( A - D ) Wide - field fluorescence images of basal views through living SCs expressing a GFP - tagged form of Btl - GFP ( yellow ) in non - acidic compartments , with SC outline approximated by dashed white circles . Acidic compartments are marked by LysoTracker Red ® ( magenta ) . Boxed non - acidic compartments in merge images are magnified in A - D , Zoom . On the right , lower magnification confocal transverse images of fixed accessory gland ( AG ) lumens are shown with dotted lines indicating the basal side of the AG epithelial layer , which contains several fluorescent SCs ( highlighted by arrows ) . ( A ) SC with no RNAi construct expressed ( control ) and AG lumen from same genotype . Btl - GFP - positive compartments containing fluorescent ILVs ( in Btl - GFP panel ) , ILV membranes inside compartments ( in Zoom panel ) and secreted fluorescent puncta ( AG lumen panel ) are marked by arrowheads . ( B ) SC also expressing RNAi construct targeting ESCRT - 0 subunit , Stam , and AG lumen from same genotype . Btl - GFP - positive ILVs ( Zoom ) and secreted puncta ( AG lumen ) are strongly reduced . ( C ) SC also expressing RNAi construct targeting ESCRT - I subunit , Vps28 , and AG lumen from same genotype . Btl - GFP - positive ILVs ( Zoom ) and secreted puncta ( AG lumen ) are strongly reduced . ( D ) SC also expressing RNAi construct targeting ESCRT - III subunit , shrb , and AG lumen from same genotype . Btl - GFP - positive ILVs ( Zoom ) and secreted puncta are strongly reduced . Genotypes are : w ; P [ w + , tub - GAL80ts ] / + ; dsx - GAL4 / P [ w + , UAS - btl - GFP ] with no knockdown construct ( A ) , UAS - Stam - RNAi ( HMS01429 ; B ) , UAS - Vps28 - RNAi ( v31894 ; C ) or UAS - shrb - RNAi ( v106823 ; D ) .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY B-SUBCELLULAR O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O B-TISSUE I-TISSUE O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O B-TISSUE I-TISSUE O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O

(A-D) Wide-field fluorescence images of basal views through living SCs expressing a GFP-tagged form of Btl-GFP (yellow) in non-acidic compartments, with SC outline approximated by dashed white circles. Acidic compartments are marked by LysoTracker Red® (magenta). Boxed non-acidic compartments in merge images are magnified in A-D, Zoom. On the right, lower magnification confocal transverse images of fixed accessory gland (AG) lumens are shown with dotted lines indicating the basal side of the AG epithelial layer, which contains several fluorescent SCs (highlighted by arrows). (A) SC with no RNAi construct expressed (control) and AG lumen from same genotype. Btl-GFP-positive compartments containing fluorescent ILVs (in Btl-GFP panel), ILV membranes inside compartments (in Zoom panel) and secreted fluorescent puncta (AG lumen panel) are marked by arrowheads. (B) SC also expressing RNAi construct targeting ESCRT-0 subunit, Stam, and AG lumen from same genotype. Btl-GFP-positive ILVs (Zoom) and secreted puncta (AG lumen) are strongly reduced. (C) SC also expressing RNAi construct targeting ESCRT-I subunit, Vps28, and AG lumen from same genotype. Btl-GFP-positive ILVs (Zoom) and secreted puncta (AG lumen) are strongly reduced. (D) SC also expressing RNAi construct targeting ESCRT-III subunit, shrb, and AG lumen from same genotype. Btl-GFP-positive ILVs (Zoom) and secreted puncta are strongly reduced. Genotypes are: w; P[w+, tub-GAL80ts]/+; dsx-GAL4/P[w+, UAS-btl-GFP] with no knockdown construct (A), UAS-Stam-RNAi (HMS01429; B), UAS-Vps28-RNAi (v31894; C) or UAS-shrb-RNAi (v106823; D).


( E ) Bar chart showing the number of large ( diameter greater than one micrometre ) non - acidic Btl - GFP - positive compartments per SC . Data from 39 SCs ( three per gland ) are shown .

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(E) Bar chart showing the number of large (diameter greater than one micrometre) non-acidic Btl-GFP-positive compartments per SC. Data from 39 SCs (three per gland) are shown.


( F ) Bar chart showing percentage of these large Btl - GFP compartments containing Btl - GFP - positive ILVs in control and ESCRT knockdown SCs . Data from 39 SCs ( three per gland ) are shown .

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(F) Bar chart showing percentage of these large Btl-GFP compartments containing Btl-GFP-positive ILVs in control and ESCRT knockdown SCs. Data from 39 SCs (three per gland) are shown.


( G ) Bar chart showing the total number of Btl - GFP fluorescent puncta in ten Z - planes from the AG lumen following ESCRT knockdown in SCs , compared to controls without knockdown . Data from at least 17 AG lumens per condition are shown .

O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY B-SUBCELLULAR O O O O O O O B-TISSUE I-TISSUE O B-SUBCELLULAR O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O

(G) Bar chart showing the total number of Btl-GFP fluorescent puncta in ten Z-planes from the AG lumen following ESCRT knockdown in SCs, compared to controls without knockdown. Data from at least 17 AG lumens per condition are shown.


( A - D ) Confocal images of fixed HCT116 cells , with boxed regions enlarged to the right . DAPI ( grey ) marks nucleus . Rab11a antibody is isoform - specific except in ( F ) . ( A ) Rab11a ( yellow ) is located in compartments distinct from the late endosomal and lysosomal marker , LAMP2 ( magenta ) . ( B ) CD63 ( magenta ) predominantly co - localises with the late endosomal and lysosomal marker , LAMP1 ( yellow ) . ( C ) Rab11a ( yellow ) is located in compartments distinct from CD63 ( magenta ) under glutamine - replete conditions . ( D ) Rab11a ( yellow ) is located in compartments distinct from CD63 ( magenta ) under glutamine - depleted conditions .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O

(A-D) Confocal images of fixed HCT116 cells, with boxed regions enlarged to the right. DAPI (grey) marks nucleus. Rab11a antibody is isoform-specific except in (F). (A) Rab11a (yellow) is located in compartments distinct from the late endosomal and lysosomal marker, LAMP2 (magenta). (B) CD63 (magenta) predominantly co-localises with the late endosomal and lysosomal marker, LAMP1 (yellow). (C) Rab11a (yellow) is located in compartments distinct from CD63 (magenta) under glutamine-replete conditions. (D) Rab11a (yellow) is located in compartments distinct from CD63 (magenta) under glutamine-depleted conditions.


( E ) Super - resolution 3D - SIM image of fixed HCT116 cell expressing GFP - Rab11a ( yellow ) , and stained with CD63 ( magenta ) . DAPI ( grey ) marks nucleus . Boxed Rab11a - positive compartments , which frequently cluster , are magnified in Merge Zoom . This panel is further magnified in Merge Zoom x 2 , revealing GFP - Rab11a ( arrows in right panel ) inside compartments .

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(E) Super-resolution 3D-SIM image of fixed HCT116 cell expressing GFP-Rab11a (yellow), and stained with CD63 (magenta). DAPI (grey) marks nucleus. Boxed Rab11a-positive compartments, which frequently cluster, are magnified in Merge Zoom. This panel is further magnified in Merge Zoom x 2, revealing GFP-Rab11a (arrows in right panel) inside compartments.


( F ) Wide - field fluorescence image of fixed HCT116 cells , stained with Rab11a ( yellow ) and Rab7 ( magenta ) , expressing constitutively active GFP - tagged Rab5 ( GFP - Rab5CA ; cyan ) , which stalls endosomal maturation and produces enlarged Rab5 - positive endosomes . One of these is boxed in the Merge and magnified in Merge Zoom , revealing internal puncta marked by Rab11a ( arrows ) and Rab7 ( arrowheads ) and limiting membrane subdomains of Rab11a ( yellow arrowhead ) and Rab7 ( magenta arrowhead ) . DAPI ( grey ) marks nuclei .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O

(F) Wide-field fluorescence image of fixed HCT116 cells, stained with Rab11a (yellow) and Rab7 (magenta), expressing constitutively active GFP-tagged Rab5 (GFP-Rab5CA; cyan), which stalls endosomal maturation and produces enlarged Rab5-positive endosomes. One of these is boxed in the Merge and magnified in Merge Zoom, revealing internal puncta marked by Rab11a (arrows) and Rab7 (arrowheads) and limiting membrane subdomains of Rab11a (yellow arrowhead) and Rab7 (magenta arrowhead). DAPI (grey) marks nuclei.


( A ) Western blot analysis of putative exosome markers in lysates from HCT116 cells cultured in glutamine - replete ( 2 . 00 mM ) and glutamine - depleted ( 0 . 15 mM ) medium for 24 h . Gel was loaded with equal amounts of protein ( total cell lysate protein was reduced by 19 ± 4 % after glutamine depletion ) . The activity of mTORC1 was assessed via phosphorylation of S6 and 4E - BP1 , using phospho - specific antibodies and a pan - 4E - BP1 antibody , where the hyper - phosphorylated form of 4E - BP1 produced by mTORC1 gives the slowest migrating γ band . Bar chart shows the abundance of putative exosome proteins relative to tubulin in these lysates .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O

(A) Western blot analysis of putative exosome markers in lysates from HCT116 cells cultured in glutamine-replete (2.00 mM) and glutamine-depleted (0.15 mM) medium for 24 h. Gel was loaded with equal amounts of protein (total cell lysate protein was reduced by 19 ± 4 % after glutamine depletion). The activity of mTORC1 was assessed via phosphorylation of S6 and 4E-BP1, using phospho-specific antibodies and a pan-4E-BP1 antibody, where the hyper-phosphorylated form of 4E-BP1 produced by mTORC1 gives the slowest migrating γ band. Bar chart shows the abundance of putative exosome proteins relative to tubulin in these lysates.


Western blot analyses of EV preparations . ( B ) EVs isolated by ultracentrifugation ( UC ) of medium from HCT116 cells cultured in glutamine - replete and - depleted conditions for 24 h , and loaded according to cell lysate protein levels to compare secretion on a per cell basis , as shown in bar chart from four biological replicates .

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Western blot analyses of EV preparations. (B) EVs isolated by ultracentrifugation (UC) of medium from HCT116 cells cultured in glutamine-replete and -depleted conditions for 24 h, and loaded according to cell lysate protein levels to compare secretion on a per cell basis, as shown in bar chart from four biological replicates.


Western blot analyses of EV preparations . ( C ) EV preparation isolated by size - exclusion chromatography ( SEC ) from glutamine - depleted HCT116 cells and then separated by high - resolution iodixanol - PBS density centrifugation . Note that exosomes ( exos ) and microvesicles ( MVs ) are found in only partially overlapping fractions and the pattern of Rab11a separation mirrors that of exosome markers ( primarily 1 . 064 - 1 . 085 g / ml ) .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

Western blot analyses of EV preparations. (C) EV preparation isolated by size-exclusion chromatography (SEC) from glutamine-depleted HCT116 cells and then separated by high-resolution iodixanol-PBS density centrifugation. Note that exosomes (exos) and microvesicles (MVs) are found in only partially overlapping fractions and the pattern of Rab11a separation mirrors that of exosome markers (primarily 1.064 - 1.085 g/ml).


Western blot analyses of EV preparations . ( D ) EVs isolated by UC under glutamine - depleted conditions as in ( B ) and then subjected to Proteinase K ( Prot K ) digestion in the presence or absence of Triton ® X - 100 ( Triton - X ) . Only membrane - associated CD81 is digested in the absence of Triton ® X - 100 , while CD63 is resistant to digestion , even in the presence of Triton ® X - 100 .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O

Western blot analyses of EV preparations. (D) EVs isolated by UC under glutamine-depleted conditions as in (B) and then subjected to Proteinase K (Prot K) digestion in the presence or absence of Triton® X-100 (Triton-X). Only membrane-associated CD81 is digested in the absence of Triton® X-100, while CD63 is resistant to digestion, even in the presence of Triton® X-100.


Western blot analyses of EV preparations . EVs isolated under glutamine - depleted conditions were immunocaptured with anti - CD63 antibodies coupled to magnetic beads . This method only pulls down a fraction of CD63 , so the protein from pull - down of eight times the input is loaded for comparison . Approximately the same low levels of Rab11a are captured by control IgG and anti - CD63 beads .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O

Western blot analyses of EV preparations. EVs isolated under glutamine-depleted conditions were immunocaptured with anti-CD63 antibodies coupled to magnetic beads. This method only pulls down a fraction of CD63, so the protein from pull-down of eight times the input is loaded for comparison. Approximately the same low levels of Rab11a are captured by control IgG and anti-CD63 beads.


Western blot analyses of EV preparations . ( F ) EVs isolated under glutamine - depleted conditions were immunocaptured with anti - CD81 antibodies coupled to magnetic beads . All or most CD63 and CD81 appears to be pulled down , but less than 10 % of Rab11a and Cav - 1 is captured . NTA analysis suggests about 10 % of all particles are pulled down with this approach .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

Western blot analyses of EV preparations. (F) EVs isolated under glutamine-depleted conditions were immunocaptured with anti-CD81 antibodies coupled to magnetic beads. All or most CD63 and CD81 appears to be pulled down, but less than 10% of Rab11a and Cav-1 is captured. NTA analysis suggests about 10% of all particles are pulled down with this approach.


Western blot analyses of EV preparations . Gel loading is normalised to cell lysate protein levels . Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein ( A ) EVs isolated by UC of medium from HeLa cells cultured in glutamine - replete ( 2 . 00 mM ) and - depleted ( 0 . 02 mM ) conditions for 24 h ( see Appendix Fig S7A ' ' for analysis of SEC - isolated EVs ) .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O 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-EXP_ASSAY O O B-SUBCELLULAR O O

Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (A) EVs isolated by UC of medium from HeLa cells cultured in glutamine-replete (2.00 mM) and -depleted (0.02 mM) conditions for 24 h (see Appendix Fig S7A'' for analysis of SEC-isolated EVs).


Western blot analyses of EV preparations . Gel loading is normalised to cell lysate protein levels . Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein ( B ) EVs isolated by size - exclusion chromatography ( SEC ; fractions two to seven ) from LNCaP cells cultured in glutamine - replete ( 2 . 00 mM ) and - depleted ( 0 . 02 mM ) conditions for

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (B) EVs isolated by size-exclusion chromatography (SEC; fractions two to seven) from LNCaP cells cultured in glutamine-replete (2.00 mM) and -depleted (0.02 mM) conditions for 24


Western blot analyses of EV preparations . Gel loading is normalised to cell lysate protein levels . Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein ( C ) EVs isolated by SEC from LNCaP cells cultured in the presence or absence of 120 nM Torin 1 for 24 h .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (C) EVs isolated by SEC from LNCaP cells cultured in the presence or absence of 120 nM Torin 1 for 24 h.


Western blot analyses of EV preparations . Gel loading is normalised to cell lysate protein levels . Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein ( D ) EVs isolated by UC from HCT116 cells cultured for 24 h following three days of raptor or non - targeting ( NT ) shRNA knockdown .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O

Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (D) EVs isolated by UC from HCT116 cells cultured for 24 h following three days of raptor or non-targeting (NT) shRNA knockdown.


Western blot analyses of EV preparations . Gel loading is normalised to cell lysate protein levels . Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein ( E ) EVs isolated by SEC from HCT116 cells cultured in the presence or absence of 3 µM Akt inhibitor AZD5363 for 24 h .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O

Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. Bar charts present changes in levels of putative exosome proteins relative to cell lysate protein (E) EVs isolated by SEC from HCT116 cells cultured in the presence or absence of 3 µM Akt inhibitor AZD5363 for 24 h.


Western blot analyses of EV preparations . Gel loading is normalised to cell lysate protein levels . ( F ) EVs isolated by SEC from HCT116 cells cultured under glutamine - replete conditions in the presence or absence of 10 µM KRAS inhibitor BI - 2852 ( or control compound Bl2853 ) for 24 h .

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Western blot analyses of EV preparations. Gel loading is normalised to cell lysate protein levels. (F) EVs isolated by SEC from HCT116 cells cultured under glutamine-replete conditions in the presence or absence of 10 µM KRAS inhibitor BI-2852 (or control compound Bl2853) for 24 h.


( A ) Growth curves for HCT116 recipient cells in reduced ( 1 % ) serum conditions following 30 min pre - incubation with EVs isolated by UC ( 104 per cell ; top ) or by SEC ( 4 x 103 per cell ; bottom ) from glutamine - replete and - depleted HCT116 cells or with vehicle ( PBS ) . Fold change in confluency is a measure of cell area occupying well relative to zero time , as measured by IncuCyte software .

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(A) Growth curves for HCT116 recipient cells in reduced (1%) serum conditions following 30 min pre-incubation with EVs isolated by UC (104 per cell; top) or by SEC (4 x 103 per cell; bottom) from glutamine-replete and -depleted HCT116 cells or with vehicle (PBS). Fold change in confluency is a measure of cell area occupying well relative to zero time, as measured by IncuCyte software.


( B ) Cumulative tube length for HUVEC recipient cells following treatment with 104 EVs isolated by UC from glutamine - replete and - depleted HCT116 cells or with vehicle ( PBS ) . Both EV preparations promote tubulation , but the network is more stable with EVs from glutamine - depleted HCT116 cells .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-CELL_LINE O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O B-CELL_LINE O O

(B) Cumulative tube length for HUVEC recipient cells following treatment with 104 EVs isolated by UC from glutamine-replete and -depleted HCT116 cells or with vehicle (PBS). Both EV preparations promote tubulation, but the network is more stable with EVs from glutamine-depleted HCT116 cells.


( 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O B-EXP_ASSAY O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O B-SUBCELLULAR 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-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O

(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 .

O O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD 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-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

(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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-GENEPROD O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O B-GENEPROD O O

(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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O B-EXP_ASSAY O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O

(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 B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O B-TISSUE O O B-CELL_LINE O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-CELL_LINE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD 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 ) .

O O O O O O B-CELL_LINE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O 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-GENEPROD O B-SMALL_MOLECULE O O O O O O O

(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 B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O B-EXP_ASSAY O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE O O B-CELL_LINE O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O B-TISSUE O O B-CELL_LINE O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD 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 .

B-CELL_LINE O 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-SUBCELLULAR O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE 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 .

B-CELL_LINE O 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-SUBCELLULAR O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD B-EXP_ASSAY 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 .

B-CELL_LINE O 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-SUBCELLULAR O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-DISEASE O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-DISEASE 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 .

B-CELL_LINE O 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-SUBCELLULAR O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD B-EXP_ASSAY 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O

(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 .

O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O 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-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O 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-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O

(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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O

(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 ) .

O O O O O O B-CELL_TYPE O O B-DISEASE O B-CELL_LINE O O O O O O O O B-CELL_TYPE O O B-DISEASE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

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 ) .

O O B-DISEASE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O B-DISEASE O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O B-DISEASE O O O O O O 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. 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 .

O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O 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-GENEPROD O O

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 .

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-GENEPROD O O

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 -

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O 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-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY 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 B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-TISSUE O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE 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 B-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-TISSUE O B-TISSUE I-TISSUE

(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 B-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE

(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 B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O B-SUBCELLULAR O O O O O O B-TISSUE O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O B-SUBCELLULAR O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-TISSUE O B-TISSUE I-TISSUE

(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 B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O 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-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE I-TISSUE O B-TISSUE O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE 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 .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O 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-GENEPROD 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-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O

(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 B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE I-TISSUE O B-TISSUE O O O B-TISSUE O O O O O O B-ORGANISM O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 B-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O 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-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O 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-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM 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 )

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-ORGANISM O

(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 B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE 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 B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE 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 B-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O B-GENEPROD 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 B-EXP_ASSAY O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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 .

O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O 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) 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O 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 .

O O O O O O O O O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O

(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 B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O 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-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O 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 B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE 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 B-EXP_ASSAY O B-SUBCELLULAR B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE 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 B-TISSUE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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.