D Re - clustered EPCAM - negative non - epithelial cells revealed seven clusters ( resolution 0 . 05 ) representing immune and stromal cell lineages . E Multidimensional scaling plot showing expression profile distances between the pseudo - bulk samples . Each dot corresponds to aggregated expression for a cell cluster for one patient . Cluster colors are overlaid from ( D , right panel ) . Distances on the plot correspond to leading log2 - fold - change , defined as the average log2 - fold - change for the 500 most differential genes between each pair of profiles . F Heat map of pseudo - bulk samples showing marker genes for each non - epithelial cluster . The top 20 marker genes were identified for each cluster by differential expression analysis of the pseudo - bulk read counts . Color bars at the top of the plot indicate the cluster and menopausal status ( blue / yellow for pre / post menopause ) of each sample . G Same t - SNE map of non - epithelial cells as in ( D ) but colored by menopausal status . Barplot shows relative cluster sizes ( percentage of total cells ) for each status condition . Clusters 1 and 2 have significantly different sizes in post - menopause samples after allowing for inter - patient variability ( P = 0 . 040 and P = 0 . 032 by quasi - binomial F - test ) . Sizes are not significantly different for other clusters ( P > 0 . 15 ) . H Relative cluster sizes as for ( G ) but by individual patients . Cluster colors correspond to ( D ) . by cluster . A quasi - multinomial F - test was used to test for differences in cluster frequencies between pre - and post - menopausal samples ( P = 0 . 007 ) . I Bar plots showing percentage of cells expressing selected immune and endothelial cell markers for pre - and post - menopausal samples and the cell clusters identified in Fig 2D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Re-clustered EPCAM-negative non-epithelial cells revealed seven clusters (resolution 0.05) representing immune and stromal cell lineages. E Multidimensional scaling plot showing expression profile distances between the pseudo-bulk samples. Each dot corresponds to aggregated expression for a cell cluster for one patient. Cluster colors are overlaid from (D, right panel). Distances on the plot correspond to leading log2-fold-change, defined as the average log2-fold-change for the 500 most differential genes between each pair of profiles. F Heat map of pseudo-bulk samples showing marker genes for each non-epithelial cluster. The top 20 marker genes were identified for each cluster by differential expression analysis of the pseudo-bulk read counts. Color bars at the top of the plot indicate the cluster and menopausal status (blue/yellow for pre/post menopause) of each sample. G Same t-SNE map of non-epithelial cells as in (D) but colored by menopausal status. Barplot shows relative cluster sizes (percentage of total cells) for each status condition. Clusters 1 and 2 have significantly different sizes in post-menopause samples after allowing for inter-patient variability (P = 0.040 and P=0.032 by quasi-binomial F-test). Sizes are not significantly different for other clusters (P > 0.15). H Relative cluster sizes as for (G) but by individual patients. Cluster colors correspond to (D). by cluster. A quasi-multinomial F-test was used to test for differences in cluster frequencies between pre- and post-menopausal samples (P = 0.007). I Bar plots showing percentage of cells expressing selected immune and endothelial cell markers for pre- and post-menopausal samples and the cell clusters identified in Fig 2D.


A Microenvironment t - SNE map separated by menopausal status and colored by expression ( red = high expression , grey = undetectable ) of selected fibroblast markers ( upper panel ) . The dotted lines indicate the pericyte subsets . Bottom panel shows percentage of cells expressing the markers for the cell

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Microenvironment t-SNE map separated by menopausal status and colored by expression (red=high expression, grey=undetectable) of selected fibroblast markers (upper panel). The dotted lines indicate the pericyte subsets. Bottom panel shows percentage of cells expressing the markers for the cell clusters


B Co - immunofluorescence staining of pre - versus post - menopausal tissue for E - cadherin ( cyan ) , PDGFRβ ( yellow ) and F - actin ( pink ) . DAPI is shown in grey . The arrowheads in ( v ) depict fibroblasts in direct contact with the myoepithelial layer . For 2D and 3D confocal imaging : n = 13 premenopausal and n = 10 postmenopausal specimens . Scale bars : wholemount and optical sections : 100 μm ( panels i - iv ) ; enlargements , 30 μm ( panel v ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Co-immunofluorescence staining of pre- versus post-menopausal tissue for E-cadherin (cyan), PDGFRβ (yellow) and F-actin (pink). DAPI is shown in grey. The arrowheads in (v) depict fibroblasts in direct contact with the myoepithelial layer. For 2D and 3D confocal imaging: n=13 premenopausal and n=10 postmenopausal specimens. Scale bars: wholemount and optical sections:100 μm (panels i-iv); enlargements, 30 μm (panel v).


C t - SNE plot of the integrated scRNA - seq profiles of fibroblasts from pre - and post - menopausal tissue Cell colors correspond to tissue specimens . D Same t - SNE plot as in ( C ) showing 5 distinct clusters ( clusters 3 and 5 were specific to one patient ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 t-SNE plot of the integrated scRNA-seq profiles of fibroblasts from pre-and post-menopausal tissue Cell colors correspond to tissue specimens. D Same t-SNE plot as in (C) showing 5 distinct clusters (clusters 3 and 5 were specific to one patient).


F Heat map of same cells as ( D ) showing expression of the top 20 marker genes in each cluster . Color bars at the top of the plot show cluster membership ( colors as in ( D ) ) and pre - or post - menopausal status ( blue and yellow , respectively ) .

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

F Heat map of same cells as (D) showing expression of the top 20 marker genes in each cluster. Color bars at the top of the plot show cluster membership (colors as in (D)) and pre- or post-menopausal status (blue and yellow, respectively).


A t - SNE map of combined scRNA - seq profiles of total cells isolated from pathologically normal preneoplastic tissue from BRCA1 mutation carriers ( BRCA1 ; n = 4 ) and non - BRCA premenopausal women ( n = 8 ) with no family history of breast cancer . Cell colors represent individual samples . B Same t - SNE map as in ( A ) but colored by cluster ( cluster resolution 0 . 12 ) . C Epithelial clusters were identified by EPCAM expression and the non - epithelial cells were re - clustered to reveal immune and stromal cell populations ( cluster resolution 0 . 08 ) . Lineage identity was determined by hierarchical clustering according to top marker

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 t-SNE map of combined scRNA-seq profiles of total cells isolated from pathologically normal preneoplastic tissue from BRCA1 mutation carriers (BRCA1; n=4) and non-BRCA premenopausal women (n=8) with no family history of breast cancer. Cell colors represent individual samples. B Same t-SNE map as in (A) but colored by cluster (cluster resolution 0.12). C Epithelial clusters were identified by EPCAM expression and the non-epithelial cells were re-clustered to reveal immune and stromal cell populations (cluster resolution 0.08). Lineage identity was determined by hierarchical clustering according to top marker genes


D t - SNE plot showing the combined single - cell transcriptomes of total tissue cells from BRCA1 preneoplastic tissue and TNBCs ( n = 4 for each ) , colored according to individual patients ( B1 = preneoplastic BRCA1 + / - tissue ; TN - B1 = BRCA1 - associated TNBCs ) . E Same t - SNE map as ( D ) but colored by cluster ( cluster resolution 0 . 15 ) . F Expression of epithelial markers indicated on the combined t - SNE plot as in ( D , E ) for BRCA1 preneoplastic and BRCA1 - associated tumor cells . G Epithelial clusters were identified by EPCAM expression and non - epithelial cells are indicated by the dotted line . H Same t - SNE map as in ( D , E ) but colored according to cancerous state : preneoplastic tissue ( blue ) and BRCA1 - associated TNBCs ( yellow ) . I Box plots of signature expression scores for the 13 cell clusters in ( D , E ) according to human breast epithelial and stromal gene signatures . Cluster 1 corresponds to tumor cells , while clusters 6 , 9 and 10 represent adjacent normal LP , basal and ML cells , respectively . Box plots show quartiles , minimum and maximum .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 t-SNE plot showing the combined single-cell transcriptomes of total tissue cells from BRCA1 preneoplastic tissue and TNBCs (n=4 for each), colored according to individual patients (B1 = preneoplastic BRCA1+/- tissue; TN-B1= BRCA1-associated TNBCs). E Same t-SNE map as (D) but colored by cluster (cluster resolution 0.15). F Expression of epithelial markers indicated on the combined t-SNE plot as in (D, E) for BRCA1 preneoplastic and BRCA1-associated tumor cells. G Epithelial clusters were identified by EPCAM expression and non-epithelial cells are indicated by the dotted line. H Same t-SNE map as in (D, E) but colored according to cancerous state: preneoplastic tissue (blue) and BRCA1-associated TNBCs (yellow). I Box plots of signature expression scores for the 13 cell clusters in (D, E) according to human breast epithelial and stromal gene signatures. Cluster 1 corresponds to tumor cells, while clusters 6, 9 and 10 represent adjacent normal LP, basal and ML cells, respectively. Box plots show quartiles, minimum and maximum.


A Reclustered EPCAM - negative cells ( excluding clusters 1 , 6 , 9 and 10 revealed immune / stromal cells in the microenvironment , identified using lineage markers . B Combined t - SNE plot as in ( A ) of the single - cell transcriptomes of immune and stromal cells from preneoplastic tissue of BRCA1 mutation carriers ( blue ; preneo ) and BRCA1 - associated TNBCs ( yellow ; tumor ) . C t - SNE plots showing expression profiles of cardinal markers of immune and stromal cells . D Bar plots showing the percentage of cells expressing typical immune cell ( including Treg ) genes for clusters in ( A ) by preneoplastic vs tumor . E Left panel , same t - SNE map as in ( A ) but separated into cells from preneoplastic ( blue ) and tumor specimens ( yellow ) . Endothelial cell ( endo ) , fibroblast cell ( fibro ) and pericytes ( peri ) clusters are marked . Right panel , proportion of clusters as in ( A ) according to tissue type . F Relative cluster sizes as in ( E ) but by individual patient .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Reclustered EPCAM-negative cells (excluding clusters 1, 6, 9 and 10 revealed immune/stromal cells in the microenvironment, identified using lineage markers. B Combined t-SNE plot as in (A) of the single-cell transcriptomes of immune and stromal cells from preneoplastic tissue of BRCA1 mutation carriers (blue; preneo) and BRCA1-associated TNBCs (yellow; tumor). C t-SNE plots showing expression profiles of cardinal markers of immune and stromal cells. D Bar plots showing the percentage of cells expressing typical immune cell (including Treg) genes for clusters in (A) by preneoplastic vs tumor. E Left panel, same t-SNE map as in (A) but separated into cells from preneoplastic (blue) and tumor specimens (yellow). Endothelial cell (endo), fibroblast cell (fibro) and pericytes (peri) clusters are marked. Right panel, proportion of clusters as in (A) according to tissue type. F Relative cluster sizes as in (E) but by individual patient.


G Co - immunofluorescence of tissue stained for the epithelial marker ( cytokeratin 19 ; yellow ) and the tumor - associated macrophage marker CX3CR1 ( magenta ) . DAPI is shown in white ( n = 2 preneoplastic samples ; n = 2 tumors ) . Scale bar , 100 μm .

G Co-immunofluorescence of tissue stained for the epithelial marker (cytokeratin 19; yellow) and the tumor-associated macrophage marker CX3CR1 (magenta). DAPI is shown in white (n=2 preneoplastic samples; n=2 tumors). Scale bar, 100 μm.


H Heat map of top differentially expressed genes in the major immune / stromal cell clusters , identified in ( A ) . BRCA1 + / - preneoplastic cells , blue ; BRCA1 - associated tumor cells , yellow .

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

H Heat map of top differentially expressed genes in the major immune/stromal cell clusters, identified in (A). BRCA1+/- preneoplastic cells, blue; BRCA1-associated tumor cells, yellow.


A - C t - SNE plots of combined scRNA - seq profiles of total cells from 8 TNBC tumors , 6 HER2 + tumors and 13 ER + tumors respectively . Integration and cluster sizes for the same cells Cells colored by cluster ( top left panels ) , EPCAM expression ( top right ) , cancer subtype marker ( bottom left ) or MKI67 expression ( bottom right ) . Dotted lines delineate epithelial cells ( top panels ) and cycling epithelial cells ( bottom panels ) . Normal epithelial subsets ( normal ) are also demarcated by dotted lines in the upper - right panels of B and C . D InferCNV plots to map inferred copy number variation ( CNV ) for the combined tumor scRNA - seq expression data for the epithelial clusters indicated in panels A - C . scRNA - seq data from normal breast epithelial cells ( N - 1105 - epi ) served as a reference for normalization . Each row represents a gene and each column cells from a cluster in a single tumor . The tumor clusters are color - coded as in A - C . Amplifications ( red ) and deletions ( blue ) are inferred from the gene expression . Tumor cells were distinguished from normal ( N ) cells by abundance of CNV .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C t-SNE plots of combined scRNA-seq profiles of total cells from 8 TNBC tumors, 6 HER2+ tumors and 13 ER+ tumors respectively. Integration and cluster sizes for the same cells Cells colored by cluster (top left panels), EPCAM expression (top right), cancer subtype marker (bottom left) or MKI67 expression (bottom right). Dotted lines delineate epithelial cells (top panels) and cycling epithelial cells (bottom panels). Normal epithelial subsets (normal) are also demarcated by dotted lines in the upper-right panels of B and C. D InferCNV plots to map inferred copy number variation (CNV) for the combined tumor scRNA-seq expression data for the epithelial clusters indicated in panels A-C. scRNA-seq data from normal breast epithelial cells (N-1105-epi) served as a reference for normalization. Each row represents a gene and each column cells from a cluster in a single tumor. The tumor clusters are color-coded as in A-C. Amplifications (red) and deletions (blue) are inferred from the gene expression. Tumor cells were distinguished from normal (N) cells by abundance of CNV.


H Combined t - SNE transcriptome profiles of two distinct ER + tumors isolated from the same breast of a patient : ER - 0029 - 7C ( blue ) and ER - 0029 - 9C ( yellow ) . The corresponding t - SNE cell clusters are shown in the middle panel and expression of EPCAM is shown in the right - hand panel .

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

H Combined t-SNE transcriptome profiles of two distinct ER+ tumors isolated from the same breast of a patient: ER-0029-7C (blue) and ER-0029-9C (yellow). The corresponding t-SNE cell clusters are shown in the middle panel and expression of EPCAM is shown in the right-hand panel.


A - C t - SNE maps of reclustered EPCAM - negative non - epithelial cells Cluster resolutions 0 . 136 , 0 . 1 and 0 . 1 respectively . The major cell clusters within the microenvironment were identified based on expression of lineage - specific genes . Heat maps of pseudo - bulk samples show marker genes for each cluster . The top 30 marker genes were identified for each cluster by differential expression analysis of the pseudo - bulk read counts . Cluster 9 in HER2 + tumors ( B ) expressed myeloid and luminal epithelial markers , suggesting phagocytosis of the latter by macrophages . Color bars at the top of the heat map indicate the cluster of each sample ; top genes that mark each cluster are 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C t-SNE maps of reclustered EPCAM-negative non-epithelial cells Cluster resolutions 0.136, 0.1 and 0.1 respectively. The major cell clusters within the microenvironment were identified based on expression of lineage-specific genes. Heat maps of pseudo-bulk samples show marker genes for each cluster. The top 30 marker genes were identified for each cluster by differential expression analysis of the pseudo-bulk read counts. Cluster 9 in HER2+ tumors (B) expressed myeloid and luminal epithelial markers, suggesting phagocytosis of the latter by macrophages. Color bars at the top of the heat map indicate the cluster of each sample; top genes that mark each cluster are indicated.


A , B Image quantification showing number of CD8 + Ki67 + cells ( A ) or CD68 + Ki67 + cells ( B ) per mm2 of tissue from TNBC , HER2 + and ER + tumors , either within the tumor region ( K8 / 18 + ) or the stroma ( defined as > 5 μm from tumor border ) . Error bars represent s . e . m . CD8 / Ki67 : n = 11 for TNBC , n = 12 for ER + , n = 5 for HER2 + . CD68 / Ki67 : n = 6 for TNBC , n = 8 for ER + , n = 5 for HER2 + .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B Image quantification showing number of CD8+Ki67+ cells (A) or CD68+Ki67+ cells (B) per mm2 of tissue from TNBC, HER2+ and ER+ tumors, either within the tumor region (K8/18+) or the stroma (defined as >5 μm from tumor border). Error bars represent s.e.m. CD8/Ki67: n=11 for TNBC, n=12 for ER+, n=5 for HER2+. CD68/Ki67: n=6 for TNBC, n=8 for ER+, n=5 for HER2+.


C - F Representative confocal images of ER + ( ER - 0032 ) and triple negative tumors ( TN - 0066 ) immunolabeled for K8 / 18 ( yellow ) , CD68 ( green ) and Ki67 ( red ) ( C , E ) or K8 / 18 ( yellow ) , CD8 ( green ) and Ki67 ( red ) ( D , F ) . DAPI is shown in blue . Arrows depict proliferative T cells ( CD8 + Ki67 + ) or macrophages ( CD68 + Ki67 + ) . Enlargements on shown in the right - hand panels . Scale bars , 200 µm for large tilescans ; 50 µm for enlargements and smaller tilescans .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Representative confocal images of ER+ (ER-0032) and triple negative tumors ( TN-0066) immunolabeled for K8/18 (yellow), CD68 (green) and Ki67 (red) (C, E) or K8/18 (yellow), CD8 (green) and Ki67 (red) (D, F). DAPI is shown in blue. Arrows depict proliferative T cells (CD8+Ki67+) or macrophages (CD68+Ki67+). Enlargements on shown in the right-hand panels. Scale bars, 200 µm for large tilescans; 50 µm for enlargements and smaller tilescans.


C Immunostaining of tumor and LN from patient ER - 0064 for expression of ER , PR and pan - cytokeratin . Insets show sections stained with control isotype antibodies . PR , progesterone receptor . Scale bar , 100 μm .

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

C Immunostaining of tumor and LN from patient ER-0064 for expression of ER, PR and pan-cytokeratin. Insets show sections stained with control isotype antibodies. PR, progesterone receptor. Scale bar, 100 μm.


C HEK293 FT cells were transfected with expression plasmids for the indicated proteins . The cells were lysed and subjected to immunoprecipitation with anti - Flag antibodies , and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as 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 O O O O O O O O O O O O O O

C HEK293 FT cells were transfected with expression plasmids for the indicated proteins. The cells were lysed and subjected to immunoprecipitation with anti-Flag antibodies, and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as indicated.


C Proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting .

C Proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting.


In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs ( E , Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein .

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

In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs (E, Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.


In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs F , Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein .

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

In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs F, Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.


In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein .

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

In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.


In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein .

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

In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein.


J In vitro translation ( IVT ) / biotinylation reactions using Hela cell lysates and the indicated constructs . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated protein . Bottom , the indicated proteins were prepared by in vitro translation using Hela cell lysates and analyzed by αFlag western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In vitro translation (IVT)/biotinylation reactions using Hela cell lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated protein. Bottom, the indicated proteins were prepared by in vitro translation using Hela cell lysates and analyzed by αFlag western blotting.


C - F Mouse kidney was lysed , treated with or without RNase I , and RNA was isolated . The RNA was reverse transcribed with the indicated primers for ( C , E ) Hifa and ( D , F ) Xbp1u mRNA , and then analyzed by Real Time PCR for disomes using the indicated primers . ∆ CT = ( ∆ CT = CT - RNase - CT + RNase ) . Biological replicates ( from three males ) , with mean ± SD are shown . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - tailed t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Mouse kidney was lysed, treated with or without RNase I, and RNA was isolated. The RNA was reverse transcribed with the indicated primers for (C,E) Hifa and (D,F) Xbp1u mRNA, and then analyzed by Real Time PCR for disomes using the indicated primers. ∆CT = (∆CT = CT-RNase - CT+RNase). Biological replicates (from three males), with mean ± SD are shown. * P < 0.05, ** P < 0.01, by unpaired two-tailed t-test.


A HEK293 FT cells were transfected with expression plasmids for the indicated proteins . The cells were lysed and subjected to immunoprecipitation with anti - Flag antibodies , and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as indicated . ZFmut = C36S / C42S .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HEK293 FT cells were transfected with expression plasmids for the indicated proteins. The cells were lysed and subjected to immunoprecipitation with anti-Flag antibodies, and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as indicated. ZFmut = C36S/C42S.


B In vitro translation / biotinylation reactions using reticulocyte lysates and the indicated constructs . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated substrate . Bottom , the indicated proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In vitro translation/biotinylation reactions using reticulocyte lysates and the indicated constructs. Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated substrate. Bottom, the indicated proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting.


C HEK293 FT cells were transfected with expression plasmids for the indicated proteins . Tib = Tibetan . The cells were lysed and subjected to immunoprecipitation with anti - Flag antibodies , and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as 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 O O O O O O O O O O O O O O O O O O

C HEK293 FT cells were transfected with expression plasmids for the indicated proteins. Tib = Tibetan. The cells were lysed and subjected to immunoprecipitation with anti-Flag antibodies, and then the immunoprecipitates and aliquots of lysate were examined by Western blotting as indicated.


E In vitro translation / biotinylation reactions using reticulocyte lysates and construct 1 ( left ) or construct 7 ( right ) . Streptavidin far western ( FW ) assesses biotinylation . Anti - HA western blot ( WB ) measures translated substrate . Bottom , the indicated proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting .

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E In vitro translation/biotinylation reactions using reticulocyte lysates and construct 1 (left) or construct 7 (right). Streptavidin far western (FW) assesses biotinylation. Anti-HA western blot (WB) measures translated substrate. Bottom, the indicated proteins were prepared by in vitro translation using reticulocyte lysates and analyzed by αFlag western blotting.


C , Olaparib and ETC - 159 synergize to prevent the growth of HPAF - II xenografts in mice . NSG mice with established HPAF - II subcutaneous xenografts were randomized into four groups . Mice were gavaged daily with ETC - 159 ( 10 mg / kg ) , Olaparib ( 50 mg / kg ) or a combination of ETC - 159 ( 10 mg / kg ) and Olaparib ( 50 mg / kg ) . Treatment was initiated after HPAF - II tumors were established . ( C ) Tumor volumes were measured starting from day 0 and during the course of treatment as shown . Data points represent the mean ± SD . n = 7 - 8 tumors / group . p - values indicate significant difference compared to the vehicle group .

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

C, Olaparib and ETC-159 synergize to prevent the growth of HPAF-II xenografts in mice. NSG mice with established HPAF-II subcutaneous xenografts were randomized into four groups. Mice were gavaged daily with ETC-159 (10 mg/kg), Olaparib (50 mg/kg) or a combination of ETC-159 (10 mg/kg) and Olaparib (50 mg/kg). Treatment was initiated after HPAF-II tumors were established. (C) Tumor volumes were measured starting from day 0 and during the course of treatment as shown. Data points represent the mean ± SD. n = 7-8 tumors/group. p-values indicate significant difference compared to the vehicle group.


DNA repair genes involved in HR and FA pathways were differentially expressed over time in response to PORCN inhibition in HPAF - II orthotopic xenografts . Comparison of log2 fold change ( FDR < 10 % ) in the expression of Wnt - activated genes over time across multiple DNA repair pathways shows that genes regulating HR and FA pathways have higher fold changes compared to genes regulating BER , NER or other pathways . BER : base excision repair ; HR : homologous recombination ; FA : Fanconi anemia ; MMR : mismatch repair ; NER : nucleotide excision repair ; NHEJ : non - homologous end joining ; TLS : translesion DNA synthesis . The horizontal line represents median of 4 - 6 replicates with the upper and the lower edges of the box representing the 75th and 25th percentile of the data respectively and the whiskers representing 1 . 5 x interquartile range .

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DNA repair genes involved in HR and FA pathways were differentially expressed over time in response to PORCN inhibition in HPAF-II orthotopic xenografts. Comparison of log2 fold change (FDR < 10%) in the expression of Wnt-activated genes over time across multiple DNA repair pathways shows that genes regulating HR and FA pathways have higher fold changes compared to genes regulating BER, NER or other pathways. BER: base excision repair; HR: homologous recombination; FA: Fanconi anemia; MMR: mismatch repair; NER: nucleotide excision repair; NHEJ: non-homologous end joining; TLS: translesion DNA synthesis. The horizontal line represents median of 4-6 replicates with the upper and the lower edges of the box representing the 75th and 25th percentile of the data respectively and the whiskers representing 1.5 x interquartile range.


H , I . Wnt inhibition reduces the expression of HR and FA pathway genes in a RNF43 - mutant pancreatic cancer patient - derived xenograft and AsPC - 1 cells . ( H ) Pancreatic canc er PDX with G371fs RNF43 mutation treated with vehicle or ETC - 159 ( 30 mg / kg ) for 21 days were analysed for changes in the expression of the indicated genes measured by qRT - PCR . Each data point represents an individual tumor . The horizontal lines represent mean of 3 tumors / group . ( I ) AsPC - 1 cells were seeded in low adherence plates , treated with DMSO or ETC - 159 ( 100 nM ) for 72 hours and the expression of indicated genes was measured by qRT - PCR .

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H, I. Wnt inhibition reduces the expression of HR and FA pathway genes in a RNF43-mutant pancreatic cancer patient-derived xenograft and AsPC-1 cells. (H) Pancreatic cancer PDX with G371fs RNF43 mutation treated with vehicle or ETC-159 (30 mg/kg) for 21 days were analysed for changes in the expression of the indicated genes measured by qRT-PCR. Each data point represents an individual tumor. The horizontal lines represent mean of 3 tumors/group. (I) AsPC-1 cells were seeded in low adherence plates, treated with DMSO or ETC-159 (100 nM) for 72 hours and the expression of indicated genes was measured by qRT-PCR.


Olaparib and ETC - 159 combination is more effective in preventing the growth of PaTu8988T xenografts in mice . NSG mice with established PaTu8988T orthotopic xenografts were randomized into four groups . Mice were gavaged daily with ETC - 159 ( 30 mg / kg ) , olaparib ( 50 mg / kg ) or a combination of ETC - 159 and olaparib . Treatment was initiated after the tumors were established . Tumor weights in the respective groups at the end of the 18 days treatment are shown . Each dot represents an individual tumor and the horizontal lines represent the mean . p - values were calculated with Mann - Whitney U test .

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Olaparib and ETC-159 combination is more effective in preventing the growth of PaTu8988T xenografts in mice. NSG mice with established PaTu8988T orthotopic xenografts were randomized into four groups. Mice were gavaged daily with ETC-159 (30 mg/kg), olaparib (50 mg/kg) or a combination of ETC-159 and olaparib. Treatment was initiated after the tumors were established. Tumor weights in the respective groups at the end of the 18 days treatment are shown. Each dot represents an individual tumor and the horizontal lines represent the mean. p-values were calculated with Mann-Whitney U test.


Wnt inhibition through ETC - 159 does not change the expression of HR and FA pathway genes in Panc 08 . 13 cells . Panc 08 . 13 cells were cultured in low adherence plates and treated with ETC - 159 for 72 hours . Total RNA was then isolated and expression of indicated genes was measured by qRT - PCR . The horizontal lines represent mean of replicates .

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Wnt inhibition through ETC-159 does not change the expression of HR and FA pathway genes in Panc 08.13 cells. Panc 08.13 cells were cultured in low adherence plates and treated with ETC-159 for 72 hours. Total RNA was then isolated and expression of indicated genes was measured by qRT-PCR. The horizontal lines represent mean of replicates.


Stabilized β - catenin prevents the downregulation of HR and FA pathway genes upon Wnt inhibition . Mice bearing HPAF - II xenografts without or with stabilized β - catenin were treated with ETC - 159 ( 37 . 5mg / kg bid ) for 56 hours before tumors were harvested and the expression of indicated genes was measured by qRT - PCR . ETC - 159 induced reduction in the expression of HR and FA pathway genes was blocked in xenografts with stabilized β - catenin . The horizontal lines represent mean of

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Stabilized β-catenin prevents the downregulation of HR and FA pathway genes upon Wnt inhibition. Mice bearing HPAF-II xenografts without or with stabilized β-catenin were treated with ETC-159 (37.5mg/kg bid) for 56 hours before tumors were harvested and the expression of indicated genes was measured by qRT-PCR. ETC-159 induced reduction in the expression of HR and FA pathway genes was blocked in xenografts with stabilized β-catenin. The horizontal lines represent mean of replicates


E , F . G007 - LK and olaparib synergistically inhibit the growth of COLO320HSR colorectal cancer cells with APC mutation . COLO320HSR cells were plated at a low density and treated with G007 - LK , olaparib or the combination of the two inhibitors at an equivalent dose as described in Figure 1A . ( E ) Representative image from two independent experiments is shown . ( F ) The Combination Index ( CI ) values of olaparib and G007 - LK calculated from two independent experiments using the Chou - Talalay CompuSyn software

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E, F. G007-LK and olaparib synergistically inhibit the growth of COLO320HSR colorectal cancer cells with APC mutation. COLO320HSR cells were plated at a low density and treated with G007-LK, olaparib or the combination of the two inhibitors at an equivalent dose as described in Figure 1A. (E) Representative image from two independent experiments is shown. (F) The Combination Index (CI) values of olaparib and G007-LK calculated from two independent experiments using the Chou-Talalay CompuSyn software


β - catenin regulates the expression of MYBL2 in HPAF - II cells . HPAF - II cells were either treated with ETC - 159 ( 100 nM ) or transfected with siRNA against β - catenin alone or in the presence of ETC - 159 for 48 hours . The relative expression of MYBL2 as measured by qRT - PCR is shown . The horizontal lines represent mean of replicates .

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β-catenin regulates the expression of MYBL2 in HPAF-II cells. HPAF-II cells were either treated with ETC-159 (100 nM) or transfected with siRNA against β-catenin alone or in the presence of ETC-159 for 48 hours. The relative expression of MYBL2 as measured by qRT-PCR is shown. The horizontal lines represent mean of replicates.


Expression of DNA repair genes in Wnt - addicted cells is regulated by MYBL2 . HPAF - II cells were transfected with two independent siRNAs against MYBL2 or treated with ETC - 159 ( 100 nM ) for 48 hours . Total RNA was isolated and expression of MYBL2 and DNA repair genes was measured by qRT - PCR . Data are representative of three independent experiments . The horizontal lines represent mean of replicates .

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Expression of DNA repair genes in Wnt-addicted cells is regulated by MYBL2. HPAF-II cells were transfected with two independent siRNAs against MYBL2 or treated with ETC-159 (100 nM) for 48 hours. Total RNA was isolated and expression of MYBL2 and DNA repair genes was measured by qRT-PCR. Data are representative of three independent experiments. The horizontal lines represent mean of replicates.


H - K . Expression of MYBL2 is downregulated upon ETC - 159 or G007 - LK treatment in multiple Wnt - high tumors . Expression of MYBL2 in AsPC - 1 xenograft tumors and colorectal cancer PDX ( as measured by RNA - seq ) or in pancreatic cancer PDX with RNF43 mutation ( measured by qRT - PCR ) is reduced with ETC - 159 treatment . G007 - LK reduces MYBL2 expression in COL320HSR cells ( measured by qRT - PCR ) . p - values were calculated with Mann - Whitney U test . Each data point represents an individual tumor or replicate . n = 4 - 6 samples / group . The horizontal lines represent median of 4 - 6 replicates with the upper and the lower edges of the boxes representing the 75th and the 25th percentile of the data respectively and the whiskers representing 1 . 5x interquartile range .

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H-K. Expression of MYBL2 is downregulated upon ETC-159 or G007-LK treatment in multiple Wnt-high tumors. Expression of MYBL2 in AsPC-1 xenograft tumors and colorectal cancer PDX (as measured by RNA-seq) or in pancreatic cancer PDX with RNF43 mutation (measured by qRT-PCR) is reduced with ETC-159 treatment. G007-LK reduces MYBL2 expression in COL320HSR cells (measured by qRT-PCR). p-values were calculated with Mann-Whitney U test. Each data point represents an individual tumor or replicate. n = 4-6 samples/group. The horizontal lines represent median of 4-6 replicates with the upper and the lower edges of the boxes representing the 75th and the 25th percentile of the data respectively and the whiskers representing 1.5x interquartile range.


F , Wnt inhibition induces senescence , which is further enhanced by co - treatment with olaparib . ( F ) Representative images of tumor sections from the four treatment groups ( treated for 21 days ) stained for SA - β - galactosidase , a senescence marker , and counterstained with nuclear fast red . Blue color indicates positive staining for senescent cells . The horizontal lines represent mean of replicates . p - values were calculated by Mann - Whitney U test .

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F, Wnt inhibition induces senescence, which is further enhanced by co-treatment with olaparib. (F) Representative images of tumor sections from the four treatment groups (treated for 21 days) stained for SA-β-galactosidase, a senescence marker, and counterstained with nuclear fast red. Blue color indicates positive staining for senescent cells. The horizontal lines represent mean of replicates. p-values were calculated by Mann-Whitney U test.


Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment . ( H ) HPAF - II cells were treated with DMSO , ETC - 159 ( 50 nM ) , olaparib ( 20 µM ) or both for 7 days . 53BP1 foci per cell were then assessed by immunofluorescence . The horizontal lines represent mean of replicates . p - values were calculated by Mann - Whitney U test .

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Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment. (H) HPAF-II cells were treated with DMSO, ETC-159 (50 nM), olaparib (20 µM) or both for 7 days. 53BP1 foci per cell were then assessed by immunofluorescence. The horizontal lines represent mean of replicates. p-values were calculated by Mann-Whitney U test.


I . Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment . ( I ) Representative immunofluorescence images of 53BP1 foci ( red ) and nuclei counterstained with DAPI ( blue ) are shown for each group .

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I. Wnt inhibition potentiates DNA double strand breaks induced by olaparib treatment. (I) Representative immunofluorescence images of 53BP1 foci (red) and nuclei counterstained with DAPI (blue) are shown for each group.


Immunohistochemical staining of sections of small intestine from C57BL / 6J mice for DNA repair proteins shows that the expression of BRCA1 , FANCD2 and RAD51 was high in the crypts ( Wnt high compartment ) whereas the villi did not show any staining . Small intestinal adenomas from APCmin / + mice displayed positive nuclear immunostaining for HR and FA pathway proteins BRCA1 , FANCD2 and RAD51 . The bottom panels are higher magnification images of the areas marked in the top panels .

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Immunohistochemical staining of sections of small intestine from C57BL/6J mice for DNA repair proteins shows that the expression of BRCA1, FANCD2 and RAD51 was high in the crypts (Wnt high compartment) whereas the villi did not show any staining. Small intestinal adenomas from APCmin/+ mice displayed positive nuclear immunostaining for HR and FA pathway proteins BRCA1, FANCD2 and RAD51. The bottom panels are higher magnification images of the areas marked in the top panels.


PORCN inhibition reduces the expression of HR and FA pathway proteins in the normal mouse intestine . C57BL / 6J mice were treated with vehicle or ETC - 159 ( 60 mg / kg ) for 3 days . Small intestines were sectioned and analyzed by immunostaining for BRCA1 , FANCD2 , RAD51 and phosphorylated - BRCA1 .

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PORCN inhibition reduces the expression of HR and FA pathway proteins in the normal mouse intestine. C57BL/6J mice were treated with vehicle or ETC-159 (60 mg/kg) for 3 days. Small intestines were sectioned and analyzed by immunostaining for BRCA1, FANCD2, RAD51 and phosphorylated-BRCA1.


( a ) Expression levels of Tcfeb mRNA in tissues isolated from 24 - h - fasted ( 24 h starved ) 6 - week - old mice . Values are expressed as fold change relative to Tcfeb expression in mice fed ad libitum ( Fed ) . Bars represent mean ± s . d . for n = 5 mice ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 .

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(a) Expression levels of Tcfeb mRNA in tissues isolated from 24-h-fasted (24 h starved) 6-week-old mice. Values are expressed as fold change relative to Tcfeb expression in mice fed ad libitum (Fed). Bars represent mean ± s.d. for n = 5 mice; *P≤0.05; **P≤0.01; ***P≤0.001.


( b ) Representative β - gal staining of liver and kidney frozen sections isolated from fed and 24 - h - fasted heterozygous Tcfeb - β - gal mice .

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(b) Representative β-gal staining of liver and kidney frozen sections isolated from fed and 24-h-fasted heterozygous Tcfeb- β-gal mice.


c ) Time - course expression analysis of TFEB in wild - type HeLa cells , MEFs and hepatocytes after the addition of starvation media ( time 0 ) . Data represent mean ± s . d . for n = 3 independent experiments .

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c) Time-course expression analysis of TFEB in wild-type HeLa cells, MEFs and hepatocytes after the addition of starvation media (time 0). Data represent mean ± s.d. for n = 3 independent experiments.


( d ) Expression levels of transfected hTFEB - FLAG , Tcfeb - β - gal fusion transcript and endogenous Tcfeb mRNAs in MEFs isolated from control ( + / + ) and heterozygous Tcfeb - β - gal / + mice . Data represent mean ± s . d . for n = 3 mice ; * P ≤ 0 . 05 .

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(d) Expression levels of transfected hTFEB-FLAG, Tcfeb- β-gal fusion transcript and endogenous Tcfeb mRNAs in MEFs isolated from control (+/+) and heterozygous Tcfeb- β-gal/+ mice. Data represent mean ± s.d. for n = 3 mice; *P≤0.05.


( e ) Time - course expression analysis of TFEB mRNA in control ( Ctrl ) or TFEB - overexpressing HeLa cells during fasting and re - feeding . Primers specific for the endogenous TFEB were used . Data represent mean ± s . d . for n = 3 .

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(e) Time-course expression analysis of TFEB mRNA in control (Ctrl) or TFEB-overexpressing HeLa cells during fasting and re-feeding. Primers specific for the endogenous TFEB were used. Data represent mean ± s.d. for n = 3.


( f ) Time - course expression analysis of Tcfeb mRNA in heterozygous Tcfeb - β - gal / + or control MEFs during fasting and re - feeding . Specific primers for the endogenous Tcfeb were used . Data represent mean ± s . d . for n = 3 mice .

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(f) Time-course expression analysis of Tcfeb mRNA in heterozygous Tcfeb- β-gal/+ or control MEFs during fasting and re-feeding. Specific primers for the endogenous Tcfeb were used. Data represent mean ± s.d. for n = 3 mice.


( g ) Expression analysis of Tcfeb mRNA in heterozygous Tcfeb - β - gal / + or control hepatocytes in fed and after 24 h fasting . Data represent mean ± s . d . for n = 3 mice .

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(g) Expression analysis of Tcfeb mRNA in heterozygous Tcfeb- β-gal/+ or control hepatocytes in fed and after 24 h fasting. Data represent mean ± s.d. for n = 3 mice.


( h ) ChIP analysis from liver of mice fed ad libitum or 24 h fasted . The CLEAR elements in the first intron of Tcfeb genomic DNA are shown as numbered black rectangles as indicated in Supplementary Table S1 . Red rectangles represent exons , and the ATG indicates the first codon ( from the mouse Tcfeb isoform b ) . The histogram shows the amount of immunoprecipitated DNA as detected by quantitative PCR assay . Values were normalized to the input and plotted as relative enrichment over a mock control . Data represent mean ± s . d of 3 independent experiments .

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(h) ChIP analysis from liver of mice fed ad libitum or 24 h fasted. The CLEAR elements in the first intron of Tcfeb genomic DNA are shown as numbered black rectangles as indicated in Supplementary Table S1. Red rectangles represent exons, and the ATG indicates the first codon (from the mouse Tcfeb isoform b). The histogram shows the amount of immunoprecipitated DNA as detected by quantitative PCR assay. Values were normalized to the input and plotted as relative enrichment over a mock control. Data represent mean ± s.d of 3 independent experiments.


( a ) ChIP analysis from liver of mice fed ad libitum ( fed ) or 24 h - fasted ( starved ) . CLEAR sites in the promoter region of Pgc - 1α are indicated by squares . Numbers indicate the distance ( bp ) of the binding element from the start codon . Bar graphs show the amount of immunoprecipitated DNA as detected by quantitative PCR assay . Values were normalized to the input and plotted as relative enrichment over a mock control . Bar graphs represent mean ± s . d of 3 independent experiments , * P ≤ 0 . 05 ; * * P ≤ 0 . 01 .

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(a) ChIP analysis from liver of mice fed ad libitum (fed) or 24 h-fasted (starved). CLEAR sites in the promoter region of Pgc-1α are indicated by squares. Numbers indicate the distance (bp) of the binding element from the start codon. Bar graphs show the amount of immunoprecipitated DNA as detected by quantitative PCR assay. Values were normalized to the input and plotted as relative enrichment over a mock control. Bar graphs represent mean ± s.d of 3 independent experiments, *P≤0.05; **P≤0.01.


( c ) Luciferase activity was measured after transfecting increasing amounts of TFEB - FLAG in combination with Pgc - 1α WT or Pgc - 1α DEL plasmids . Bar graphs represent mean ± s . d . of n = 3independent experiments , * * * * P ≤ 0 . 0001 compared with mock transfected cells .

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(c) Luciferase activity was measured after transfecting increasing amounts of TFEB-FLAG in combination with Pgc-1α WT or Pgc-1α DEL plasmids. Bar graphs represent mean ± s.d. of n = 3independent experiments, ****P≤0.0001 compared with mock transfected cells.


( d ) Luciferase activity was measured in cells stably overexpressing TFEB cultured in normal and starved media . Bar graphs represent mean ± s . d . of n = 3independent experiments * * P ≤ 0 . 01 compared with mock transfected cells .

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(d) Luciferase activity was measured in cells stably overexpressing TFEB cultured in normal and starved media. Bar graphs represent mean ± s.d. of n = 3independent experiments **P≤0.01 compared with mock transfected cells.


( e ) Quantification of mRNA levels of PGC1α in liver and hepatocytes from control , HDAd - TFEB and Tcfeb - LiKO ( liver KO ) mice treated as indicated . Bar graphs show mean ± s . d . for n = 4 mice . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 .

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

(e) Quantification of mRNA levels of PGC1α in liver and hepatocytes from control, HDAd-TFEB and Tcfeb-LiKO (liver KO) mice treated as indicated. Bar graphs show mean ± s.d. for n = 4 mice. *P≤0.05; **P≤0.01.


( f ) Expression analysis of TFEB target genes in liver of mice with indicated genotypes . Bar graphs show mean ± s . d . for n = 3 mice . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression analysis of TFEB target genes in liver of mice with indicated genotypes. Bar graphs show mean ± s.d. for n = 3 mice. *P≤0.05;**P≤0.01; ***P≤0.001.


( g ) Expression analysis of PGC1α / PPARα target genes in liver from either fasted or fed mice with indicated genotypes . Bar graphs show mean ± s . d . for n = 4 . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 compared with the respective controls ( fed or fasted ) .

(g) Expression analysis of PGC1α/PPARα target genes in liver from either fasted or fed mice with indicated genotypes. Bar graphs show mean ± s.d. for n = 4. *P≤0.05; **P≤0.01 compared with the respective controls (fed or fasted).


( a ) Oil red O staining of liver sections isolated from mice with the indicated genotype fed ad libitum and 24 h fasted . Original magnification × 40 . ( b ) Toluidin blue staining of liver sections isolated from fed and 24 - h - fasted Tcfeb - LiKO and control mice ( Tcfeb flox / flox mice ) . Arrows indicate lipid droplets . Original magnification × 100 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Oil red O staining of liver sections isolated from mice with the indicated genotype fed ad libitum and 24 h fasted. Original magnification ×40. (b) Toluidin blue staining of liver sections isolated from fed and 24-h-fasted Tcfeb-LiKO and control mice (Tcfeb flox/flox mice). Arrows indicate lipid droplets. Original magnification ×100.


( c ) Bar graphs show the quantification of the number of lipid droplets per hepatocyte from electron microscopy analysis . A representative image is shown on the right . Values are mean ± s . d . of at least 10 cells per mouse ( n = 3 mice per group ) . * P ≤ 0 . 05 .

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

(c) Bar graphs show the quantification of the number of lipid droplets per hepatocyte from electron microscopy analysis. A representative image is shown on the right. Values are mean ± s.d. of at least 10 cells per mouse (n = 3 mice per group). *P≤0.05.


( d ) The oxygen consumption rate ( OCR ) in primary hepatocytes isolated from control and Tcfeb - LiKO mice was measured with an XF24 analyser ( Seahorse ) before and after the addition of palmitic acid ( 0 . 2 mM ) conjugated with BSA . The vertical red line indicates the time at which palmitate was added to cells . Values are mean ± s . d . for 3 independent experiments * P ≤ 0 . 05 .

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

(d) The oxygen consumption rate (OCR) in primary hepatocytes isolated from control and Tcfeb-LiKO mice was measured with an XF24 analyser (Seahorse) before and after the addition of palmitic acid (0.2 mM) conjugated with BSA. The vertical red line indicates the time at which palmitate was added to cells. Values are mean ± s.d. for 3 independent experiments *P≤0.05.


( e , f ) Total FFA ( e ) and glycerol ( f ) in the serum isolated from 6 - h - fasted Tcfeb - LiKO and control mice . Values are mean ± s . d . ( n = 5 mice per group ) * P ≤ 0 . 05 compared with controls .

O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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,f) Total FFA (e) and glycerol (f) in the serum isolated from 6-h-fasted Tcfeb-LiKO and control mice. Values are mean ± s.d. (n = 5 mice per group) *P≤0.05 compared with controls.


( g ) Total serum ketones in fed and fasted Tcfeb - LiKO and control mice . Bars are mean ± s . d . for n = 10 mice per group . * * P ≤ 0 . 01 compared with fed control mice .

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

(g) Total serum ketones in fed and fasted Tcfeb-LiKO and control mice. Bars are mean ± s.d. for n = 10 mice per group. **P≤0.01 compared with fed control mice.


( h ) EchoMRI measurement of fat and lean mass in fed and in 24 - and 48 - h - fasted mice expressed as relative percentage to fed ( 100 % in the graph ) . Indicated values are mean ± s . d . for n = 5 mice per group . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 compared with fed control mice .

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

(h) EchoMRI measurement of fat and lean mass in fed and in 24- and 48-h-fasted mice expressed as relative percentage to fed (100% in the graph). Indicated values are mean ± s.d. for n = 5 mice per group. *P≤0.05;**P≤0.01 compared with fed control mice.


( i ) Visceral fat pad mass isolated from 2 - month - old - mice with indicated genotypes . Indicated values are mean ± s . d . for n = 5 mice per group . * P ≤ 0 . 05 ; compared with fed control mice .

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

(i) Visceral fat pad mass isolated from 2-month-old-mice with indicated genotypes. Indicated values are mean ± s.d. for n = 5 mice per group. *P≤0.05; compared with fed control mice.


( a ) Mice with the indicated genotype were kept on a high - fat diet ( HFD ) for 12 weeks when indicated . Gross liver morphology ( upper panel ) , haematoxylin - eosin ( H / E ) ( middle panel ) , and oil red O staining of liver sections ( bottom panel ) . Ctrl , control .

(a) Mice with the indicated genotype were kept on a high-fat diet (HFD) for 12 weeks when indicated. Gross liver morphology (upper panel), haematoxylin-eosin (H/E) (middle panel), and oil red O staining of liver sections (bottom panel). Ctrl, control.


( b , c ) Bar graph showing normalized liver weights ( mean ± s . d . for n = 10 mice per group ; b ) and total lipid content in mice with indicated genotype ( mean ± s . d . for n = 10 ; c ) . * P ≤ 0 . 05 ; * * * P ≤ 0 . 001 compared with control .

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

(b,c) Bar graph showing normalized liver weights (mean ± s.d. for n = 10 mice per group; b) and total lipid content in mice with indicated genotype (mean ± s.d. for n = 10; c). *P≤0.05;***P≤0.001 compared with control.


( d ) Toluidin blue staining of liver sections isolated from Atg7 - LiKO mice injected with HDAd - Ctrl or HDAd - TFEB vector .

O O 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) Toluidin blue staining of liver sections isolated from Atg7-LiKO mice injected with HDAd-Ctrl or HDAd-TFEB vector.


( e , f ) Bar graph showing normalized liver weights ( mean ± s . d . for n = 10 mice per group * * * P ≤ 0 . 001 ; e ) and total lipid content in mice with the indicated genotype ( mean ± s . d . for n = 5 mice per group ; * * * P ≤ 0 . 001 ; NS , not significant ; f ) . Mice injected with an empty HDAd virus behaved as wild - type untreated mice ; therefore , data are not represented in the figure .

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

(e,f) Bar graph showing normalized liver weights (mean ± s.d. for n = 10 mice per group ***P≤0.001; e) and total lipid content in mice with the indicated genotype (mean ± s.d. for n = 5 mice per group; ***P≤0.001; NS, not significant; f). Mice injected with an empty HDAd virus behaved as wild-type untreated mice; therefore, data are not represented in the figure.


( a , b ) Body weight ( a ) and visceral fat mass ( b ) isolated from 2 - month - old - mice with the indicated genotypes . n = 5 mice per group .

O O O O O O O O O O O O O O O O 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,b) Body weight (a) and visceral fat mass (b) isolated from 2-month-old-mice with the indicated genotypes. n = 5 mice per group.


( c - i ) Serum metabolic profile in HDad - TFEB mice compared with control mice .

(c-i) Serum metabolic profile in HDad-TFEB mice compared with control mice.


( j , k ) Respiratory exchange ratio ( RER ; VCO2 / VO2 ; j ) and fatty acid utilization calculated from data in j ( k ) . Values are mean ± s . d ( n = 10 mice per group ) * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; compared with controls . VLDL , very - low - density lipoprotein ; HDL , high - density lipoprotein .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,k) Respiratory exchange ratio (RER; VCO2/VO2; j) and fatty acid utilization calculated from data in j (k). Values are mean ± s.d (n = 10 mice per group) *P≤0.05;**P≤0.01; compared with controls. VLDL, very-low-density lipoprotein; HDL, high-density lipoprotein.


( a ) Body weight curves of male mice fed with the high - fat diet ( HFD ; 40 % calories from fat ) for 10 weeks starting from 5 weeks of age ( 0 on the x axis ) . Mice were injected with HDAd - TFEB either 1 week before ( early inj ) , or 4 weeks after , ( late inj ) being placed on the high - fat diet , as indicated by the arrows . Values are represented as percentages of weight increase . Ctrl , control . ( b ) Whole - body composition analysis ( EchoMRI ) of the same mice as in a after 10 weeks of the high - fat diet . In a , b n = 10 mice per group ; bars represent mean ± s . d . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 compared with control high - fat diet group .

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(a) Body weight curves of male mice fed with the high-fat diet (HFD; 40% calories from fat) for 10 weeks starting from 5 weeks of age (0 on the x axis). Mice were injected with HDAd-TFEB either 1 week before (early inj), or 4 weeks after, (late inj) being placed on the high-fat diet, as indicated by the arrows. Values are represented as percentages of weight increase. Ctrl, control. (b) Whole-body composition analysis (EchoMRI) of the same mice as in a after 10 weeks of the high-fat diet. In a,b n = 10 mice per group; bars represent mean ± s.d. *P≤0.05;**P≤0.01; ***P≤0.001 compared with control high-fat diet group.


( c - f ) Total serum insulin , leptin , triglyceride and cholesterol levels in control and HDAd - TFEB mice kept on the high - fat diet for 10 weeks . Value are mean ± s . d . n = 10 add per group . * P ≤ 0 . 05 ; * * * * P ≤ 0 . 0001 compared with control . ( g - i ) Glucose and insulin tolerance tests in control and HDAd - TFEB mice challenged with the high - fat diet for 10 weeks .

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

(c-f) Total serum insulin, leptin, triglyceride and cholesterol levels in control and HDAd-TFEB mice kept on the high-fat diet for 10 weeks. Value are mean ± s.d. n = 10 add per group. *P≤0.05; ****P≤0.0001 compared with control. (g-i) Glucose and insulin tolerance tests in control and HDAd-TFEB mice challenged with the high-fat diet for 10 weeks.


( g , h ) Glucose ( g ) and serum insulin ( h ) levels at the indicated time points after glucose challenge . ( i ) Glucose levels at the indicated time points after insulin challenge . In g - i , values are mean ± s . d . n = 7 mice per group ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 compared with control . Mice injected with an empty HDAd virus behaved as wild - type untreated mice ; therefore , data are not represented in the figure .

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

(g,h) Glucose (g) and serum insulin (h) levels at the indicated time points after glucose challenge. (i) Glucose levels at the indicated time points after insulin challenge. In g-i, values are mean ± s.d. n = 7 mice per group; *P≤0.05;**P≤0.01 compared with control. Mice injected with an empty HDAd virus behaved as wild-type untreated mice; therefore, data are not represented in the figure.


( a ) hlh - 30 quantitative rtPCR showing increased expression of the C . elegans TFEB gene hlh - 30 over a time course of starvation in wild - type animals followed by a rapid decrease to the basal level following re - feeding of the animals ( mean ± s . e . m . of n = 3 ) . * P ≤ 0 . 05 ( t - test , compared with wild - type at t = 0 h ) .

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(a) hlh-30 quantitative rtPCR showing increased expression of the C. elegans TFEB gene hlh-30 over a time course of starvation in wild-type animals followed by a rapid decrease to the basal level following re-feeding of the animals (mean±s.e.m. of n = 3). *P≤0.05 (t-test, compared with wild-type at t = 0 h).


( b ) hlh - 30 3 ' UTR quantitative rtPCR showing expression of hlh - 30 after 12 h starvation in wild - type and hlh - 30 ( tm1978 ) animals ( mean ± s . e . m . of n = 3 ) . * P ≤ 0 . 05 ( t - test , compared with wild - type starved ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) hlh-30 3' UTR quantitative rtPCR showing expression of hlh-30 after 12 h starvation in wild-type and hlh-30(tm1978) animals (mean±s.e.m. of n = 3). *P≤0.05 (t-test, compared with wild-type starved).


( c ) Quantification of oil red O stain in starved animals relative to well - fed counterparts ( mean ± s . e . m . of n = 3 ) . * * P ≤ 0 . 01 ( t - test , compared with wild - type starved ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of oil red O stain in starved animals relative to well-fed counterparts (mean±s.e.m. of n = 3). **P≤0.01 (t-test, compared with wild-type starved).


( d - g ) Representative micrographs of wild - type and hlh - 30 animals after 8 h starvation and stained with oil red O .

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(d-g) Representative micrographs of wild-type and hlh-30 animals after 8 h starvation and stained with oil red O.


( h - k ) Representative TEMs of wild - type and hlh - 30 animals after 24 h starvation . IEC , intestinal epithelial cell ; EPI , epidermis ; BB , brush border ; GON , gonad ; BWM , body wall muscles , aj , apical junction ; ld , lipid droplet . Scale bar , 2 μm .

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

(h-k) Representative TEMs of wild-type and hlh-30 animals after 24 h starvation. IEC, intestinal epithelial cell; EPI, epidermis; BB, brush border; GON, gonad; BWM, body wall muscles, aj, apical junction; ld, lipid droplet. Scale bar, 2 μm.


( l ) Quantitative rtPCR of starvation - induced genes in wild - type and hlh - 30 animals after 12 h starvation ( means ± s . e . m of n = 3 ) . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ( t - test , compared with wild - type starved ) .

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

(l) Quantitative rtPCR of starvation-induced genes in wild-type and hlh-30 animals after 12 h starvation (means ± s.e.m of n = 3). *P≤0.05; **P≤0.01; ***P≤0.001 (t-test, compared with wild-type starved).


( m , n ) hlh - 30 is required for starvation - induced lifespan extension . One representative experiment of 2 independent trials ; error bars represent mean ± s . e . m . Median survival ( MS ) ( wild - type fed ) = 10 d ; MS ( wild - type starved ) = 17 d , P0 . 0001 versus fed using the Log - rank test ; MS ( hlh - 30 fed ) = 7 d ; MS ( hlh - 30 starved ) = 9 d , P0 . 0001 versus fed .

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(m,n) hlh-30 is required for starvation-induced lifespan extension. One representative experiment of 2 independent trials; error bars represent mean±s.e.m. Median survival (MS) (wild-type fed) = 10 d; MS (wild-type starved) = 17 d, P0.0001 versus fed using the Log-rank test; MS (hlh-30 fed) = 7 d; MS (hlh-30 starved) = 9 d, P0.0001 versus fed.


( o ) L1 arrest assay showing survival of wild - type and hlh - 30 starved animals relative to non - starved conditions ( mean ± s . e . m . of n = 3 ) . * * * P ≤ 0 . 001 ( t - test , compared with wild - type starved ) .

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

(o) L1 arrest assay showing survival of wild-type and hlh-30 starved animals relative to non-starved conditions (mean±s.e.m. of n = 3). ***P≤0.001 (t-test, compared with wild-type starved).


( A - B ) Coronal brain section , bregma : - 2 . 88 from The Mouse Brain Atlas shows the site of injection in the CA1 cell body layer of the right hippocampus ( A ) . 8 h post - injection , majority of the injected Fib - Tau - 1N3R - ATTO550 ( red ) is taken up by the corpus callosum of the injected side ( B ) . Images are shown at low ( left , 10X ) and high ( right , 63X ) magnification . Data information : Scale bar , 100µm in the left panel in ( B ) , 10µm everywhere else .

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(A-B) Coronal brain section, bregma: - 2.88 from The Mouse Brain Atlas shows the site of injection in the CA1 cell body layer of the right hippocampus (A). 8 h post-injection, majority of the injected Fib-Tau-1N3R-ATTO550 (red) is taken up by the corpus callosum of the injected side (B). Images are shown at low (left, 10X) and high (right, 63X) magnification. Data information: Scale bar, 100µm in the left panel in (B), 10µm everywhere else.


Representative images of the distribution of Fib - Tau ( red ) 8 h and 24 h after injection . Dendrites are immunolabeled using MAP2 antibody ( green ) . 8 h after injection , both diffused and clustered distribution of exogenous Fib - Tau is observed in stratum oriens and CA1 cell body region No exogenous Fib - Tau is detected in the adjoining cortex , stratum radiatum and entrorhinal cortex 24 h after injection , clustered distribution is primarily restricted to the pyramidal ( CA ) cell body layer ( D ) . Uptake of Fib - Tau is seen in some cells in both stratum oriens and pyramidal cell body layer ( D ) . Paired - t - test performed between ' 8 / 24h - right ' and ' 8 / 24h - left ' ; Unpaired t - test performed between ' 8h - left ' and ' 24h - left ' columns ( n is number of animals : 6 for 8h and 5 for 24h ) ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns = not significant . Note : The images presented in panels C and D were independently acquired images under non - identical exposure setting at low magnification for display purpose Data information : Scale bar ,

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Representative images of the distribution of Fib-Tau (red) 8 h and 24 h after injection. Dendrites are immunolabeled using MAP2 antibody (green). 8 h after injection, both diffused and clustered distribution of exogenous Fib-Tau is observed in stratum oriens and CA1 cell body region No exogenous Fib-Tau is detected in the adjoining cortex, stratum radiatum and entrorhinal cortex 24 h after injection, clustered distribution is primarily restricted to the pyramidal (CA) cell body layer (D). Uptake of Fib-Tau is seen in some cells in both stratum oriens and pyramidal cell body layer (D). Paired-t-test performed between '8/24h-right' and '8/24h-left'; Unpaired t-test performed between '8h-left' and '24h-left' columns (n is number of animals: 6 for 8h and 5 for 24h); ***p<0.001, **p<0.01, *p<0.05, ns= not significant. Note: The images presented in panels C and D were independently acquired images under non-identical exposure setting at low magnification for display purpose Data information: Scale bar, 10µm


Representative images of the distribution of Fib - Tau ( red ) 8 h and 24 h after injection . Dendrites are immunolabeled using MAP2 antibody ( green ) . 8 h after injection , both diffused and clustered distribution of exogenous Fib - Tau is observed in stratum oriens The number of Fib - Tau clusters per µm2 within stratum oriens decreases between 8 h and 24 h post - injection ( G ) . A similar decrease in the total fluorescence ( clusters + diffused ) is observed indicating the diffusion and / or clearance of exogenous Fib - Tau ( H ) . Paired - t - test performed between ' 8 / 24h - right ' and ' 8 / 24h - left ' ; Unpaired t - test performed between ' 8h - left ' and ' 24h - left ' columns ( n is number of animals : 6 for 8h and 5 for 24h ) ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns = not significant . Panels ( E and F ) are shown for same exposure and are used for quantification . Data information : Scale bar

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

Representative images of the distribution of Fib-Tau (red) 8 h and 24 h after injection. Dendrites are immunolabeled using MAP2 antibody (green). 8 h after injection, both diffused and clustered distribution of exogenous Fib-Tau is observed in stratum oriens The number of Fib-Tau clusters per µm2 within stratum oriens decreases between 8 h and 24 h post-injection (G). A similar decrease in the total fluorescence (clusters + diffused) is observed indicating the diffusion and/or clearance of exogenous Fib-Tau (H). Paired-t-test performed between '8/24h-right' and '8/24h-left'; Unpaired t-test performed between '8h-left' and '24h-left' columns (n is number of animals: 6 for 8h and 5 for 24h); ***p<0.001, **p<0.01, *p<0.05, ns= not significant. Panels (E and F) are shown for same exposure and are used for quantification. Data information: Scale bar 10µm


( A - C ) Time and concentration dependent clustering of Fib - Tau in primary neurons . Representative images are shown for certain conditions to illustrate Fib - Tau clustering time dependence ( A , top - row ) and concentration dependence ( A , bottom - row ) . Quantification of the number of Fib - Tau clusters per µm2 ( B ) or fluorescence intensity of clusters At low concentrations ( up to 0 . 72nM ) the density of clusters increased with time ( between 10 and 60 min ) but the increase in intensity was small . At high concentrations of Fib - Tau ( ≥ 1 . 8nM ) , both density and size increased with increasing time . Box - plot represents : median , interquartile range and 10 - 90 % distribution ; One - way ANOVA with Dunnett post - hoc test , number of images analysed from 3 cultures ( from left to right : 25 , 25 , 25 , 25 , 70 , 45 , 45 , 45 , 45 , 45 , 30 , 40 , 40 , 40 , 40 and 40 images ) . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant . Scale bar

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Time and concentration dependent clustering of Fib-Tau in primary neurons. Representative images are shown for certain conditions to illustrate Fib-Tau clustering time dependence (A, top-row) and concentration dependence (A, bottom-row). Quantification of the number of Fib-Tau clusters per µm2 (B) or fluorescence intensity of clusters At low concentrations (up to 0.72nM) the density of clusters increased with time (between 10 and 60 min) but the increase in intensity was small. At high concentrations of Fib-Tau (≥1.8nM), both density and size increased with increasing time. Box-plot represents: median, interquartile range and 10-90% distribution; One-way ANOVA with Dunnett post-hoc test, number of images analysed from 3 cultures (from left to right: 25, 25, 25, 25, 70, 45, 45, 45, 45, 45, 30, 40, 40, 40, 40 and 40 images). Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar 2µm


( D - F ) Single Particle Tracking using Quantum Dots ( SPT - QD ) of biotin tagged Fib - Tau . Representative single molecule trajectories of Fib - Tau following 10 or 60 min exposure are shown ( D ) . Note after 60min exposure ( 0 . 36nM ) , single molecules are more confined suggesting they are trapped and clustered . Quantification of diffusion coefficient ( E ) and explored area ( F , extracted from mean squared displacement ( MSD ) , see methods ) shows that both these parameters decrease after 60min exposure to Fib - Tau . Unpaired t - test , n is averaged value per cells imaged in 3 experiments ( 10 min : 22 , 60 min : 23 ) . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant . Scale bar ,

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(D-F) Single Particle Tracking using Quantum Dots (SPT-QD) of biotin tagged Fib-Tau. Representative single molecule trajectories of Fib-Tau following 10 or 60 min exposure are shown (D). Note after 60min exposure (0.36nM), single molecules are more confined suggesting they are trapped and clustered. Quantification of diffusion coefficient (E) and explored area (F, extracted from mean squared displacement (MSD), see methods) shows that both these parameters decrease after 60min exposure to Fib-Tau. Unpaired t-test, n is averaged value per cells imaged in 3 experiments (10 min: 22, 60 min: 23). Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar, 2µm


( G ) Neurons were exposed for 60 min to Fib - Tau ( 0 . 36nM ) labeled both with biotin and ATTO488 ( red ) . Cell surface exposed biotin was labelled using streptavidin - 550 ( green ) followed by live imaging . Note that most of the clusters of ATTO488 ( red ) are co - labelled with Streptavidin 550 ( green ) indicating that the clusters are at the cell surface . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant . Scale bar ,

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(G) Neurons were exposed for 60 min to Fib-Tau (0.36nM) labeled both with biotin and ATTO488 (red). Cell surface exposed biotin was labelled using streptavidin-550 (green) followed by live imaging. Note that most of the clusters of ATTO488 (red) are co-labelled with Streptavidin 550 (green) indicating that the clusters are at the cell surface. Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar, 5µm


( H - J ) Clearance of Tau clusters from neurons . Neurons were exposed ( 0 . 36nM ) to ATTO 550 labeled Fib - Tau for 10 min and the unbound fibrils were washed . Cells were fixed immediately ( 10min ) or allowed to recover in culture medium for 60 min . Two representative images ( H ) and quantifications ( I , J ) show that following 60 min recovery most of the Tau clusters disappear / dissociate as indicated by a decrease in their density . Box - plot represents : median , interquartile range and 10 - 90 % distribution , Unpaired t - test , n is number of images analyzed from 3 cultures ( 49 images ) . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant . Scale bar

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(H-J) Clearance of Tau clusters from neurons. Neurons were exposed (0.36nM) to ATTO 550 labeled Fib-Tau for 10 min and the unbound fibrils were washed. Cells were fixed immediately (10min) or allowed to recover in culture medium for 60 min. Two representative images (H) and quantifications (I, J) show that following 60 min recovery most of the Tau clusters disappear/dissociate as indicated by a decrease in their density. Box-plot represents: median, interquartile range and 10-90% distribution, Unpaired t-test, n is number of images analyzed from 3 cultures (49 images). Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar 2µm


( A ) Super - resolution STORM images of neuronal membrane after exposure to ATTO647N labeled Fib - Tau ( 0 . 36nM ) . Primary neurons were fixed 10 , 60 , 120 and 240 min after exposure to ATTO647N labeled Fib - Tau . Images rendered with a pixel size of 5nm show both clustered and diffused ( non - clustered ) distribution of exogenous Fib - Tau molecules . Scale bar , 1µm . ( B - F ) Quantification of various parameters from the super - resolution images . Averaged values per image is plotted . Increased " total detections per µm2 " ( B ) and " cluster - density " ( C ) of Fib - Tau bound to neurons with increasing exposure time is observed . No change in the " size of cluster " ( D ) or " number of molecules within a cluster " ( E ) is seen with increased exposure time . The proportion of single molecule events detected within clusters remained nearly constant ( ~ 50 % ) in all conditions ( F ) indicating an equilibrium between clustered and non - clustered Fib - Tau single molecules . Unpaired t - test to compare difference with 10 min , n is number of images ( 8 images ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Super-resolution STORM images of neuronal membrane after exposure to ATTO647N labeled Fib-Tau (0.36nM). Primary neurons were fixed 10, 60, 120 and 240 min after exposure to ATTO647N labeled Fib-Tau. Images rendered with a pixel size of 5nm show both clustered and diffused (non-clustered) distribution of exogenous Fib-Tau molecules. Scale bar, 1µm. (B-F) Quantification of various parameters from the super-resolution images. Averaged values per image is plotted. Increased "total detections per µm2" (B) and "cluster-density" (C) of Fib-Tau bound to neurons with increasing exposure time is observed. No change in the "size of cluster" (D) or "number of molecules within a cluster" (E) is seen with increased exposure time. The proportion of single molecule events detected within clusters remained nearly constant (~ 50%) in all conditions (F) indicating an equilibrium between clustered and non-clustered Fib-Tau single molecules. Unpaired t-test to compare difference with 10 min, n is number of images (8 images). *p<0.05; **p<0.01; ***p<0.001; ns= not significant.


( D ) List of synaptic and plasma membrane proteins with extracellular domains significantly enriched in pull - downs from neurons exposed to Fib - Tau . For each identified protein , the name of the protein , the gene name , the p - value ( t - test with Benjamini - Hochberg correction ) , and the fold change corresponding to the ratio of spectral counts between exposed neuron and control samples are given . In an independent analysis , after 10 min exposure of neurons to biotinylated Fib - Tau , a cross - linking step was performed during 20 min using 1mM of DTSSP added in the culture medium , in order to cross - link the protein complexes formed at the cell surface using a membrane impermeable cross - linker . After cross - linking , proteins were analyzed and identified exactly as non - cross - linked samples . Proteins identified with at least 2 peptides are labeled " + " , the other are labeled " - " .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) List of synaptic and plasma membrane proteins with extracellular domains significantly enriched in pull-downs from neurons exposed to Fib-Tau. For each identified protein, the name of the protein, the gene name, the p-value (t-test with Benjamini-Hochberg correction), and the fold change corresponding to the ratio of spectral counts between exposed neuron and control samples are given. In an independent analysis, after 10 min exposure of neurons to biotinylated Fib-Tau, a cross-linking step was performed during 20 min using 1mM of DTSSP added in the culture medium, in order to cross-link the protein complexes formed at the cell surface using a membrane impermeable cross-linker. After cross-linking, proteins were analyzed and identified exactly as non-cross-linked samples. Proteins identified with at least 2 peptides are labeled "+", the other are labeled "-".


( E ) . Co - immunoprecipitation of exogenous biotin - labelled Fib - Tau with α3 - NKA , GluA2 and GluN1 . α3 - NKA , GluA2 and GluN1 were immunoprecipitated using specific antibodies as described in the methods section . The presence of Fib - Tau in the immunoprecipitate was assessed using a slot blot apparatus and nitrocellulose membranes probed with streptavidin - HRP . A 2 . 4 - , 2 . 3 - , and 1 . 8 - fold enrichment in Tau band - intensity is observed in α3 - NKA , GluA2 and GluN1 immunoprecipitates , respectively , compared to controls performed with pre - immune goat or rabbit IgGs . Co - immunoprecipitates of exogenous biotin - labelled Fib - Tau with anti α3 - NKA , GluA2 and GluN1 specific antibodies were also subjected to SDS - PAGE and Western blot analysis . The presence of Fib - Tau in the immunoprecipitates was assessed by probing the nitrocellulose membranes with streptavidin - HRP . Fib - Tau co - immunoprecipitates with α3 - NKA and GluA2 - AMPA receptor but not with GluN1 - NMDA receptor .

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

(E). Co-immunoprecipitation of exogenous biotin-labelled Fib-Tau with α3-NKA, GluA2 and GluN1. α3-NKA, GluA2 and GluN1 were immunoprecipitated using specific antibodies as described in the methods section. The presence of Fib-Tau in the immunoprecipitate was assessed using a slot blot apparatus and nitrocellulose membranes probed with streptavidin-HRP. A 2.4-, 2.3-, and 1.8- fold enrichment in Tau band-intensity is observed in α3-NKA, GluA2 and GluN1 immunoprecipitates, respectively, compared to controls performed with pre-immune goat or rabbit IgGs. Co-immunoprecipitates of exogenous biotin-labelled Fib-Tau with anti α3-NKA, GluA2 and GluN1 specific antibodies were also subjected to SDS-PAGE and Western blot analysis. The presence of Fib-Tau in the immunoprecipitates was assessed by probing the nitrocellulose membranes with streptavidin-HRP. Fib-Tau co-immunoprecipitates with α3-NKA and GluA2-AMPA receptor but not with GluN1-NMDA receptor.


( A , B ) Primary neurons were exposed to Fib - Tau - ATTO550 ( 0 . 36nM , red ) for 10 ( top row ) or 60 min ( bottom row ) and excitatory ( Homer , green ) and inhibitory synapses ( Gephyrin , blue ) were immuno - labelled . Fib - Tau clusters co - localized and / or apposed to Homer ( arrowheads ) but not Gephyrin ( A ) . Quantitative analysis shows that 10 to 20 % of Fib - Tau clusters are localized at Homer containing synapses ( B ) . Box - plot represents : median , interquartile range and 10 - 90 % distribution , Unpaired t - test to compare difference between 10 and 60 min , n is number of images analyzed from 3 - experiments ( 10 min : 49 ; 60 min : 50 ) . Scale bar , 5µm .

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(A, B) Primary neurons were exposed to Fib-Tau-ATTO550 (0.36nM, red) for 10 (top row) or 60 min (bottom row) and excitatory (Homer, green) and inhibitory synapses (Gephyrin, blue) were immuno-labelled. Fib-Tau clusters co-localized and/or apposed to Homer (arrowheads) but not Gephyrin (A). Quantitative analysis shows that 10 to 20% of Fib-Tau clusters are localized at Homer containing synapses (B). Box-plot represents: median, interquartile range and 10-90% distribution, Unpaired t-test to compare difference between 10 and 60 min, n is number of images analyzed from 3-experiments (10 min: 49; 60 min: 50). Scale bar, 5µm.


Exposure of neurons to Fib - Tau - ATTO550 ( 0 . 36nM , 60 min , red ) and immuno - labelling of excitatory synapse ( Anti - Rabbit - Homer or Anti - Mouse - PSD , blue ) and α3 - NKA or GluA1 - AMPA or GluA2 - AMPA or GluN1 - NMDA or GluN2B - NMDA subunits ( green , post - permeabilization ) . Arrows indicate excitatory synapses where Fib - Tau and α3 - NKA / AMPA / NMDA are co - localized ( C ) . Mann - Whitney - test , n is number of images analyzed from 3 - 4 experiments ( α3 - NKA : 75 ; GluA1 : 95 ; GluA2 : 50 ; GluN1 / N2B : 45 ) .

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

Exposure of neurons to Fib-Tau-ATTO550 (0.36nM, 60 min, red) and immuno-labelling of excitatory synapse (Anti-Rabbit-Homer or Anti-Mouse-PSD, blue) and α3-NKA or GluA1-AMPA or GluA2-AMPA or GluN1-NMDA or GluN2B-NMDA subunits (green, post-permeabilization). Arrows indicate excitatory synapses where Fib-Tau and α3-NKA/AMPA/NMDA are co-localized (C). Mann-Whitney-test, n is number of images analyzed from 3-4 experiments (α3-NKA: 75; GluA1: 95; GluA2: 50; GluN1/N2B: 45).