chain (hc). The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control. Bar graphs represent mean values ± s.e.m. of at least n = 6 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05, ***p ≤ 0.005.


( D ) Representative images of RFP - GFP - LC3 assay in ARPE - 19 cells transiently transfected with RFP - GFP - LC3 and treated with DMSO or NSC668394 . Nuclei were counterstained with DAPI ( blue ) . Scale bars : 5 μm . ( E ) Box plots showing quantitative analysis of RFP + GFP + puncta ( Autophagosome ) and RFP + GFP - puncta ( Autolysosome ) ( n ≥ 100 cells ) from 3 independent experiments . Box limits represent 25th percentile and 75th percentile ; horizontal lines represent medians ; whiskers display min . to max . values . Student ' s t - test ( NSC668394 vs DMSO ) * p ≤ 0 . 05 .

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

(D) Representative images of RFP-GFP-LC3 assay in ARPE-19 cells transiently transfected with RFP-GFP-LC3 and treated with DMSO or NSC668394. Nuclei were counterstained with DAPI (blue). Scale bars: 5 μm. (E) Box plots showing quantitative analysis of RFP+GFP+ puncta (Autophagosome) and RFP+GFP- puncta (Autolysosome ) (n ≥ 100 cells) from 3 independent experiments. Box limits represent 25th percentile and 75th percentile; horizontal lines represent medians; whiskers display min. to max. values. Student's t-test (NSC668394 vs DMSO) *p ≤ 0.05.


( F ) Endolysosomal Cathepsin activity was revealed by incubation with cathepsin B ( CTSB ) Magic Red substrate for 20 min and images of the living cell collected on the HC analysis . Scale bar 10 μm . ( G ) Graph showing the mean values of intensity of cresyl violet fluorescence ± s . e . m . of representative data ( n ≥ 100 cells ) from 3 independent experiments . Mann and Whitney test ( NSC668394 vs DMSO ) * p ≤ 0 . 05 .

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

(F) Endolysosomal Cathepsin activity was revealed by incubation with cathepsin B (CTSB) Magic Red substrate for 20 min and images of the living cell collected on the HC analysis. Scale bar 10 μm. (G) Graph showing the mean values of intensity of cresyl violet fluorescence ± s.e.m. of representative data (n ≥ 100 cells) from 3 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05.


( H ) Cathepsin B ( CTSB ) activity in lysates from NSC668394 - treated and DMSO - control ARPE - 19 cells . Graph showing the percentage of values ± s . e . m . of CTSB activity from 4 independent experiments . Mann and Whitney test ( NSC668394 vs DMSO ) * p ≤ 0 . 05 .

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

(H) Cathepsin B (CTSB) activity in lysates from NSC668394-treated and DMSO-control ARPE-19 cells. Graph showing the percentage of values ± s.e.m. of CTSB activity from 4 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05.


( A ) Representative images from HC assay of HeLaTFEB - GFP cells transfected with control ( siCTRL ) or siPPP3CB , and subjected to the indicated conditions . Analysis of TFEB nuclear translocation in HeLaTFEB - GFP cells transfected with siCTRL or siPPP3CB and treated with DMSO or NSC668394 , silenced for EZRIN ( siEZR ) or serum - starved ( stv ) . Both pharmacological inhibition and silencing of Ezrin induced TFEB nuclear localization in stable HeLaTFEB - GFP cells . Starvation ( stv ) was used as control . Nuclear translocation of TFEB in stable HeLaTFEB - GFP cells subjected to the indicated conditions is rescue after silencing of PPP3CB . Scale bar 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O 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) Representative images from HC assay of HeLaTFEB-GFP cells transfected with control (siCTRL) or siPPP3CB, and subjected to the indicated conditions. Analysis of TFEB nuclear translocation in HeLaTFEB-GFP cells transfected with siCTRL or siPPP3CB and treated with DMSO or NSC668394, silenced for EZRIN (siEZR) or serum-starved (stv). Both pharmacological inhibition and silencing of Ezrin induced TFEB nuclear localization in stable HeLaTFEB-GFP cells. Starvation (stv) was used as control. Nuclear translocation of TFEB in stable HeLaTFEB-GFP cells subjected to the indicated conditions is rescue after silencing of PPP3CB. Scale bar 5 μm.


( B ) The graph shows the mean ± s . e . m . of the percentage of nuclear TFEB translocation in Ezrin - inhibited cells compared to control ; n = 3 independent experiments were performed . Mann and Whitney test ( NSC668394 and stv vs DMSO ; siEZR vs siCTRL ) * p ≤ 0 . 05 .

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

(B) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells compared to control; n=3 independent experiments were performed. Mann and Whitney test (NSC668394 and stv vs DMSO; siEZR vs siCTRL) *p ≤ 0.05.


( C ) The graph shows the mean ± s . e . m . of the percentage of nuclear TFEB translocation in Ezrin - inhibited cells and subjected to the silencing of PPP3CB . At least 3 independent experiments were performed . Mann and Whitney test ( siPPP3CB vs siCTRL ) * p ≤ 0 . 05 .

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(C) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells and subjected to the silencing of PPP3CB. At least 3 independent experiments were performed. Mann and Whitney test (siPPP3CB vs siCTRL) *p ≤ 0.05.


( D ) qRT - PCR assay for PPP3CB from HeLaTFEB - GFP cells transfected with siPPP3CB and siCTRL . The graph shows the reduction in PPP3CB expression level normalized to the Hprt . Bar graphs represent mean values ± s . e . m . Mann and Whitney test ( siPPP3CB vs siCTRL ) , * * * p ≤ 0 . 005 ( n = 6 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 O O O O O O O O O O O O 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) qRT-PCR assay for PPP3CB from HeLaTFEB-GFP cells transfected with siPPP3CB and siCTRL. The graph shows the reduction in PPP3CB expression level normalized to the Hprt. Bar graphs represent mean values ± s.e.m. Mann and Whitney test (siPPP3CB vs siCTRL), ***p ≤ 0.005 (n=6 independent experiments).


( E ) Both pharmacological inhibition and silencing of EZRIN induce downshift of endogenous TFEB electrophoretic mobility in Western blot analysis . Torin treatment was used as control .

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(E) Both pharmacological inhibition and silencing of EZRIN induce downshift of endogenous TFEB electrophoretic mobility in Western blot analysis. Torin treatment was used as control.


( F ) Representative traces of ML1 - GCaMP3 normalized fluorescence recorded in transiently transfected ARPE - 19 cells . During time - lapse recording , cells were stimulated with ML - SA1 ( 20 µM ) after addition ( arrowhead ) of DMSO or NSC668394 . Where indicated , the specific ML1 inhibitor ML - SI3 ( 10 µM ) was added . Bar graph reports the mean values ± s . e . m . of the time required by fluorescence to decay to half of the ML - SA1 - induced peak . n = 50 cells from 3 independent experiments . * * * p < 0 . 005 by Mann and Whitney test .

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

(F) Representative traces of ML1-GCaMP3 normalized fluorescence recorded in transiently transfected ARPE-19 cells. During time-lapse recording, cells were stimulated with ML-SA1 (20 µM) after addition (arrowhead) of DMSO or NSC668394. Where indicated, the specific ML1 inhibitor ML-SI3 (10 µM) was added. Bar graph reports the mean values ± s.e.m. of the time required by fluorescence to decay to half of the ML-SA1-induced peak. n = 50 cells from 3 independent experiments. ***p<0.005 by Mann and Whitney test.


( G ) Nuclear translocation of TFEB in stable HeLaTFEB - GFP cells subjected to the indicated conditions is reduced after Ca2 + chelator BAPTA treatment . Scale bar 5 μm .

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

(G) Nuclear translocation of TFEB in stable HeLaTFEB-GFP cells subjected to the indicated conditions is reduced after Ca2+ chelator BAPTA treatment. Scale bar 5 μm.


H ) The graph shows the mean ± s . e . m . of the percentage of nuclear TFEB translocation in Ezrin - inhibited cells compared with DMSO under Ca2 + chelator BAPTA treatment . At least 3 independent experiments were performed . Mann and Whitney test ( stv , stv + BAPTA , BAPTA , NSC668394 , NSC668394 + BAPTA vs DMSO ) * p ≤ 0 . 05 .

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

H) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells compared with DMSO under Ca2+ chelator BAPTA treatment. At least 3 independent experiments were performed. Mann and Whitney test (stv, stv+BAPTA, BAPTA, NSC668394, NSC668394+BAPTA vs DMSO) *p ≤ 0.05.


( I ) qRT - PCR analysis for TFEB target genes ( TRPML1 , BECLIN , MAPLC3B and LAMP1 ) was performed on ARPE - 19 cells treated with DMSO or NSC668394 . Bar graphs represent mean values ± s . e . m . of at least 3 independent experiments . Mann and Whitney * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 005 .

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

(I) qRT-PCR analysis for TFEB target genes (TRPML1, BECLIN, MAPLC3B and LAMP1) was performed on ARPE-19 cells treated with DMSO or NSC668394. Bar graphs represent mean values ± s.e.m. of at least 3 independent experiments. Mann and Whitney *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005.


( A ) Confocal images of representative eye cryosections immunostained with anti - Ezrin ( green ) / anti - ZO - 1 ( red ) , anti - Ezrin - pT567 ( green ) / anti - ZO - 1 ( red ) , anti - Lamp1 ( green ) and anti - Sqstm1 / p62 ( green ) antibodies from 6 - month - old miR - 211 - / - sacrificed five months after DMSO or NSC668394 treatment . Nuclei are counterstained with DAPI ( blue ) . At least n = 6 mice per group . Scale bar 100 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer nuclear layer .

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

(A) Confocal images of representative eye cryosections immunostained with anti-Ezrin (green) /anti-ZO-1 (red), anti-Ezrin-pT567 (green) /anti-ZO-1 (red), anti-Lamp1 (green) and anti-Sqstm1/p62 (green) antibodies from 6-month-old miR-211-/- sacrificed five months after DMSO or NSC668394 treatment. Nuclei are counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 100 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer.


( B ) RPE was isolated 5 months after DMSO or NSC668394 treatment from 6 - month - old miR - 211 - / - mice . Representative Western blots proteins from these tissues were performed to determine the expression levels of Lamp1 , Ezrin , Ezrin - pT567 , Sqstm1 / p62 and LC3 . The plots show the quantification of the indicated proteins normalized to the β - Actin loading control . Bar graphs represent mean values ± s . e . m . of independent experiments ( n = 6 mice ) . Mann and Whitney test ( DMSO vs WT ; NSC668394 vs DMSO ) , * p ≤ 0 . 05 , * * * p ≤ 0 . 005 .

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

(B) RPE was isolated 5 months after DMSO or NSC668394 treatment from 6-month-old miR-211-/- mice. Representative Western blots proteins from these tissues were performed to determine the expression levels of Lamp1, Ezrin, Ezrin-pT567, Sqstm1/p62 and LC3. The plots show the quantification of the indicated proteins normalized to the β-Actin loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=6 mice). Mann and Whitney test (DMSO vs WT; NSC668394 vs DMSO), *p ≤ 0.05, ***p≤0.005.


( C ) Cathepsin B ( CtsB ) activity in RPE lysates from miR - 211 - / - and WT mice sacrificed 3h after light on at 10 AM ( diurnal condition ) . RPE was isolated 1 week after DMSO or NSC668394 treatment from 2 - month - old miR - 211 - / - mice . CtsB was rescued in NSC668394 - treated miR - 211 - / - compared to DMSO - control miR - 211 - / - mice . Bar graphs represent percentage of CtsB activity ± s . e . m . of independent experiments ( n = 3 mice ) Mann and Whitney test ( DMSO - miR - 211 - / - vs WT ; NSC668394 - miR - 211 - / - vs DMSO - miR - 211 - / - ) , * p ≤ 0 . 05 . ( D ) GUSB activity from retina from miR - 211 - / - and control mice sacrificed 3h after light on at 10 AM ( diurnal condition ) . Retina was isolated 1 week after DMSO or NSC668394 treatment from 2 - month - old miR - 211 - / - mice . GUSB was rescued in NSC668394 - treated miR - 211 - / - compared to DMSO - control miR - 211 - / - mice . Bar graphs represent percentage of GUSB activity ± s . e . m . of independent experiments ( n = 3 mice ) . Mann and Whitney test ( DMSO - miR - 211 - / - vs WT ; NSC668394 - miR - 211 - / - vs DMSO - miR - 211 - / - ) .

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

(C) Cathepsin B (CtsB) activity in RPE lysates from miR-211-/- and WT mice sacrificed 3h after light on at 10 AM (diurnal condition). RPE was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. CtsB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of CtsB activity ± s.e.m. of independent experiments (n=3 mice) Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-), *p ≤ 0.05. (D) GUSB activity from retina from miR-211-/- and control mice sacrificed 3h after light on at 10 AM (diurnal condition). Retina was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. GUSB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of GUSB activity ± s.e.m. of independent experiments (n=3 mice). Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-).


( A ) Representative images of conventional TEM analysis of RPE of WT , DMSO - treated miR - 211 - / - mice and NSC668394 - treated miR - 211 - / - three - month - old mice . NSC668394 - treated miR - 211 - / - mice show rescue of accumulation of double - membrane phagolysosome - like structures containing poorly processed POS ( red arrows ) compared to DMSO - treated miR - 211 - / - mice . Scale bar 1 mm . Data information : ( OS ) outer segment ( RPE ) Retinal Pigment Epithelium .

O 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 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

(A) Representative images of conventional TEM analysis of RPE of WT, DMSO-treated miR-211-/- mice and NSC668394-treated miR-211-/- three-month-old mice. NSC668394-treated miR-211-/- mice show rescue of accumulation of double-membrane phagolysosome-like structures containing poorly processed POS (red arrows) compared to DMSO-treated miR-211-/- mice. Scale bar 1 mm. Data information: (OS) outer segment (RPE) Retinal Pigment Epithelium.


( B ) Confocal images of representative eye cryosections immunostained with anti - Rhodopsin ( green ) antibody from WT and miR - 211 - / - mice at three months of age after DMSO or NSC668394 treatment , which are sacrificed 3h after light on at 10 AM ( diurnal condition ) . Nuclei are counterstained with DAPI ( blue ) . At least n = 6 mice per group . Scale bar 100 μm . The enlarged box highlights rhodopsin accumulation in the RPE ( green spots ) . Scale bar 5 μm . Autofluorescence from lipofuscin granules from WT and miR - 211 - / - mice at three months of age after DMSO or NSC668394 treatment . Representative images of retina cryosections immunostained with anti - Cone Arrestin ( green ) antibody from WT and miR - 211 - / - mice at three months of age after DMSO or NSC668394 treatment . Nuclei are counterstained with DAPI ( blue ) . N = at least 6 mice per group . Scale bar 100 μm . Data information : ( OS ) outer segment ; ( ONL ) outer nuclear layer ; ( RPE ) Retinal Pigment Epithelium .

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

(B) Confocal images of representative eye cryosections immunostained with anti-Rhodopsin (green) antibody from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment, which are sacrificed 3h after light on at 10 AM (diurnal condition). Nuclei are counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 100 μm. The enlarged box highlights rhodopsin accumulation in the RPE (green spots). Scale bar 5 μm. Autofluorescence from lipofuscin granules from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment. Representative images of retina cryosections immunostained with anti-Cone Arrestin (green) antibody from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment. Nuclei are counterstained with DAPI (blue). N = at least 6 mice per group. Scale bar 100 μm. Data information: (OS) outer segment; (ONL) outer nuclear layer; (RPE) Retinal Pigment Epithelium.


( C ) Graphs showing number of lipofuscin granules from the RPE of mice as in ( B ) . Bar graphs represent mean values ± s . e . m . Mann and Whitney test ( miR - 211 - / - DMSO vs WT and miR - 211 - / - NSC668394 vs DMSO treated mice ) * * * p ≤ 0 . 005 . * p ≤ 0 . 05 ( n = 6 mice ) .

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(C) Graphs showing number of lipofuscin granules from the RPE of mice as in (B). Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- DMSO vs WT and miR-211-/- NSC668394 vs DMSO treated mice) ***p ≤ 0.005. *p ≤ 0.05 (n=6 mice).


( D ) Graphs show cone percentage ( cones / area ) from the retina of mice treated as in ( B ) . Error bars represent s . e . m . Mann and Whitney test ( DMSO vs WT and NSC668394 vs DMSO treated mice ) * * * p ≤ 0 . 005 ( n = 6 mice ) .

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

(D) Graphs show cone percentage (cones/area) from the retina of mice treated as in (B). Error bars represent s.e.m. Mann and Whitney test (DMSO vs WT and NSC668394 vs DMSO treated mice) ***p ≤ 0.005 (n=6 mice).


( E ) Representative flicker traces at three months of age show the rescue of flicker responses of NSC668394 - treated miR - 211 - / - mice ( green lines ) compared to DMSO - treated miR - 211 - / - control mice ( red lines ) . WT mice were used as a control ( black lines ) . Flicker recordings were performed with light intensities ranging from 10 − 4 to 15 cd s / m2 in steps of 0 . 6 logarithmic units at 6 Hz frequency . ( F ) Flicker responses , plotted as a function of stimulus intensity , from WT ( black lines ) , DMSO - treated miR - 211 - / - ( red lines ) and NSC668394 - treated miR - 211 - / - ( green lines ) mice , at three months of age . The amplitude of the recordings from NSC668394 miR - 211 - / - treated mice is significantly rescued compared to DMSO - treated miR - 211 - / - mice . WT mice were used as a control . Error bars represent s . e . m . ANOVA test ( DMSO vs WT and NSC668394 vs DMSO treated mice ) * * p ≤ 0 . 01 , * * * p ≤ 0 . 005 .

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

(E) Representative flicker traces at three months of age show the rescue of flicker responses of NSC668394-treated miR-211-/- mice (green lines) compared to DMSO-treated miR-211-/- control mice (red lines). WT mice were used as a control (black lines). Flicker recordings were performed with light intensities ranging from 10−4 to 15 cd s/m2 in steps of 0.6 logarithmic units at 6 Hz frequency. (F) Flicker responses, plotted as a function of stimulus intensity, from WT (black lines), DMSO-treated miR-211-/- (red lines) and NSC668394-treated miR-211-/- (green lines) mice, at three months of age. The amplitude of the recordings from NSC668394 miR-211-/- treated mice is significantly rescued compared to DMSO-treated miR-211-/- mice. WT mice were used as a control. Error bars represent s.e.m. ANOVA test (DMSO vs WT and NSC668394 vs DMSO treated mice) **p ≤ 0.01, ***p ≤ 0.005.


( B ) Met - Phe formation monitored upon rapidly mixing initiation complexes ( 80S IC ) with ternary complexes eEF1A - GTP - [ 14C ] Phe - tRNAPhe in the absence ( orange , 0 . 13 ± 0 . 02 s - 1 ) or presence ( cyan , 0 . 15 ± 0 . 02 s - 1 ) of eEF3 ( 2 µM ) in a quench - flow apparatus , and the extent of peptide formation was analyzed by HPLC and radioactivity counting . Data are normalized to Met - Phe formation in the absence of eEF3 with the maximum value in the dataset set to 1 . Data are presented as mean ± SEM of n = 3 biological replicates . ( C ) Met - Phe - Val formation monitored upon rapidly mixing 80S complexes carrying MetPhe - tRNAPhe ( 80S 2C ) with ternary complexes eEF1A - GTP - [ 14C ] Val - tRNAVal in the presence of eEF2 and eEF3 ( green , 0 . 15 ± 0 . 01 s - 1 ) , eEF2 ( red ) or eEF3 ( blue ) . Data presented as mean ± SEM of n = 3 biological replicates . ( D ) Met - Phe - Val formation monitored with 80S 2C incubated with eEF2 and eEF3 ( green , 0 . 4 ± 0 . 14 s - 1 ) , eEF2 ( red , 1 . 0 ± 0 . 5 s - 1 ) or eEF3 ( blue ) , or in the absence of eEF2 and eEF3 ( brown ) for 15 min before adding with ternary complexes eEF1A - GTP - [ 14C ] Val - tRNAVal and monitoring the kinetics of Val incorporation . Data presented as mean ± SEM of n = 3 . Data information : Datasets in ( C ) and ( D ) are normalized to Met - Phe - Val formation in presence of eEF2 and eEF3 with the maximum value set to 1 .

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

(B) Met-Phe formation monitored upon rapidly mixing initiation complexes (80S IC) with ternary complexes eEF1A-GTP-[14C]Phe-tRNAPhe in the absence (orange, 0.13±0.02 s-1) or presence (cyan, 0.15±0.02 s-1) of eEF3 (2 µM) in a quench-flow apparatus, and the extent of peptide formation was analyzed by HPLC and radioactivity counting. Data are normalized to Met-Phe formation in the absence of eEF3 with the maximum value in the dataset set to 1. Data are presented as mean ± SEM of n = 3 biological replicates. (C) Met-Phe-Val formation monitored upon rapidly mixing 80S complexes carrying MetPhe-tRNAPhe (80S 2C) with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal in the presence of eEF2 and eEF3 (green, 0.15±0.01 s-1), eEF2 (red) or eEF3 (blue). Data presented as mean ± SEM of n = 3 biological replicates. (D) Met-Phe-Val formation monitored with 80S 2C incubated with eEF2 and eEF3 (green, 0.4±0.14 s-1), eEF2 (red, 1.0±0.5 s-1) or eEF3 (blue), or in the absence of eEF2 and eEF3 (brown) for 15 min before adding with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal and monitoring the kinetics of Val incorporation. Data presented as mean ± SEM of n = 3. Data information: Datasets in (C) and (D) are normalized to Met-Phe-Val formation in presence of eEF2 and eEF3 with the maximum value set to 1.


( B ) mRNA - tRNA translocation monitored in a time - resolved Pmn assay upon mixing 80S 2C with Pmn in the absence ( brown triangles ) or presence of eEF2 and eEF3 ( green , 0 . 13 ± 0 . 01 min - 1 ) , eEF2 ( red , 0 . 030 ± 0 . 005 min - 1 ) or eEF3 ( blue , 0 . 10 ± 0 . 01 min - 1 ) . Data are normalized to Met - Phe - Pmn formation in presence of eEF2 with the maximum value set to 1 . Data presented as mean ± SEM of n = 3 .

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 B-MEASURED_VAR 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

(B) mRNA-tRNA translocation monitored in a time-resolved Pmn assay upon mixing 80S 2C with Pmn in the absence (brown triangles) or presence of eEF2 and eEF3 (green, 0.13±0.01 min-1), eEF2 (red, 0.030±0.005 min-1) or eEF3 (blue, 0.10±0.01 min-1). Data are normalized to Met-Phe-Pmn formation in presence of eEF2 with the maximum value set to 1. Data presented as mean ± SEM of n = 3.


( C ) Dissociation of deacylated tRNA from the E site by the fluorescence change of tRNAfMet ( Flu ) upon rapidly mixing of 80S 2C with buffer ( grey ) , or with ternary complexes eEF1A - GTP - [ 14C ] Val - tRNAVal in the presence of eEF2 and eEF3 ( green ) , eEF2 ( red ) , eEF3 ( blue ) , or eEF2 and eEF3 without the ternary complex ( magenta ) in a stopped - flow apparatus . Each trace is an average of 5 - 7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction .

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 B-CONTROLLED_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 O O O O O O O O O O O O O O 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) Dissociation of deacylated tRNA from the E site by the fluorescence change of tRNAfMet(Flu) upon rapidly mixing of 80S 2C with buffer (grey), or with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal in the presence of eEF2 and eEF3 (green), eEF2 (red), eEF3 (blue), or eEF2 and eEF3 without the ternary complex (magenta) in a stopped-flow apparatus. Each trace is an average of 5-7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction.


( D ) tRNAfMet ( Flu ) co - eluting with 80S 2C incubated without factors ( orange ) , with eEF2 ( red ) , with eEF2 and eEF3 ( magenta ) , or with eEF2 , eEF3 and TC - Val ( green ) for 15 min before loading on a BioSuite 450 size - exclusion column to separate ribosome - bound and - unbound tRNA . Data are normalized to tRNAfMet ( Flu ) bound to 80S 2C in the absence of eEF2 / eEF3 with the maximum value set to 100 % . Data presented as mean ± SEM of n = 3 .

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 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

(D) tRNAfMet(Flu) co-eluting with 80S 2C incubated without factors (orange), with eEF2 (red), with eEF2 and eEF3 (magenta), or with eEF2, eEF3 and TC-Val (green) for 15 min before loading on a BioSuite 450 size-exclusion column to separate ribosome-bound and -unbound tRNA. Data are normalized to tRNAfMet(Flu) bound to 80S 2C in the absence of eEF2/eEF3 with the maximum value set to 100%. Data presented as mean ± SEM of n = 3.


( E ) Dissociation of tRNAfMet ( Flu ) from 80S 2C deacylated tRNA incubated with eEF2 and eEF3 ( green ) or eEF2 ( red ) for 15 min before adding with ternary complexes . Each trace is an average of 5 - 7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction .

O O O O O B-MEASURED_VAR 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

(E) Dissociation of tRNAfMet(Flu) from 80S 2C deacylated tRNA incubated with eEF2 and eEF3 (green) or eEF2 (red) for 15 min before adding with ternary complexes. Each trace is an average of 5-7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction.


( F ) Codon - specific ribosome occupancies for 21 nt RPFs from WT and eEF3d cells ( two biological replicates ) . CGA and AGG codons are shown in red and purple , respectively . Variance for each dataset is indicated .

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

(F) Codon-specific ribosome occupancies for 21 nt RPFs from WT and eEF3d cells (two biological replicates). CGA and AGG codons are shown in red and purple, respectively. Variance for each dataset is indicated.


( G ) Scatter plot of codon - specific occupancies for 28 nt RPFs comparing eEF3d to WT cells from libraries prepared with CHX + ANS . Trend line ( blue ) and its slope are shown . Codons that encode proline and glycine are colored in purple and green , respectively .

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

(G) Scatter plot of codon-specific occupancies for 28 nt RPFs comparing eEF3d to WT cells from libraries prepared with CHX+ANS. Trend line (blue) and its slope are shown. Codons that encode proline and glycine are colored in purple and green, respectively.


Telomeric lncRNA levels in the indicated deletion strains as analyzed by dot blot ( top ) or Northern blot hybridization ( bottom ) using a double ‐ stranded telomeric probe . Telomeric dot blot signals are expressed as fold increase relative to wtA after normalization through U6 snRNA . Bars and error bars are averages and s . d . from 4 independent experiments . U6 and 18S rRNA are shown as loading controls for Northern blotting . The asterisk indicates unspliced U6 . Molecular weights are on the left in kilobases .

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

Telomeric lncRNA levels in the indicated deletion strains as analyzed by dot blot (top) or Northern blot hybridization (bottom) using a double‐stranded telomeric probe. Telomeric dot blot signals are expressed as fold increase relative to wtA after normalization through U6 snRNA. Bars and error bars are averages and s.d. from 4 independent experiments. U6 and 18S rRNA are shown as loading controls for Northern blotting. The asterisk indicates unspliced U6. Molecular weights are on the left in kilobases.


ARRET and αARRETNorthern blot analysis of the indicated strains using strand ‐ specific probes .

ARRET and αARRETNorthern blot analysis of the indicated strains using strand‐specific probes.


Telomere length analysis of ApaI ‐ digested DNA from the indicated strains .

Telomere length analysis of ApaI‐digested DNA from the indicated strains.


Quantification of subtelomeric DNA from ChIP experiments using strains carrying the indicated Myc ‐ tagged proteins ( 2A9 and 8B7 : SPBC2A9 . 02 ‐ Myc and SPBP8B7 . 08c ‐ Myc , respectively ) . Immunoprecipitated DNA is expressed as fraction of input DNA . An untagged strain ( − ) served as a negative control . Bars and error bars are averages and s . d . from three independent experiments . Statistical significance was assayed using the unpaired , two ‐ tailed Student ' s t ‐ test . * * * P 0 . 001 relative to untagged strain .

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

Quantification of subtelomeric DNA from ChIP experiments using strains carrying the indicated Myc‐tagged proteins (2A9 and 8B7: SPBC2A9.02‐Myc and SPBP8B7.08c‐Myc, respectively). Immunoprecipitated DNA is expressed as fraction of input DNA. An untagged strain (−) served as a negative control. Bars and error bars are averages and s.d. from three independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to untagged strain.


Chromosome maps of differentially expressed genes in cay1Δ cells shown as fold enrichment over wt . Red dots are overexpressed genes and Tf2 retrotransposons , blue dots are overexpressed LTR sequences .

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

Chromosome maps of differentially expressed genes in cay1Δ cells shown as fold enrichment over wt. Red dots are overexpressed genes and Tf2 retrotransposons, blue dots are overexpressed LTR sequences.


Northern blot analysis of tlh1 / 2 + , Tf2 retrotransposons , and 18S rRNA ( loading control ) in wt , cay1Δ , and trichostatin A ( TSA ) ‐ treated wt cells . Black arrowhead indicates tlh1 / 2 + mRNA ; red arrowhead unprocessed Tf2 transcripts ; and blue arrowhead processed Tf2 transcripts .

O 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

Northern blot analysis of tlh1/2+, Tf2 retrotransposons, and 18S rRNA (loading control) in wt, cay1Δ, and trichostatin A (TSA)‐treated wt cells. Black arrowhead indicates tlh1/2+ mRNA; red arrowhead unprocessed Tf2 transcripts; and blue arrowhead processed Tf2 transcripts.


ChIP analysis of Cay1 ‐ Myc binding to the indicated genomic loci . subtel : subtelomeres ; Tf2 : Tf2 retrotransposon genes ; LTR : Tf2 LTR sequences ; Tlh : tlh1 / 2 + genes ; cen : centromeres . Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over untagged strains ( unt ) .

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

ChIP analysis of Cay1‐Myc binding to the indicated genomic loci. subtel: subtelomeres; Tf2: Tf2 retrotransposon genes; LTR: Tf2 LTR sequences; Tlh: tlh1/2+ genes; cen: centromeres. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over untagged strains (unt).


ChIP analysis of H3K9ac , H3K9me3 , and total H3 at the indicated genomic loci . Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only .

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

ChIP analysis of H3K9ac, H3K9me3, and total H3 at the indicated genomic loci. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only.


Western blot analysis of endogenous Rap1 and Taz1 in the indicated strains . Act1 was used as a loading control . Asterisks indicate cross ‐ reacting bands .

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

Western blot analysis of endogenous Rap1 and Taz1 in the indicated strains. Act1 was used as a loading control. Asterisks indicate cross‐reacting bands.


RT - PCR analysis of splicing efficiencies of the indicated pre ‐ mRNAs in wt and cay1 ∆ cells . The prp1 ‐ 1 strain carries a thermosensitive allele of the splicing factor Prp1 and was used as a positive control for splicing impairment . prp1 ‐ 1 cells were grown at 36 ° C for 4 h before harvesting . Amplification products corresponding to unspliced and spliced mRNAs are indicated by asterisks and gene names , respectively . rap1T indicates amplification products obtained with oligonucleotides spanning rap1 + exon 3 and thereby amplifying both spliced and unspliced mRNA . Intronless act1 + was used as a loading control . Numbers at the bottom of each panel are ratios between spliced and unspliced forms ( s / u ) and spliced and act1 + ( s / act1 ) . Values are expressed as fold increase over wt .

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

RT-PCR analysis of splicing efficiencies of the indicated pre‐mRNAs in wt and cay1∆ cells. The prp1‐1 strain carries a thermosensitive allele of the splicing factor Prp1 and was used as a positive control for splicing impairment. prp1‐1 cells were grown at 36°C for 4 h before harvesting. Amplification products corresponding to unspliced and spliced mRNAs are indicated by asterisks and gene names, respectively. rap1T indicates amplification products obtained with oligonucleotides spanning rap1+ exon 3 and thereby amplifying both spliced and unspliced mRNA. Intronless act1+ was used as a loading control. Numbers at the bottom of each panel are ratios between spliced and unspliced forms (s/u) and spliced and act1+ (s/act1). Values are expressed as fold increase over wt.


Western blot analysis of Rap1 - YFP protein levels in the indicated strains upon nmt1 promoter shutoff by thiamine addition . Membranes were probed with antibodies against GFP and Act1 ( loading control ) . Quantification of Rap1 - YFP protein levels in experiments as in ( C ) . Rap1 - YFP levels are expressed as fold increase over time 0 after normalization through Act1 . Data points and error bars are averages and s . d . from at least four independent experiments . Statistical significance was assayed using the unpaired , two - tailed Student ' s t - test . * P 0 . 05 , * * P 0 . 01 for cay1 + versus cay1 ∆ samples .

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

Western blot analysis of Rap1-YFP protein levels in the indicated strains upon nmt1 promoter shutoff by thiamine addition. Membranes were probed with antibodies against GFP and Act1 (loading control).Quantification of Rap1-YFP protein levels in experiments as in (C). Rap1-YFP levels are expressed as fold increase over time 0 after normalization through Act1. Data points and error bars are averages and s.d. from at least four independent experiments. Statistical significance was assayed using the unpaired, two-tailed Student's t-test. *P 0.05, **P 0.01 for cay1+ versus cay1∆ samples.


Telomere length analysis of ApaI ‐ digested DNA from cay1 ∆ trt1 ∆ cells harvested at increasing generation doublings ( gen ) .

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

Telomere length analysis of ApaI‐digested DNA from cay1∆trt1∆ cells harvested at increasing generation doublings (gen).


Telomere length analysis of ApaI ‐ digested DNA from the indicated strains . dh : centromeric dh repeats shown as loading control .

Telomere length analysis of ApaI‐digested DNA from the indicated strains. dh: centromeric dh repeats shown as loading control.


PFGE analysis of telomeric fusions in strains grown to logarithmic phase ( log ) or G1 ‐ arrested by nitrogen starvation ( G1 ) . Genomic DNA was digested with NotI and hybridized to C , I , L , and M probes detecting terminal fragments of chromosomes I and II . Bands corresponding to chromosome end fusions are indicated ( fused ) .

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

PFGE analysis of telomeric fusions in strains grown to logarithmic phase (log) or G1‐arrested by nitrogen starvation (G1). Genomic DNA was digested with NotI and hybridized to C, I, L, and M probes detecting terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated (fused).


Northern blot analysis of ARIA , ARRET , Tf2 retrotransposons , and 18S rRNA ( loading control ) in the indicated strains .

Northern blot analysis of ARIA, ARRET, Tf2 retrotransposons, and 18S rRNA (loading control) in the indicated strains.


qRT - PCR quantification of TERRA levels expressed as fold increase over wt after normalization through act1 + mRNA . Bars and error bars are averages and s . d . from 3 independent experiments . Statistical significance was assayed using the unpaired , two ‐ tailed Student ' s t ‐ test . * * P 0 . 01 relative to wt .

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

qRT-PCR quantification of TERRA levels expressed as fold increase over wt after normalization through act1+ mRNA. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. **P 0.01 relative to wt.


ChIP analysis of H3K9ac and total H3 for the indicated strains . Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only . Bars and error bars are averages and s . d . from at least four independent experiments . Statistical significance was assayed using the unpaired , two ‐ tailed Student ' s t ‐ test . * * * P 0 . 001 relative to wt .

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

ChIP analysis of H3K9ac and total H3 for the indicated strains. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only. Bars and error bars are averages and s.d. from at least four independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to wt.


Serial dilutions of the indicated strains were spotted on complete medium and grown at 30 ° C or 20 ° C . Otrt1 ∆ and Otrt1 ∆ cay1 ∆ are strains with circularized chromosomes lacking telomeric sequence ( Supplementary Fig S7 ) .

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

Serial dilutions of the indicated strains were spotted on complete medium and grown at 30°C or 20°C. Otrt1∆ and Otrt1∆cay1∆ are strains with circularized chromosomes lacking telomeric sequence (Supplementary Fig S7).


Representative images of wt and cay1 ∆ cells grown for 24 h at 30 ° C or 20 ° C . Scale bar , 20 μm .

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

Representative images of wt and cay1∆ cells grown for 24 h at 30°C or 20°C. Scale bar, 20 μm.


Quantification of cell length for strains grown in complete medium for 24 h at 30 ° C or 20 ° C . Each dot represents a single cell and median cell length ( m ) is indicated for each sample . Data are from three independent experiments and at least 100 cells were scored per sample in each experiment .

Quantification of cell length for strains grown in complete medium for 24 h at 30°C or 20°C. Each dot represents a single cell and median cell length (m) is indicated for each sample. Data are from three independent experiments and at least 100 cells were scored per sample in each experiment.


Top : Examples of DAPI ‐ stained wt and cay1 ∆ cells grown in complete medium for 24 h at 30 ° C or 20 ° C . The white arrowhead indicates a chromosome bridge . Scale bar , 10 μm . Bottom : Quantification of chromosome bridges . Bars and error bars are averages and s . d . from three independent experiments and at least 200 cells were scored per sample in each experiment . Statistical significance was assayed using the unpaired , two ‐ tailed Student ' s t ‐ test . * * * P 0 . 001 relative to 30 ° C .

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

Top: Examples of DAPI‐stained wt and cay1∆ cells grown in complete medium for 24 h at 30°C or 20°C. The white arrowhead indicates a chromosome bridge. Scale bar, 10 μm. Bottom: Quantification of chromosome bridges. Bars and error bars are averages and s.d. from three independent experiments and at least 200 cells were scored per sample in each experiment. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to 30°C.