D Scatter plot showing correlation between CDK12 expression its copy number alteration ( CNA ) in METABRIC ( left ) . r value was calculated as Pearson ' s correlation coefficient . Box plots showing mRNA of CDK12 in indicated subtypes from METABRIC ( right ) . P - values were calculated using one - way ANOVA , followed by post - hoc LSD .

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D Scatter plot showing the correlation between CDK12 expression and its copy number alteration (CNA) in METABRIC (left). The r value was calculated as the Pearson's correlation coefficient. Box plots showing the mRNA levels of CDK12 in the indicated subtypes from METABRIC (right). P-values were calculated using one-way ANOVA, followed by post-hoc LSD test.


E Survival of breast cancer according to expression of CDK12 in METABRIC ( top ) KM plotter ( bottom ) using Kaplan - Meier method with log - rank .

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E Survival analysis of breast cancer patients according to the expression of CDK12 in METABRIC (top) and KM plotter (bottom) using the Kaplan-Meier method with the log-rank test.


A FACS of percentages of CD44 + / CD24 − / ESA + breast CSC - like populations in CDK12 - overexpressing ZR - 75 - 30 HCC - 1419 CDK12 - siRNA - transfected BT474 HCC - 1954 . Top , representative dot plot of CD44 - APC versus CD24 - PE expression for viable ( red ) ESA - positive ( blue ) from viable . Bottom , of CD44 + / CD24 - / ESA + population ( blue dots at lower right quadrant in plots ) was quantified in indicated lines . Mean ± s . d . of three independent samples . P - values were on two - Student ' s t - ( ZR - 75 - 30 HCC - 1419 ) one - way ANOVA with a post - hoc LSD ( BT474 HCC - 1954 ) . Amp , amplification ; non - amp , non - amplification ; Trz , trastuzumab .

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

A FACS analysis of the percentages of CD44+/CD24−/ESA+ breast CSC-like populations in CDK12-overexpressing ZR-75-30 and HCC-1419 cells and CDK12-siRNA-transfected BT474 and HCC-1954 cells. Top, representative dot plot of CD44-APC versus CD24-PE expression for viable cells (red) and ESA-positive cells (blue) from the viable cells. Bottom, the percentage of CD44+/CD24-/ESA+ cell population (blue dots at lower right quadrant in the plots) was quantified in the indicated cell lines. Mean ± s.d. of three independent samples. P-values were based on two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) and one-way ANOVA with a post-hoc LSD test (BT474 and HCC-1954 cells). Amp, amplification; non-amp, non-amplification; Trz, trastuzumab.


B ALDEFLUOR for analysing population with a high ALDH activity in indicated lines . either treated with ALDH inhibitor DEAB ( with DEAB ; negative control ) or untreated ( without DEAB ) were incubated with ALDEFLURO substrate ( BAAA ) . In FACS , of ALDEFLUOR - positive population was determined on shift of seen in dot plots in absence of DEAB . Mean ± s . d . . P - values were calculated on a two - Student ' s t - information was indicated as follows : a / a , CDK12 - amplified / HER2 - amplified ; n / a , CDK12 non - amplified / HER2 - amplified S , trastuzumab - sensitive .

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B ALDEFLUOR assay for analysing cell population with a high ALDH activity in the indicated cell lines. Cells either treated with ALDH inhibitor DEAB (with DEAB; negative control) or untreated (without DEAB) were incubated with ALDEFLURO substrate (BAAA). In the FACS analysis, the percentage of ALDEFLUOR-positive population was determined based on the shift of fluorescent cells seen in the dot plots in the absence of DEAB. Mean ± s.d. . P-values were calculated based on a two-tailed Student's t-test cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.


C self - renewal of CSCs in indicated stable lines was analysed using a tumorsphere - formation . number of tumorsphere ( > 100 μm in diameter ) was counted after 3 , 5 , 7 days . Data represent mean ± s . d . ( n = 3 ) . P - value was calculated on a two - Student ' s t - ( ZR - 75 - 30 HCC - 1419 ) or ANOVA with a post - hoc LSD ( BT474 HCC - 1954 ) . information was indicated as follows : a / a , CDK12 - amplified / HER2 - amplified ; n / a , CDK12 non - amplified / HER2 - amplified ; R , trastuzumab - resistant ; PR , trastuzumab - partial responsive ; S , trastuzumab - sensitive .

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

C The self-renewal of CSCs in the indicated stable cell lines was analysed using a tumorsphere-formation assay. The number of tumorsphere cells (> 100 μm in diameter) was counted after 3, 5, and 7 days. Data represent the mean ± s.d. (n = 3). P-value was calculated based on a two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) or ANOVA with a post-hoc LSD test (BT474 and HCC-1954 cells). cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive.


D growth of indicated stable lines treated with trastuzumab ( Trz ) or vehicle ( veh ) as measured by SRB . Trastuzumab - sensitive HER2 + breast cancer lines ( S ) trastuzumab - insensitive HER2 + breast cancer lines ( partial responsive , PR ; resistant , R ) were treated with 1 µg / mL or 100 µg / mL trastuzumab , respectively . Data represent mean ± s . d . P - values were calculated using RM ANOVA with a post - hoc LSD . information was indicated as follows : a / a , CDK12 - amplified / HER2 - amplified ; n / a , CDK12 non - amplified / HER2 - amplified ; R , trastuzumab - resistant ; PR , trastuzumab - partial responsive ; S , trastuzumab - sensitive .

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

D Cell growth of the indicated stable cell lines treated with trastuzumab (Trz) or vehicle (veh) as measured by SRB assay. Trastuzumab-sensitive HER2+ breast cancer cell lines (S) and trastuzumab-insensitive HER2+ breast cancer cell lines (partial responsive, PR; resistant, R) were treated with 1 µg/mL or 100 µg/mL trastuzumab, respectively. Data represent the mean ± s.d. P-values were calculated using RM ANOVA with a post-hoc LSD test. cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive.


E of effect of CDK12 deficiency on in vivo trastuzumab response of HER2 + breast cancer . Mice were orthotopically xenografted with indicated HER2 + breast cancer treated with 20 mg / kg trastuzumab . growth curve of each group was analysed twice weekly for 5 to 6 ( n = 8 / group ; mean ± s . e . m . ) . P - values were calculated using one - way ANOVA with a post - hoc LSD for last day of measurement . information was indicated as follows : a / a , CDK12 - amplified / HER2 - amplified R , trastuzumab - resistant S , trastuzumab - sensitive .

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E Analysis of the effect of CDK12 deficiency on the in vivo trastuzumab response of HER2+ breast cancer. Mice were orthotopically xenografted with the indicated HER2+ breast cancer cells and treated with 20 mg/kg trastuzumab. The growth curve of each group was analysed twice weekly for 5 to 6 weeks (n = 8/group; mean ± s.e.m.). P-values were calculated using one-way ANOVA with a post-hoc LSD test for the last day of tumor measurement. cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells R, trastuzumab-resistant S, trastuzumab-sensitive.


A presents alteration of gene expression ( ≥ 1 . 5 - fold ) between control ( CON ) CDK12 - overexpressing ( CDK12 ) ZR - 75 - 30 obtained from - seq .

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A Heatmap presents the alteration of gene expression (≥1.5-fold) between control (CON) and CDK12-overexpressing (CDK12) ZR-75-30 cells obtained from RNA-seq analysis.


B GSEA displaying correlations of gene - expression profiles between control CDK12 - overexpressing on - seq results .

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B GSEA displaying correlations of the gene-expression profiles between control and CDK12-overexpressing cells based on RNA-seq results.


D CDK12 - target genes identified by - seq were validated in CDK12 - overexpressing ZR - 75 - 30 using qRT - PCR . Mean ± s . d . ( n = 3 ) . P - values by two - Student ' s t - .

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D The CDK12-target genes identified by RNA-seq were validated in CDK12-overexpressing ZR-75-30 cells using qRT-PCR analysis. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test.


E ChIP - qPCR displaying fold - enrichment of indicated histone H3 acetylation at TSS of CDK12 - target genes in indicated lines . Mean ± s . d . ( n = 3 ) . P - values by two - Student ' s t - . information : a / a , CDK12 - amplified / HER2 - amplified ; n / a , CDK12 non - amplified / HER2 - amplified S , trastuzumab - sensitive .

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E ChIP-qPCR analysis displaying the fold-enrichment of the indicated proteins and histone H3 acetylation at the TSS of CDK12-target genes in the indicated cell lines. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.


F Effect of CDK12 kinase inhibition on expression of WNT1 , WNT3 , IRS1 . were treated with different doses of dinaciclib for 12 h , expression of candidate targets of CDK12 were analysed by immunoblot qRT - PCR Mean ± s . d . ( n = 3 ) . P - values by ANOVA with a post - hoc LSD . information : a / a , CDK12 - amplified / HER2 - amplified ; R , trastuzumab - resistant ; S , trastuzumab - sensitive .

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F Effect of CDK12 kinase inhibition on the expression of WNT1, WNT3, and IRS1. Cells were treated with different doses of dinaciclib for 12 h, and the expression of candidate targets of CDK12 were analysed by immunoblot and qRT-PCR Mean ± s.d. (n = 3). P-values by ANOVA with a post-hoc LSD test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells ; R, trastuzumab-resistant; S, trastuzumab-sensitive.


G viability was measured by SRB in trastuzumab - sensitive BT474 trastuzumab - resistant HCC - 1954 treated with 1 µg / mL or 100 µg / mL trastuzumab ( Trz ) / or 5 nM or 10 nM dinaciclib ( Dina ) , respectively . Mean ± s . d . ( n = 3 ) . P - values , RM ANOVA with a post - hoc LSD . information : a / a , CDK12 - amplified / HER2 - amplified R , trastuzumab - resistant ; S , trastuzumab - sensitive .

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G Cell viability was measured by SRB assay in trastuzumab-sensitive BT474 cells and trastuzumab-resistant HCC-1954 cells treated with 1 µg/mL or 100 µg/mL trastuzumab (Trz) and/or 5 nM or 10 nM dinaciclib (Dina), respectively. Mean ± s.d. (n = 3). P-values, RM ANOVA with a post-hoc LSD test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells R, trastuzumab-resistant; S, trastuzumab-sensitive.


H growth curves for mice xenografted with indicated HER2 + breast cancer treated with 20 mg / kg trastuzumab / or 20 mg / kg dinaciclib ( n = 8 / group ; mean ± s . e . m . ) . P - values , one - way ANOVA with a post - hoc LSD ( BT474 ) or Dunnett T3 ( HCC - 1954 ) for last day of measurement . information : a / a , CDK12 - amplified / HER2 - amplified R , trastuzumab - resistant ; S , trastuzumab - sensitive .

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H Tumor growth curves for mice xenografted with the indicated HER2+ breast cancer cells treated with 20 mg/kg trastuzumab and/or 20 mg/kg dinaciclib (n = 8/group; mean ± s.e.m.). P-values, one-way ANOVA with a post-hoc LSD (BT474) or Dunnett T3 (HCC-1954) test for the last day of tumor measurement. Cell line information: a/a, CDK12-amplified/HER2-amplified cells R, trastuzumab-resistant; S, trastuzumab-sensitive.


A Western blot qRT - PCR results showing phosphorylation of GSK - 3β WNT1 WNT3 expression , respectively , in indicated lines . Mean ± s . d . ( n = 3 ) . P - values by two - Student ' s t - . a / a , CDK12 - amplified / HER2 - amplified ; n / a , CDK12 non - amplified / HER2 - amplified ; S , trastuzumab -

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A Western blot and qRT-PCR results showing phosphorylation levels of GSK-3β and WNT1 and WNT3 expression, respectively, in the indicated cell lines. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test. a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; S, trastuzumab-sensitive


Subcellular localization of β - catenin in indicated stable lines were analysed by fractionation ( B )

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Subcellular localization of β-catenin in the indicated stable cell lines were analysed by cellular fractionation (B)


Subcellular localization of β - catenin in indicated stable lines were analysed by immunofluorescence ( C ) . translocation of β - catenin in control , CDK12 - overexpressing , CDK12 - knockdown stable lines was quantified by counting harboring exclusively β - catenin ( N ) from immunofluorescence image . Mean ± s . d . ( n = 3 ) . P - value was calculated using a two - Student ' s t - . M + C , containing / or β - catenin ; N , dominantly expressing β - catenin . Green , β - catenin ; Blue , DAPI for visualization of nucleus ; Scale bar = 10 μ . a / a , CDK12 - amplified / HER2 - amplified ; n / a , CDK12 non - amplified / HER2 - amplified ; S , trastuzumab - sensitive .

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Subcellular localization of β-catenin in the indicated stable cell lines were analysed by immunofluorescence staining (C). Nuclear translocation of β-catenin in control, CDK12-overexpressing, and CDK12-knockdown stable cell lines was quantified by counting the cells harboring exclusively nuclear β-catenin (N) from the immunofluorescence image. Mean ± s.d. (n = 3). P-value was calculated using a two-tailed Student's t-test. M+C, cells containing membrane and/or cytoplasmic β-catenin; N, cells dominantly expressing nuclear β-catenin. Green, β-catenin staining; Blue, DAPI staining for visualization of nucleus; Scale bar = 10 μ. a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; S, trastuzumab-sensitive.


D Luciferase - reporter measuring TCF / lymphoid enhancer - binding factor - promoter activity in indicated lines . TOP , pTOP - flash ( wild - type TCF - binding sites ) , FOP , pFOP - flash ( mutant TCF - binding sites ) . Data represent mean ± s . d . P - values were calculated using a two - Student ' s t - .

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D Luciferase-reporter assay measuring the TCF/ lymphoid enhancer-binding factor-promoter activity in the indicated cell lines. TOP, pTOP-flash (wild-type TCF-binding sites), FOP, pFOP-flash (mutant TCF-binding sites). Data represent the mean ± s.d. P-values were calculated using a two-tailed Student's t-test.


E mRNA of WNT / β - catenin / TCF - target genes in CDK12 - overexpressing ZR - 75 - 30 CDK12 - knockdown BT474 were analysed by qRT - PCR . Mean ± s . d . ( n = 3 ) . P - values were calculated using a two - Student ' s t - .

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E The mRNA levels of the WNT/β-catenin/TCF-target genes in CDK12-overexpressing ZR-75-30 cells and CDK12-knockdown BT474 cells were analysed by qRT-PCR. Mean ± s.d. (n = 3). P-values were calculated using a two-tailed Student's t-test.


F Effect of WNT / β - catenin / TCF pathway on CDK12 regulation of breast CSCs . were treated with DMSO ( vehicle ) or 10 nM FH535 , a β - catenin / TCF inhibitor , for 24 h , changes in CD44 + / CD24 - / ESA + population were measured using FACS . seen at lower right quadrant in dot plots indicate CD44 + / CD24 - population ( red ) CD44 + / CD24 - / ESA + population ( blue ) . Data represent mean ± s . d . ofP - value was calculated using one - way ANOVA with a post - hoc LSD .

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F Effect of WNT/ β-catenin/TCF pathway on the CDK12 regulation of breast CSCs. Cells were treated with DMSO (vehicle) or 10 nM FH535, a β-catenin/TCF inhibitor, for 24 h, and changes in the CD44+/CD24-/ESA+ cell population were measured using FACS analysis. Cells seen at lower right quadrant in the dot plots indicate CD44+/CD24- population (red) and CD44+/CD24-/ESA+ population (blue). Data represent the mean ± s.d. ofP-value was calculated using one-way ANOVA with a post-hoc LSD test.


A Human phospho - RTK array displaying activation of phosphokinase - related in CDK12 - knockdown BT474 . bar graph shows fold change of spot . information : a / a , CDK12 - amplified / HER2 - amplified S , trastuzumab - sensitive .

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A Human phospho-RTK array displaying the activation of phosphokinase-related proteins in CDK12-knockdown BT474 cells. The bar graph shows the fold change of spot intensity. Cell line information: a/a, CDK12-amplified/HER2-amplified cells S, trastuzumab-sensitive.


B Immunoblots showing of indicated in different types of HER2 + breast cancer stably expressing CDK12 or with CDK12 knocked down . information : a / a , CDK12 - amplified / HER2 - amplified ; n / a , CDK12 non - amplified / HER2 - amplified ; R , trastuzumab - resistant ; PR , trastuzumab - partial responsive ; S , trastuzumab - sensitive .

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B Immunoblots showing the levels of the indicated proteins in different types of HER2+ breast cancer stably expressing CDK12 or with CDK12 knocked down. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive.


Co - IP showing amounts of bindings between ErbB receptors . lysates from CDK12 - overexpressing ZR - 75 - 30 were immunoprecipitated with anti - HER2 antibody immunoblotted using indicated antibodies . IgG , negative control . information : ; n / a , CDK12 non - amplified / HER2 - amplified S , trastuzumab - sensitive .

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Co-IP analysis showing the amounts of bindings between ErbB receptors. Cell lysates from CDK12-overexpressing ZR-75-30 cells were immunoprecipitated with anti-HER2 antibody and immunoblotted using the indicated antibodies. IgG, negative control. Cell line information: ; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.


D Western blot qRT - PCR of phosphorylation expression of IRS1 , respectively , in indicated lines . Mean ± s . d . ( n = 3 ) . P - values were calculated using a two - Student ' s t - . information : a / a , CDK12 - amplified / HER2 - amplified ; n / a , CDK12 non - amplified / HER2 - amplified ; R , trastuzumab - resistant S , trastuzumab - sensitive .

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

D Western blot and qRT-PCR analysis of the phosphorylation levels and expression of IRS1, respectively, in the indicated cell lines. Mean ± s.d. (n = 3). P-values were calculated using a two-tailed Student's t-test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant S, trastuzumab-sensitive.


E Co - IP indicating amounts of IRS1 interaction with ErbB receptors p85 in CDK12 - overexpressing ZR - 75 - 30 . information n / a , CDK12 non - amplified / HER2 - amplified S , trastuzumab - sensitive .

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E Co-IP analysis indicating the amounts of IRS1 protein interaction with ErbB receptors and p85 in CDK12-overexpressing ZR-75-30 cells. Cell line information n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.


F IRS1 effect on CDK12 - induced phosphorylation of ErbB receptors . Lysates from treated with either control siRNA or IRS1 siRNA ( siIRS1 ) for 24 h were subjected to immunoblotting with indicated antibodies . information : n / a , CDK12 non - amplified / HER2 - amplified S , trastuzumab - sensitive .

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F IRS1 effect on CDK12-induced phosphorylation of ErbB receptors. Lysates from cells treated with either control siRNA or IRS1 siRNA (siIRS1) for 24 h were subjected to immunoblotting with the indicated antibodies. Cell line information: n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.


( B - F ) Basal views through living SCs , with dashed circles approximating outline of a single SC , acidic compartments marked by vital dye LysoTracker ® Red ( magenta ) . In merge , a single non - acidic ( C , D , F ) acidic ( E ) compartment containing intraluminal vesicles ( ILVs ) is boxed magnified in right panel ( Zoom ) . ILVs appear as - delineated vesicles , using super - resolution 3D - structured illumination ( 3D - SIM ) microscopy for brighter overexpressed GFP - tagged constructs ( yellow ; C F ) . However , ILVs appear only as , using lower resolution wide - field microscopy for fainter endogenously expressed YFP - tagged Rab GTPases ( yellow ; D E ) . ( B ) Wide - field image , including differential interference contrast ( DIC ) , of SC expressing a GFP - tagged version of human CD63 ( CD63 - GFP ) . CD63 - GFP expression is apparent on limiting membranes of non - acidic compartments their ILVs , also on limiting membranes of enlarged acidic compartments . Most large non - acidic compartments are Rab11 - positive ( D ) contain dense - granules , which have a ' fried egg ' appearance with DIC ( arrowheads ) ( Corrigan et al . , 2014 ; Redhai et al . , 2016 ) . ( C ) 3D - SIM image of CD63 - GFP - expressing SC . Arrow highlights CD63 - GFP - marked ILVs ( Zoom ) . Many more ILVs are apparent in non - acidic compartments in a complete Z - stack of a non - acidic compartment ( Movie EV1 ) . ( D ) Wide - field image of an SC expressing a YFP - Rab11 gene trap . YFP - Rab11 marks limiting membranes of most non - acidic compartments internal ( arrow in Zoom ) , but not surface of acidic compartments ( Appendix Fig S1B ) . ( E ) Wide - field image of SC expressing a YFP - Rab7 gene trap . YFP - Rab7 marks limiting membranes of acidic compartments internal ( arrow in Zoom ) . Enlarged acidic compartments are also present in adjacent main . ( F ) 3D - SIM image of SC expressing a GFP - tagged version of Breathless ( Btl - GFP ) . Btl - GFP marks limiting membranes of non - acidic compartments their ILVs ( arrow in Zoom ) , but not surface of acidic compartments ( Appendix Fig S1C ) . from six - day - old flies shifted to 29 ° C at eclosion . This induces GAL4 / UAS - dependent SC transgene expression in ( B ) , ( C ) ( F ) . genotypes of flies carrying multiple transgenes are : w ; P [ w + , UAS - CD63 - GFP ] P [ w + , tub - GAL80ts ] / + ; dsx - GAL4 / + ( B , C ) ; w ; P [ w + , tub - GAL80ts ] / + ; dsx - GAL4 / P [ w + , UAS - btl - GFP ] ( F ) .

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

(B-F) Basal views through living SCs, with dashed white circles approximating the outline of a single SC, and acidic compartments marked by the vital dye LysoTracker® Red (magenta). In merge images, a single non-acidic (C, D, F) and acidic (E) compartment containing intraluminal vesicles (ILVs) is boxed and magnified in the right panel (Zoom). ILVs appear as membrane-delineated vesicles, using super-resolution 3D-structured illumination (3D-SIM) microscopy for the brighter overexpressed GFP-tagged constructs (yellow; C and F). However, ILVs appear only as puncta, using lower resolution wide-field microscopy for the fainter endogenously expressed YFP-tagged Rab GTPases (yellow; D and E). (B) Wide-field fluorescence image, including differential interference contrast (DIC), of SC expressing a GFP-tagged version of human CD63 (CD63-GFP). CD63-GFP expression is apparent on the limiting membranes of non-acidic compartments and their ILVs, and also on the limiting membranes of the enlarged acidic compartments. Most large non-acidic compartments are Rab11-positive (D) and contain dense-core granules, which have a 'fried egg' appearance with DIC (arrowheads) (Corrigan et al., 2014; Redhai et al., 2016). (C) 3D-SIM image of CD63-GFP-expressing SC. Arrow highlights CD63-GFP-marked ILVs (Zoom). Many more ILVs are apparent in non-acidic compartments in a complete Z-stack of a non-acidic compartment (Movie EV1). (D) Wide-field fluorescence image of an SC expressing a YFP-Rab11 gene trap. YFP-Rab11 marks the limiting membranes of most non-acidic compartments and internal puncta (arrow in Zoom), but not the surface of acidic compartments (Appendix Fig S1B). (E) Wide-field fluorescence image of SC expressing a YFP-Rab7 gene trap. YFP-Rab7 marks the limiting membranes of acidic compartments and internal puncta (arrow in Zoom). Enlarged acidic compartments are also present in adjacent main cells. (F) 3D-SIM image of SC expressing a GFP-tagged version of Breathless (Btl-GFP). Btl-GFP marks the limiting membranes of non-acidic compartments and their ILVs (arrow in Zoom), but not the surface of acidic compartments (Appendix Fig S1C). Images from six-day-old male flies shifted to 29°C at eclosion. This induces GAL4/UAS-dependent SC transgene expression in (B), (C) and (F). The genotypes of flies carrying multiple transgenes are: w; P[w+, UAS-CD63-GFP] P[w+, tub-GAL80ts]/+; dsx-GAL4/+ (B, C); w; P[w+, tub-GAL80ts]/+; dsx-GAL4/P[w+, UAS-btl-GFP] (F).


( A - D ) Wide - field 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 circles . Acidic compartments are marked by LysoTracker Red ® ( magenta ) . Boxed non - acidic compartments in merge are magnified in A - D , Zoom . On right , lower magnification confocal transverse of fixed accessory ( AG ) lumens are shown with dotted lines indicating basal side of AG epithelial layer , which contains several SCs ( highlighted by arrows ) . ( A ) SC with no RNAi construct expressed ( control ) AG lumen from same genotype . Btl - GFP - positive compartments containing ILVs ( in Btl - GFP panel ) , ILV membranes inside compartments ( in Zoom panel ) secreted ( AG lumen panel ) are marked by arrowheads . ( B ) SC also expressing RNAi construct targeting ESCRT - 0 subunit , Stam , AG lumen from same genotype . Btl - GFP - positive ILVs ( Zoom ) secreted ( AG lumen ) are strongly reduced . ( C ) SC also expressing RNAi construct targeting ESCRT - I subunit , Vps28 , AG lumen from same genotype . Btl - GFP - positive ILVs ( Zoom ) secreted ( AG lumen ) are strongly reduced . ( D ) SC also expressing RNAi construct targeting ESCRT - III subunit , shrb , AG lumen from same genotype . Btl - GFP - positive ILVs ( Zoom ) secreted 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 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 B-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 B-EXP_ASSAY O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-TISSUE I-TISSUE 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 I-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O 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-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 B-TISSUE I-TISSUE O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O 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 B-TISSUE I-TISSUE O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O 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 B-TISSUE I-TISSUE O O O 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 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 number of large ( diameter greater than one micrometre ) non - acidic Btl - GFP - positive compartments per SC . Data from 39 SCs ( three per ) 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 of these large Btl - GFP compartments containing Btl - GFP - positive ILVs in control ESCRT knockdown SCs . Data from 39 SCs ( three per ) 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 total number of Btl - GFP in ten Z - planes from AG lumen following ESCRT knockdown in SCs , compared to controls without knockdown . Data from at least 17 AG lumens per condition are shown .

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(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 of fixed HCT116 , with boxed regions enlarged to right . DAPI ( grey ) marks nucleus . Rab11a antibody is isoform - specific except in ( F ) . ( A ) Rab11a ( yellow ) is located in compartments distinct from late endosomal lysosomal marker , LAMP2 ( magenta ) . ( B ) CD63 ( magenta ) predominantly co - localises with late endosomal 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 .

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

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

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(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 cultured in glutamine - replete - depleted conditions for 24 h , loaded according to lysate to compare secretion on a per basis , as shown in bar chart from four biological replicates .

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

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 - exclusion chromatography ( SEC ) from glutamine - depleted HCT116 then separated by high - resolution iodixanol - PBS density centrifugation . Note that exosomes ( exos ) microvesicles ( MVs ) are found in only partially overlapping fractions 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 O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR 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 ) then subjected to Proteinase K ( Prot K ) digestion in presence or absence of Triton ® X - 100 ( Triton - X ) . Only - associated CD81 is digested in absence of Triton ® X - 100 , while CD63 is resistant to digestion , even in 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 B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD 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 B-GENEPROD 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 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 from pull - down of eight times input is loaded for comparison . Approximately same low of Rab11a are captured by control IgG 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD 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 . or most CD63 CD81 appears to be pulled down , but less than 10 % of Rab11a Cav - 1 is captured . NTA suggests about 10 % of 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 B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY 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 lysate . Bar charts present changes in of putative exosome relative to lysate ( A ) EVs isolated by UC of medium from HeLa cultured in glutamine - replete ( 2 . 00 mM ) - depleted ( 0 . 02 mM ) conditions for 24 h ( see Appendix Fig S7A ' ' for of SEC - isolated EVs ) .

B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR 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-SUBCELLULAR O O B-EXP_ASSAY O O O B-CELL_LINE 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 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 lysate . Bar charts present changes in of putative exosome relative to lysate ( B ) EVs isolated by - exclusion chromatography ( SEC ; fractions two to seven ) from LNCaP cultured in glutamine - replete ( 2 . 00 mM ) - 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 O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-CELL_LINE O O B-SMALL_MOLECULE 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 lysate . Bar charts present changes in of putative exosome relative to lysate ( C ) EVs isolated by SEC from LNCaP cultured in 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 O O O O O O O B-SUBCELLULAR 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 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 lysate . Bar charts present changes in of putative exosome relative to lysate ( D ) EVs isolated by UC from HCT116 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 O O O O O O O B-SUBCELLULAR 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 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 lysate . Bar charts present changes in of putative exosome relative to lysate ( E ) EVs isolated by SEC from HCT116 cultured in 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 O O O O O O O B-SUBCELLULAR 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 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 lysate . ( F ) EVs isolated by SEC from HCT116 cultured under glutamine - replete conditions in presence or absence of 10 µM KRAS inhibitor BI - 2852 ( or control compound Bl2853 ) for 24 h .

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

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

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

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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O B-CELL_LINE 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 of EVs isolated by UC from HCT116 cultured in glutamine - replete - 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 in EVs secreted from Rab11a knockdown relative to NT - treated in glutamine - depleted conditions , following initial normalisation to lysate . ( C ' ) Growth curves are for HCT116 recipient pre - treated with EVs isolated as in ( C ) or with vehicle ( PBS ) . * * * * P colour denotes significant increase relative to EVs from glutamine - replete ( black ) Rab11a knockdown ( red ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-EXP_ASSAY O B-CELL_LINE O O B-SMALL_MOLECULE 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 B-SUBCELLULAR O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE 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 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 B-GENEPROD 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 group of four show grown in glutamine - replete ( 2 . 00 mM ) 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 imaged . Right group of four show grown in glutamine - replete 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 imaged .

O O O O O O O O O O B-SMALL_MOLECULE 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 I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE 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 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 of anti - CD63 heavy chain biotin - conjugated Tfn in HCT116 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 ( 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 B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O B-CELL_LINE 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 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 of EVs isolated by UC from HCT116 in glutamine - replete medium , transduced with a Rab7 or non - targeting ( NT ) control shRNA knockdown construct . Bar charts show changes in putative exosome in EVs isolated by ultracentrifugation , following normalisation to lysate . ( F ' ) Growth curves are for HCT116 recipient pre - treated with EVs isolated as in ( F ) or with vehicle ( PBS ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-EXP_ASSAY O B-CELL_LINE 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 B-SUBCELLULAR O O B-EXP_ASSAY 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 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 of ERK phosphorylation ( p - ERK ) in recipient serum - deprived HCT116 pre - treated with EVs isolated by SEC from glutamine - replete or - depleted HCT116 or vehicle ( PBS ) . Bar chart shows of p - ERK to ERK from triplicate independent experiments .

O O O B-EXP_ASSAY I-EXP_ASSAY 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 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 B-GENEPROD O B-GENEPROD 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 growth over 120 h , after pre - treatment with EV preparations isolated as in ( A ) or with PBS , then incubation for first 24 h of culture in presence or absence of ERK inhibitor SCH772984 ( 1 . 00 µM ) .

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

O O O B-EXP_ASSAY I-EXP_ASSAY 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 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-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 B-SUBCELLULAR 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 O O O O O B-TISSUE O O B-CELL_LINE 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 B-GENEPROD 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 B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 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 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 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 . Tumours were excised 24 h after last of four injections for . Panels show representative immunostained histological sections of tumour tissue quantified using Visiopharm Integrator System . ( A ) Sections immunostained for CD31 , which labels endothelial blood vessels , with blood vessel number ( upper ) total ( 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 B-EXP_ASSAY I-EXP_ASSAY 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 B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE 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 . Tumours were excised 24 h after last of four injections for . Panels show representative immunostained histological sections of tumour tissue quantified using Visiopharm Integrator System . ( B ) Sections immunostained for Ki67 , which stains proliferative . Proportion of tumour with Ki69 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 B-EXP_ASSAY I-EXP_ASSAY 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 O O O O O B-GENEPROD 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 . Tumours were excised 24 h after last of four injections for . Panels show representative immunostained histological sections of tumour tissue quantified using Visiopharm Integrator System . ( C ) Sections stained with haematoxylin eosin , which highlights necrotic regions ( pale ) . Proportion of tumour 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-DISEASE O O O O O O O B-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 . Tumours were excised 24 h after last of four injections for . Panels show representative immunostained histological sections of tumour tissue quantified using Visiopharm Integrator System . ( D ) Sections immunostained for CA9 , which is expressed in hypoxic regions . Proportion of tumour with CA9 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 B-EXP_ASSAY I-EXP_ASSAY 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 B-GENEPROD 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 of a expressing TurboID - HA - Emc6 , Bpl1 - AID * - 9myc OsTIR1 which was grown overnight in SD media with or without auxin . were then diluted into fresh SD media grown to mid logarithmic phase ( ~ 4hours ) with or without auxin ( respectively ) . When required , biotin was added either 30min before harvesting , or for entire 4hours . H3 ( histone H3 ) is used as a loading control .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD 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 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 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 four distinct LC - MS / MS experiments highlighted in Figure 2A showing - log ( p - value ) against log2 ( fold - change ) for identified in appropriate Emc6 / Sbh1 samples . enriched in 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 ; yellow fill for streptavidin - AP of TurboID - HA - Emc6 / ABOLISH . Grey dots represent that passed p - value fold - change criteria , but were only identified by one peptide . Only EMC components or with EMC client features are named .

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 B-GENEPROD O B-GENEPROD 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 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 B-SUBCELLULAR 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 ) microscopy 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 Gnp1 - GFP , 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 ) . are from two independent biological repeats are digitally coloured using ' fire ' lookup table ( LUT ) on Fiji . same contrast settings were used for each image pair .

O O O B-EXP_ASSAY 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 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

(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 conditions described in Figure 1E . For each , blots showing both ' lower ' ( L ) ' higher ' ( H ) contrast settings are shown side - by - side . expected molecular in kDa for each tested including AviTag is written in parentheses after 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 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 B-GENEPROD 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 . lysates from melanocytic - like melanoma 501mel , MM011 , MM074 MM117 or mesenchymal - like melanoma MM029 , MM047 MM099 were immuno - blotted for as indicated . Molecular mass of is indicated ( kDa ) .

O O O O B-CELL_TYPE O O B-DISEASE B-CELL_LINE O O O O O O B-CELL_TYPE O O B-DISEASE 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

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 were treated with increasing concentrations of THZ1 as indicated for 72h . Mean growth is shown relative to vehicle ( DMSO ) - treated . IC50 for each is indicated . Melanocytic - type ( MITF - High , proliferative ) melanoma are shown in red , while mesenchymal - like ( MITF - low , invasive ) melanoma 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 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 B-CELL_TYPE O O O B-GENEPROD O O O O O B-DISEASE 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

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

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

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

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

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

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

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 showing average TBP - normalized fold expression of MITF ( C ) , SOX10 ( D ) 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 ) . p - value ( Student ' s t - ) is indicated , * < 0 . 05 .

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

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 showing average TBP - normalized fold expression of SOX10 , MITF 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 ) . p - value ( Student ' s t - ) is indicated , * < 0 . 05 .

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

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 lysates were immuno - blotted for indicated . numbers below gel lanes represent relative level , which was determined from band using ImageJ software , 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 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 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 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 . p - value ( Student ' s t - ) is indicated , * < 0 . 05 , * * < 0 . 01 ; * * * < 0 . 001 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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

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

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

O O O O O O O B-EXP_ASSAY 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 O O O O O O O O O O O O O O O O O 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 of representative eye cryosections immunostained with anti - Lamp1 antibody from 2 - month - old miR - 211 - / - control mice sacrificed 3h after on , at 10 AM ( diurnal condition ) . Enlarged boxes highlight Lamp1 - positive structures ( arrowheads ) in RPE . Autofluorescence from lipofuscin granules are visible in cryosections of miR - 211 - / - compared to control . 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 layer .

O O O B-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 B-ORGANISM 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 B-TISSUE O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O 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 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 miR - 211 - / - mice . Representative Western blot of Lamp1 , Cln5 , Trpml1 , Sqstm1 / p62 , Cathepsin D ( CtsD ) LC3 from WT miR - 211 - / - mice . Note a decrease of both proCtsD its maturation CtsD heavy chain ( hc ) . plots show of indicated normalized to Gapdh loading control . Bar graphs represent mean values ± s . e . m . of independent experiments ( n = 6 mice ) Mann Whitney ( 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 B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O B-GENEPROD O O B-GENEPROD 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 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 - / - control mice sacrificed 3h after on at 10 AM ( diurnal condition ) . CtsB was reduced in miR - 211 - / - compared to control mice . Bar graphs represent of CtsB activity ± s . e . m . of independent experiments ( n = 3 mice ) Mann Whitney ( 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 B-ORGANISM 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 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 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