Wnt inhibition through ETC - 159 does not change expression of HR FA pathway genes in Panc 08 . 13 . Panc 08 . 13 were cultured in low adherence plates treated with ETC - 159 for 72 hours . Total was then isolated expression of indicated genes was measured by qRT - PCR . 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 downregulation of HR 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 expression of indicated genes was measured by qRT - PCR . ETC - 159 induced reduction in expression of HR FA pathway genes was blocked in xenografts with stabilized β - catenin . 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 olaparib synergistically inhibit growth of COLO320HSR colorectal cancer with APC mutation . COLO320HSR were plated at a low density treated with G007 - LK , olaparib or combination of two inhibitors at an equivalent dose as described in Figure 1A . ( E ) Representative image from two independent experiments is shown . ( F ) Combination Index ( CI ) values of olaparib G007 - LK calculated from two independent experiments using 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 expression of MYBL2 in HPAF - II . HPAF - II were either treated with ETC - 159 ( 100 nM ) or transfected with siRNA against β - catenin alone or in presence of ETC - 159 for 48 hours . relative expression of MYBL2 as measured by qRT - PCR is shown . 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 repair genes in Wnt - addicted is regulated by MYBL2 . HPAF - II were transfected with two independent siRNAs against MYBL2 or treated with ETC - 159 ( 100 nM ) for 48 hours . Total was isolated expression of MYBL2 repair genes was measured by qRT - PCR . Data are representative of three independent experiments . 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 colorectal cancer PDX ( as measured by - 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 ( measured by qRT - PCR ) . p - values were calculated with Mann - Whitney U . Each data point represents an or replicate . n = 4 - 6 samples / group . horizontal lines represent median of 4 - 6 replicates with upper lower edges of boxes representing 75th 25th percentile of data respectively 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 of sections from four treatment groups ( treated for 21 days ) stained for SA - β - galactosidase , a senescence marker , counterstained with fast red . Blue color indicates positive for senescent . horizontal lines represent mean of replicates . p - values were calculated by Mann - Whitney U .

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

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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 double strand breaks induced by olaparib treatment . ( I ) Representative immunofluorescence of 53BP1 foci ( red ) 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 of sections of small intestine from C57BL / 6J mice for repair shows that expression of BRCA1 , FANCD2 RAD51 was high in crypts ( Wnt high compartment ) whereas villi did not show any . Small intestinal adenomas from APCmin / + mice displayed positive immunostaining for HR FA pathway BRCA1 , FANCD2 RAD51 . bottom panels are higher magnification of areas marked in 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 expression of HR FA pathway in normal mouse intestine . C57BL / 6J mice were treated with vehicle or ETC - 159 ( 60 mg / kg ) for 3 days . Small intestines were sectioned analyzed by immunostaining for BRCA1 , FANCD2 , RAD51 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 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 of liver kidney frozen sections isolated from fed 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 of TFEB in wild - type HeLa , MEFs hepatocytes after 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 of transfected hTFEB - FLAG , Tcfeb - β - gal fusion transcript endogenous Tcfeb mRNAs in MEFs isolated from control ( + / + ) 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 of TFEB mRNA in control ( Ctrl ) or TFEB - overexpressing HeLa during fasting re - feeding . Primers specific for 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 of Tcfeb mRNA in heterozygous Tcfeb - β - gal / + or control MEFs during fasting re - feeding . Specific primers for 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 of Tcfeb mRNA in heterozygous Tcfeb - β - gal / + or control hepatocytes in fed 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 from liver of mice fed ad libitum or 24 h fasted . CLEAR elements in first intron of Tcfeb genomic are shown as numbered black rectangles as indicated in Supplementary Table S1 . Red rectangles represent exons , ATG indicates first codon ( from mouse Tcfeb isoform b ) . histogram shows amount of immunoprecipitated as detected by quantitative PCR . Values were normalized to input 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 from liver of mice fed ad libitum ( fed ) or 24 h - fasted ( starved ) . CLEAR sites in promoter region of Pgc - 1α are indicated by squares . Numbers indicate ( bp ) of binding element from start codon . Bar graphs show amount of immunoprecipitated as detected by quantitative PCR . Values were normalized to input 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 .

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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 stably overexpressing TFEB cultured in normal starved media . Bar graphs represent mean ± s . d . of n = 3independent experiments * * P ≤ 0 . 01 compared with mock transfected .

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

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(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 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 .

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(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 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 respective controls ( fed or fasted ) .

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

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

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

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

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

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

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(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 .

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

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(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 ) total lipid content in mice with indicated genotype ( mean ± s . d . for n = 10 ; c ) . * P ≤ 0 . 05 ; * * * P ≤ 0 . 001 compared with control .

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(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 of liver sections isolated from Atg7 - LiKO mice injected with HDAd - Ctrl or HDAd - TFEB vector .

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

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

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(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 .

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


( j , k ) Respiratory exchange ( RER ; VCO2 / VO2 ; j ) 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 .

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(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 ) curves of mice fed with high - fat diet ( HFD ; 40 % calories from fat ) for 10 starting from 5 of ( 0 on x axis ) . Mice were injected with HDAd - TFEB either 1 week before ( early inj ) , or 4 after , ( late inj ) being placed on high - fat diet , as indicated by arrows . Values are represented as percentages of increase . Ctrl , control . ( b ) Whole - composition ( EchoMRI ) of same mice as in a after 10 of 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 .

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

(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 cholesterol in control HDAd - TFEB mice kept on high - fat diet for 10 . Value are mean ± s . d . n = 10 add per group . * P ≤ 0 . 05 ; * * * * P ≤ 0 . 0001 compared with control . ( g - i ) Glucose insulin tolerance in control HDAd - TFEB mice challenged with high - fat diet for 10 .

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(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 ) serum insulin ( h ) at indicated time points after glucose challenge . ( i ) Glucose at 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 behaved as wild - type untreated mice ; therefore , data are not represented in 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 C . elegans TFEB gene hlh - 30 over a time course of starvation in wild - type followed by a rapid decrease to basal level following re - feeding of ( mean ± s . e . m . of n = 3 ) . * P ≤ 0 . 05 ( t - , compared with wild - type at t = 0 h ) .

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

(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 hlh - 30 ( tm1978 ) ( mean ± s . e . m . of n = 3 ) . * P ≤ 0 . 05 ( t - , compared with wild - type starved ) .

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

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

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(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 of wild - type hlh - 30 after 8 h starvation 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 hlh - 30 after 24 h starvation . IEC , intestinal epithelial ; EPI , epidermis ; BB , brush border ; GON , gonad ; BWM , wall , aj , apical junction ; ld , lipid droplet . Scale bar , 2 μm .

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(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 hlh - 30 after 12 h starvation ( means ± s . e . m of n = 3 ) . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ( t - , compared with wild - type starved ) .

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(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 Log - rank ; MS ( hlh - 30 fed ) = 7 d ; MS ( hlh - 30 starved ) = 9 d , P0 . 0001 versus fed .

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

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

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(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 Mouse Brain Atlas shows site of injection in CA1 layer of right hippocampus ( A ) . 8 h post - injection , majority of injected Fib - Tau - 1N3R - ATTO550 ( red ) is taken up by corpus callosum of injected side ( B ) . are shown at low ( left , 10X ) high ( right , 63X ) magnification . Data information : Scale bar , 100µm in 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 of distribution of Fib - Tau ( red ) 8 h 24 h after injection . Dendrites are immunolabeled using MAP2 antibody ( green ) . 8 h after injection , both diffused clustered distribution of exogenous Fib - Tau is observed in stratum oriens CA1 region No exogenous Fib - Tau is detected in adjoining cortex , stratum radiatum entrorhinal cortex 24 h after injection , clustered distribution is primarily restricted to pyramidal ( CA ) layer ( D ) . Uptake of Fib - Tau is seen in some in both stratum oriens pyramidal layer ( D ) . Paired - t - performed between ' 8 / 24h - right ' ' 8 / 24h - left ' ; Unpaired t - performed between ' 8h - left ' ' 24h - left ' columns ( n is number of : 6 for 8h 5 for 24h ) ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns = not significant . Note : presented in panels C D were independently acquired under non - identical exposure setting at low magnification for display purpose Data information : Scale bar ,

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

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

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

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(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 clustered . of diffusion coefficient ( E ) explored ( F , extracted from mean squared displacement ( MSD ) , see methods ) shows that both these parameters decrease after 60min exposure to Fib - Tau . Unpaired t - , n is averaged value per 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 ,

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

(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 ATTO488 ( red ) . surface exposed biotin was labelled using streptavidin - 550 ( green ) followed by live imaging . Note that most of clusters of ATTO488 ( red ) are co - labelled with Streptavidin 550 ( green ) indicating that clusters are at 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 unbound fibrils were washed . were fixed immediately ( 10min ) or allowed to recover in culture medium for 60 min . Two representative ( H ) quantifications ( I , J ) show that following 60 min recovery most of Tau clusters disappear / dissociate as indicated by a decrease in their density . Box - plot represents : median , interquartile range 10 - 90 % distribution , Unpaired t - , n is number of analyzed from 3 cultures ( 49 ) . 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 of neuronal after exposure to ATTO647N labeled Fib - Tau ( 0 . 36nM ) . neurons were fixed 10 , 60 , 120 240 min after exposure to ATTO647N labeled Fib - Tau . rendered with a pixel of 5nm show both clustered diffused ( non - clustered ) distribution of exogenous Fib - Tau molecules . Scale bar , 1µm . ( B - F ) of various parameters from super - resolution . Averaged values per image is plotted . Increased " total detections per µm2 " ( B ) " cluster - density " ( C ) of Fib - Tau bound to neurons with increasing exposure time is observed . No change in " of cluster " ( D ) or " number of molecules within a cluster " ( E ) is seen with increased exposure time . proportion of single molecule events detected within clusters remained nearly constant ( ~ 50 % ) in conditions ( F ) indicating an equilibrium between clustered non - clustered Fib - Tau single molecules . Unpaired t - to compare difference with 10 min , n is number of ( 8 ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant .

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(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 plasma with extracellular domains significantly enriched in pull - downs from neurons exposed to Fib - Tau . For each identified , name of , gene name , p - value ( t - with Benjamini - Hochberg correction ) , fold change corresponding to of spectral between exposed neuron control samples are given . In an independent , 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 culture medium , in order to cross - link complexes formed at surface using a impermeable cross - linker . After cross - linking , were analyzed identified exactly as non - cross - linked samples . identified with at least 2 peptides are labeled " + " , other are labeled " - " .

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(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 GluN1 . α3 - NKA , GluA2 GluN1 were immunoprecipitated using specific antibodies as described in methods section . presence of Fib - Tau in immunoprecipitate was assessed using a slot blot apparatus nitrocellulose membranes probed with streptavidin - HRP . A 2 . 4 - , 2 . 3 - , 1 . 8 - fold enrichment in Tau band - is observed in α3 - NKA , GluA2 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 GluN1 specific antibodies were also subjected to SDS - PAGE Western blot . presence of Fib - Tau in immunoprecipitates was assessed by probing nitrocellulose membranes with streptavidin - HRP . Fib - Tau co - immunoprecipitates with α3 - NKA GluA2 - AMPA receptor but not with GluN1 - NMDA receptor .

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(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 ) neurons were exposed to Fib - Tau - ATTO550 ( 0 . 36nM , red ) for 10 ( top row ) or 60 min ( bottom row ) excitatory ( Homer , green ) inhibitory synapses ( Gephyrin , blue ) were immuno - labelled . Fib - Tau clusters co - localized / or apposed to Homer ( arrowheads ) but not Gephyrin ( A ) . Quantitative shows that 10 to 20 % of Fib - Tau clusters are localized at Homer containing synapses ( B ) . Box - plot represents : median , interquartile range 10 - 90 % distribution , Unpaired t - to compare difference between 10 60 min , n is number of 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 ) immuno - labelling of excitatory synapse ( Anti - Rabbit - Homer or Anti - Mouse - PSD , blue ) 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 α3 - NKA / AMPA / NMDA are co - localized ( C ) . Mann - Whitney - , n is number of analyzed from 3 - 4 experiments ( α3 - NKA : 75 ; GluA1 : 95 ; GluA2 : 50 ; GluN1 / N2B : 45 ) .

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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).


Exposure of neurons to Fib - Tau - ATTO550 ( 0 . 36nM , 60 min , red ) immuno - labelling of excitatory synapse ( Anti - Rabbit - Homer or Anti - Mouse - PSD , blue ) α3 - NKA or GluA1 - AMPA or GluA2 - AMPA or GluN1 - NMDA or GluN2B - NMDA subunits ( green , post - permeabilization ) . of ( indicating of clusters of synaptic α3 - NKA / AMPA / NMDA spots ( obtained after thresholding ) following exposure to Fib - Tau showed a reduction in of α3 - NKA increase in of GluA2 - subunit containing AMPA receptors ( D ) . Box - plot represents : median , interquartile range 10 - 90 % distribution , * * p < 0 . 01 ; ns = not significant . Scale bar , 5µm .

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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). Quantification of the fluorescence intensity (indicating size of clusters of synaptic α3-NKA/AMPA/NMDA spots (obtained after thresholding) following exposure to Fib-Tau showed a reduction in the size of α3-NKA and increase in the size of GluA2-subunit containing AMPA receptors (D). Box-plot represents: median, interquartile range and 10-90% distribution, **p<0.01; ns= not significant. Scale bar, 5µm.


( A - C ) SPT - QD of pHluorin tagged AMPA ( GluA1 GluA2 ) NMDA ( GluN1 GluN2B ) receptor subunits using GFP - antibody pre - coupled to QD655 . neurons were exposed or not to Fib - Tau ( 0 . 36nM , 60 min ) . Cumulative frequency of diffusion coefficient of trajectories ( extracted from MSD plot , inset , A ) show a slow - down in diffusion of GluA2 - subunit but not GluA1 , GluN1 GluN2B subunits ( A ) . To compute statistical differences , diffusion coefficient of trajectories per movie ( from 3 independent experiments ) were averaged ( B ) . To measure statistical differences in MSD , explored ( under curve during 600 - 900ms of MSD plot ) per movie ( from 3 independent experiments ) were averaged ( C ) . Decreased diffusion coefficient explored for GluA2 - AMPA was observed indicating increased confinement concomitant with increased clustering ( Fig 5C ) following exposure to Fib - Tau . Mann - Whitney - , n is number of movies ( left to right ) : 14 , 15 , 14 , 13 , 15 , 15 , 15 , 15 . * * p < 0 . 01 ; ns = not significant .

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(A-C) SPT-QD of pHluorin tagged AMPA (GluA1 and GluA2) and NMDA (GluN1 and GluN2B) receptor subunits using GFP-antibody pre-coupled to QD655. Primary neurons were exposed or not to Fib-Tau (0.36nM, 60 min). Cumulative frequency of diffusion coefficient of all trajectories (extracted from MSD plot, inset, A) show a slow-down in diffusion of GluA2-subunit but not GluA1, GluN1 and GluN2B subunits (A). To compute statistical differences, the diffusion coefficient of all trajectories per movie (from 3 independent experiments) were averaged (B). To measure statistical differences in MSD, the explored area (area under curve during 600-900ms of MSD plot) per movie (from 3 independent experiments) were averaged (C). Decreased diffusion coefficient and area explored for GluA2-AMPA was observed indicating increased confinement concomitant with increased clustering (Fig 5C) following exposure to Fib-Tau. Mann-Whitney-test, n is number of movies (left to right): 14, 15, 14, 13, 15, 15, 15, and 15. **p<0.01; ns= not significant.


( A ) Single Particle Tracking using PALM ( SPT - PALM ) was performed on α3 - NKA - Dendra expressing neurons . A representative image shows an epifluorescence image trajectories ( strands of different colors ) of minimum 5 points in . Scale bars : 1 µm in panel ( A )

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(A) Single Particle Tracking using PALM (SPT-PALM) was performed on α3-NKA-Dendra expressing primary neurons. A representative image shows an epifluorescence image and trajectories (strands of different colors) of minimum 5 points in length. Scale bars: 1 µm in panel (A)


( B , C ) Extraction of diffusion coefficient using mean squared displacement ( MSD ) approach using minimum 5 - point trajectory . Spine shaft were visually identified . An upward shift of MSD curve , indicating reduced confinement was observed following exposure to Fib - Tau ( 0 . 36nM , 60 min ) ( B ) . As expected , an increased diffusion coefficient ( i . e . increased mobility ) ( C ) of α3 - NKA is observed in dendritic shaft following exposure to Fib - Tau . Mann - Whitney - , n = 21 , obtained from 4 independent experiments ; Each dot represents average diffusion coefficient of trajectories value per . Data information : * p < 0 . 05 ; ns = not significant .

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(B, C) Extraction of diffusion coefficient using mean squared displacement (MSD) approach using minimum 5-point trajectory length. Spine and shaft were visually identified. An upward shift of MSD curve, indicating reduced confinement was observed following exposure to Fib-Tau (0.36nM, 60 min) (B). As expected, an increased diffusion coefficient (i.e. increased mobility) (C) of α3-NKA is observed in dendritic shaft following exposure to Fib-Tau. Mann-Whitney-test, n=21 cells, obtained from 4 independent experiments; Each dot represents average diffusion coefficient of all trajectories value per cell. Data information: *p<0.05; ns= not significant.


Extraction of number of diffusive states of α3 - NKA transition rates between these diffusive states using thousands of short ( minimum 2 - point ) trajectories using Hidden Markov Model occupancy time of α3 - NKA in bound - state also decreased whereas that in free - state increased upon neuron exposure to Fib - Tau ( E ) . Box - plot represents : median , interquartile range entire distribution , Mann - Whitney - on averaged value per movie from 4 experiments ( n = 21 ) . Data information : * p < 0 . 05 ; ns = not significant .

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Extraction of the number of diffusive states of α3-NKA and the transition rates between these diffusive states using thousands of short (minimum 2-point) trajectories using Hidden Markov Model The occupancy time of α3-NKA in bound-state also decreased whereas that in free-state increased upon neuron exposure to Fib-Tau (E). Box-plot represents: median, interquartile range and entire distribution, Mann-Whitney-test on averaged value per movie from 4 experiments (n=21 cells). Data information: *p<0.05; ns= not significant.


Extraction of number of diffusive states of α3 - NKA transition rates between these diffusive states using thousands of short ( minimum 2 - point ) trajectories using Hidden Markov Model Dwell - time of α3 - NKA is also reduced in bound state indicating destabilization of native α3 - NKA - complexes following interaction with Fib - Tau ( F ) . Box - plot represents : median , interquartile range entire distribution , Mann - Whitney - on averaged value per movie from 4 experiments ( n = 21 ) . Data information : * p < 0 . 05 ; ns = not significant .

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Extraction of the number of diffusive states of α3-NKA and the transition rates between these diffusive states using thousands of short (minimum 2-point) trajectories using Hidden Markov Model Dwell-time of α3-NKA is also reduced in bound state indicating destabilization of native α3-NKA-complexes following interaction with Fib-Tau (F). Box-plot represents: median, interquartile range and entire distribution, Mann-Whitney-test on averaged value per movie from 4 experiments (n=21 cells). Data information: *p<0.05; ns= not significant.


( G - H ) - surface biotinylation on neurons exposed or not to Fib - Tau ( 1 . 8nM , 60 min ) . amount of α - NKA at surface decreased upon exposure of neurons to Fib - Tau ( G ) . was performed by normalizing western blot band with input . Paired - t - , 3 independent experiments . Data information : * p < 0 . 05 ; ns = not significant .

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(G-H) Cell-surface biotinylation on primary neurons exposed or not to Fib-Tau (1.8nM, 60 min). The amount of α-NKA at cell surface decreased upon exposure of neurons to Fib-Tau (G). Quantification was performed by normalizing the western blot band intensity with input. Paired-t-test, 3 independent experiments. Data information: *p<0.05; ns= not significant.


( I ) Paraformaldehyde fixation α3 - NKA - pHluorin ( green , top row ) reveals endocytic spots otherwise not visible in live neurons ( green , bottom row ) . Many Fib - Tau - ATTO647 ( red ) clusters overlapped ( arrow ) with α3 - NKA - pHluorin spots ( exposure , 0 . 36nM , 60 min ) . Scale bars

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(I) Paraformaldehyde fixation α3-NKA-pHluorin (green, top row) reveals endocytic spots otherwise not visible in live neurons (green, bottom row). Many Fib-Tau-ATTO647 (red) clusters overlapped (arrow) with α3-NKA-pHluorin spots (exposure, 0.36nM, 60 min). Scale bars 2µm


( J ) majority of overlapping Fib - Tau - ATTO647 clusters ( red ) α3 - NKA - pHluorin spots ( green ) localized on top of Clathrin - mRFP ( blue ) . Scale bars :

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(J) The majority of the overlapping Fib-Tau-ATTO647 clusters (red) and α3-NKA-pHluorin spots (green) localized on top of Clathrin-mRFP (blue) puncta. Scale bars: 2µm


DIV21 neurons were co - exposed to fixed of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) increasing concentrations ( given in monomer - equivalent ) of α - Syn fibrils [ in total 5 - experiments were performed with either ATTO647 labeled α - Syn ( shown here in green ) or unlabeled α - Syn A - dependent increase in binding clustering of α - Syn fibrils is seen . Notably , Fib - Tau clustering dramatically increased with increasing concentrations of α -

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DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of α-Syn fibrils [in total 5-experiments were performed with either ATTO647 labeled α-Syn (shown here in green) or unlabeled α-Syn A concentration-dependent increase in the binding and clustering of α-Syn fibrils is seen. Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn


DIV21 neurons were co - exposed to fixed of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) increasing Aβ ( given in monomer - equivalent ) of Aβ ( B ) fibrils [ in total 5 - experiments were performed with either ATTO647 labeled Aβ ( shown here in green ) or unlabeled Aβ fibrils ] . - dependent increase in binding clustering of Aβ fibrils is seen . Notably , Fib - Tau clustering dramatically increased with increasing concentrations of α - Syn , but not Aβ

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DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing Aβ (given in monomer-equivalent) of Aβ (B) fibrils [in total 5-experiments were performed with either ATTO647 labeled Aβ (shown here in green) or unlabeled Aβ fibrils]. concentration-dependent increase in the binding and clustering of Aβ fibrils is seen. Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn, but not Aβ fibrils


DIV21 neurons were co - exposed to fixed of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) increasing concentrations ( given in monomer - equivalent ) of α - Syn fibrils [ in total 5 - experiments were performed with either ATTO647 labeled α - Syn or unlabeled α - Syn A - dependent increase in binding clustering of both α - Syn fibrils is seen . One - Way ANOVA with Dunnett ' s post - hoc was performed to compare different condition to reference condition ( 100nM ) . number of analyzed is 27 , 25 , 25 , 26 28 in ( C ) , from left to right . data originate from 3 to 4 independent experiments . Data information : Box - plot represents : median , interquartile range 10 - 90 % distribution , Mann - Whitney - is performed , n = 40 from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of α-Syn fibrils [in total 5-experiments were performed with either ATTO647 labeled α-Syn or unlabeled α-Syn A concentration-dependent increase in the binding and clustering of both α-Syn fibrils is seen. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the different condition to the reference condition (100nM).The number of images analyzed is 27, 25, 25, 26 and 28 in (C) , from left to right. The data originate from 3 to 4 independent experiments. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


DIV21 neurons were co - exposed to fixed of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) increasing concentrations ( given in monomer - equivalent ) of Aβ fibrils [ in total 5 - experiments were performed with either ATTO647 labeled Aβ or unlabeled Aβ fibrils A - dependent increase in binding clustering of Aβ fibrils is seen . One - Way ANOVA with Dunnett ' s post - hoc was performed to compare different condition to reference condition ( 100nM ) . number of analyzed is 18 , 50 , 50 , 50 , 25 in ( D ) , from left to right . data originate from 3 to 4 independent experiments . Data information : Box - plot represents : median , interquartile range 10 - 90 % distribution , Mann - Whitney - is performed , n = 40 from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of Aβ fibrils [in total 5-experiments were performed with either ATTO647 labeled Aβ or unlabeled Aβ fibrils A concentration-dependent increase in the binding and clustering of Aβ fibrils is seen. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the different condition to the reference condition (100nM).The number of images analyzed is 18, 50, 50, 50, 25 in (D), from left to right. The data originate from 3 to 4 independent experiments. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


Notably , Fib - Tau clustering dramatically increased with increasing concentrations of α - Syn , but not Aβ fibrils in ( E ) includes 5 - experiments ( n = number of analyzed , left to right : 58 , 54 , 50 , 62 , 63 , 65 , 43 , 35 , 60 , 60 , 60 , 35 ; One - Way ANOVA with Dunnette ' s post - hoc was performed to compare of Fib - Tau clusters ) . Data information : Box - plot represents : median , interquartile range 10 - 90 % distribution , Mann - Whitney - is performed , n = 40 from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn, but not Aβ fibrils The quantification in (E) includes all 5-experiments (n = number of images analyzed, left to right: 58, 54, 50, 62, 63, 65, 43, 35, 60, 60, 60, 35; One-Way ANOVA with Dunnette's post-hoc test was performed to compare the size of Fib-Tau clusters). Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


A perfect co - localization between Fib - Tau α - Syn clusters is also observed . proportion of Fib - Tau clusters co - localizing with α - Syn fibrils increased with an increasing of α - Syn fibrils reaching 60 - 70 % overlap A weaker , independent , overlap ( 10 - 20 % ) was observed with Aβ fibrils in ( F ) is derived from 3 to 4 independent experiments . number of analyzed is 27 , 25 , 25 , 26 , 28 for α - Syn 18 , 50 , 50 , 50 , 25 for Aβ from left to right . One - Way ANOVA with Dunnett ' s post - hoc was performed to compare co - localization differences from 100nM . Box - plot represents : median , interquartile range 10 - 90 % distribution . Data information : Box - plot represents : median , interquartile range 10 - 90 % distribution , Mann - Whitney - is performed , n = 40 from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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A perfect co-localization between Fib-Tau and α-Syn clusters is also observed. The proportion of Fib-Tau clusters co-localizing with α-Syn fibrils increased with an increasing concentration of α-Syn fibrils reaching 60-70% overlap A weaker, concentration independent, overlap (10-20%) was observed with Aβ fibrils The quantification in (F) is derived from 3 to 4 independent experiments. The number of images analyzed is 27, 25, 25, 26, 28 for α-Syn and 18, 50, 50, 50, 25 for Aβ from left to right. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the co-localization differences from 100nM concentration. Box-plot represents: median, interquartile range and 10-90% distribution. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


( G - I ) neurons were co - exposed to Fib - Tau ( 0 . 36nM , ATTO550 labelled ) Fib - α - Syn ( 50nM , monomer - equivalent , unlabeled ) for 60min immunolabelled for homer ( G ) or α3 - NKA ( H ) . of Fib - Tau clusters co - localized with homer or α3 - NKA ( I ) . Note an increased co - localization of Fib - Tau with homer in presence of Fib - α - Syn . Data information : Box - plot represents : median , interquartile range 10 - 90 % distribution , Mann - Whitney - is performed , n = 40 from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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(G-I) Primary neurons were co-exposed to Fib-Tau (0.36nM, ATTO550 labelled) and Fib-α-Syn (50nM, monomer-equivalent, unlabeled) for 60min and immunolabelled for homer (G) or α3-NKA (H). Quantification of Fib-Tau clusters co-localized with homer or α3-NKA (I). Note an increased co-localization of Fib-Tau with homer in presence of Fib-α-Syn. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


( A ) A boxplot showing log2 TPM values of HSPA5 gene expression compared between tumors matched normal samples across 23 types from TCGA with normal tissue data available . boxplot indicates median ( central band ) interquartile range ( boxes ) of expression value whiskers indicates values that outside of middle 50 % ( interquartile ) . barplot on top shows number of normal tissue samples for each corresponding type . Asterisks indicate significant differences by student ' s t - after Benjamini - Hochberg multiple testing correction ( FDR < 0 . 05 ) .

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(A) A boxplot showing log2 TPM values of HSPA5 gene expression compared between tumors and matched normal samples across 23 tumor types from TCGA with normal tissue data available. The boxplot indicates the median (central band) and the interquartile range (boxes) of the expression value and whiskers indicates values that outside of the middle 50% (interquartile). The barplot on the top shows the number of normal tissue samples for each corresponding tumor type. Asterisks indicate significant differences by student's t-test after Benjamini-Hochberg multiple testing correction (FDR < 0.05).


( B ) of Xbp1 splicing in DLD1 SKOV3 treated with varying concentrations of Reversine ( Rv ) for 12 , 24 , 48 , 72 hours . Thapsigargin ( Tg ) is shown as positive control .

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(B) Analysis and quantification of Xbp1 splicing in DLD1 and SKOV3 cells treated with varying concentrations of Reversine (Rv) for 12, 24, 48, and 72 hours. Thapsigargin (Tg) is shown as positive control.


( D ) Xbp1 splicing in B16 melanoma eight B16 : MEF fused clonal lines .

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(D) Xbp1 splicing in B16 melanoma cells and eight B16:MEF fused clonal cell lines.


( E ) Detection of mRNA expression ( by RT - PCR ) of UPR - associated genes ( Ern1 , Af4 , Ddit3 Atf6 ) in treated with Reversine ( Rv ) at 4µM ( DLD1 ) or 0 . 5µM ( SKOV3 ) for 6 hours . Data points refer to triplicate samples collected at same time point , run in duplicate , expressed as means ± SD .

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(E) Detection of mRNA expression (by RT-PCR) of UPR-associated genes (Ern1, Af4, Ddit3 and Atf6) in tumor cells treated with Reversine (Rv) at 4µM (DLD1 cells) or 0.5µM (SKOV3 cells) for 6 hours. Data points refer to triplicate samples collected at the same time point, run in duplicate, and expressed as means ± SD.


( F ) Western Blot of activation of IRE1 , PERK ATF branches of UPR in DLD1 SKOV3 treated or not with Rv at 4µM for 24H ( DLD1 ) or 0 . 5µM for 3 days ( SKOV3 ) . Thapsigargin ( Tg ) was used as positive control .

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(F) Western Blot analysis of the activation of IRE1, PERK and ATF branches of the UPR in DLD1 and SKOV3 cells treated or not with Rv at 4µM for 24H (DLD1 cells) or 0.5µM for 3 days (SKOV3 cells). Thapsigargin (Tg) was used as positive control.


( G ) Western Blot of phosphorylation of GCN2 PKR defining integrated stress response ( ISR ) in DLD1 SKOV3 treated or not with Rv as in F ) . starved for 24 hours then treated for 30 min with Calyculin - A ( Cal - A ) were used as positive control .

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(G) Western Blot analysis of phosphorylation of GCN2 and PKR defining the integrated stress response (ISR) in DLD1 and SKOV3 cells treated or not with Rv as in F). Cells starved for 24 hours then treated for 30 min with Calyculin-A (Cal-A) were used as positive control.


( A ) Xbp1 splicing ( left panel ) ( right panel ) of bone marrow - derived macrophages ( BMDM ) cultured in conditioned medium ( CM ) of Rv - treated DLD1 SKOV3 or respective control medium ( culture medium of cancer not treated with Rv ) , CM of fused B16 melanoma or their nonfused parental .

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(A) Xbp1 splicing analysis (left panel) and quantification (right panel) of bone marrow-derived macrophages (BMDM) cultured in conditioned medium (CM) of Rv-treated DLD1 and SKOV3 cells or respective control medium (culture medium of cancer cells not treated with Rv), and CM of fused B16 melanoma cells or their nonfused parental cells.


( B , C ) mRNA expression ( RT - PCR ) of Il6 ( B ) Arg1 ( C ) in BMDM cultured in CM of Rv - treated SKOV3 ( 0 . 5µM for 3 days ) or control , CM of fused B16 or their nonfused parental . Data points refer to triplicate experiments expressed as means ± SD .

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(B, C) mRNA expression (RT-PCR) of Il6 (B) and Arg1 (C) in BMDM cultured in the CM of Rv-treated SKOV3 cells (0.5µM for 3 days) or control cells, and CM of fused B16 cells or their nonfused parental cells. Data points refer to triplicate experiments expressed as means ± SD.


( E ) Flow cytometry of IFNγ Granzyme B production by human T activated by anti - CD3 / anti - CD28 Dynabeads in presence of CM from Rv - treated SKOV3 or control SKOV3 ( cultured without Rv ) . Column graphs represent expression of IFNγ ( upper panel ) Granzyme B ( lower panel ) . Percentages in parenthesis refer to change from control . Data representative triplicate samples at same time point repeated at least twice . Data are expressed as means ± SD .

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(E) Flow cytometry analysis of IFNγ and Granzyme B production by human T cells activated by anti-CD3/anti-CD28 Dynabeads in the presence of CM from Rv-treated SKOV3 cells or control SKOV3 cells (cultured without Rv). Column graphs represent the percentage expression of IFNγ (upper panel) and Granzyme B (lower panel). Percentages in parenthesis refer to change from control. Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.


( F ) Flow cytometry of IFNγ Granzyme B production by human T activated by anti - CD3 / anti - CD28 Dynabeads in complete medium spiked with Rv ( 0 . 5µM ) . Column graphs represent expression of IFNγ ( upper panel ) Granzyme B ( lower panel ) . Data representative triplicate samples at same time point repeated at least twice . Data are expressed as means ± SD .

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(F) Flow cytometry analysis of IFNγ and Granzyme B production by human T cells activated by anti-CD3/anti-CD28 Dynabeads in complete medium spiked with Rv (0.5µM). Column graphs represent the percentage expression of IFNγ (upper panel) and Granzyme B (lower panel). Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.


( G ) IFNγ ( left ) Granzyme B ( right ) production by human T activated by anti - CD3 / anti - CD28 Dynabeads cultured in CM from fused B16 melanoma or their parental non - fused . Percentages in parenthesis refer to change from parental non - fused . Data representative triplicate samples at same time point repeated at least twice . Data are expressed as means ± SD .

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(G) IFNγ (left) and Granzyme B (right) production by human T cells activated by anti-CD3/anti-CD28 Dynabeads cultured in CM from fused B16 melanoma cells or their parental non-fused cells. Percentages in parenthesis refer to change from parental non-fused cells. Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.


A Kinetics of intracellular Ca2 + after EDN stimulation . UACC257 A2058 ( WT ENDRB - KO ) were seeded on 96 well plates , loaded with Fluo - 4 dye stimulated with 100 nM EDN or PBS . Fluo - 4 was monitored over ten minutes ( n = 6 ) . Representative example of three independent experiments .

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A Kinetics of intracellular Ca2+ after EDN stimulation. UACC257 and A2058 (WT and ENDRB-KO) were seeded on 96 well plates, loaded with Fluo-4 dye and stimulated with 100 nM EDN or PBS. Fluo-4 fluorescence was monitored over ten minutes (n = 6). Representative example of three independent experiments.


B Time course of AKT CREB phosphorylation . Melanoma lines were stimulated with 100 nM EDN for 2 - 90 min . AKT CREB phosphorylation expression was evaluated by Western blot . Representative example of three independent experiments .

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B Time course of AKT and CREB phosphorylation. Melanoma cell lines were stimulated with 100 nM EDN for 2-90 min. AKT and CREB phosphorylation and expression was evaluated by Western blot. Representative example of three independent experiments.


D High reproducibility in UACC257 A2058 datasets . CV values were grouped according to to represent dataset specific CV distributions . Numbers lines indicate median .

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D High reproducibility in the UACC257 and A2058 datasets. All CV values were grouped according to cell line to represent dataset specific CV distributions. Numbers and lines indicate the median.


A Phosphopeptide MS1 intensities in UACC257 samples were analysed by PCA . Colours indicate experimental groups shading is mapped to time .

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A Phosphopeptide MS1 intensities in UACC257 samples were analysed by PCA. Colours indicate experimental groups and shading is mapped to time.


C Comparison of kinetics of 242 shared target phosphopeptides . Log2 FC of EDN / Ctr is plotted from top to bottom for each phosphopeptide for both lines with increases shown in green decreases shown in red .

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C Comparison of the kinetics of the 242 shared target phosphopeptides. Log2 FC of EDN/Ctr is plotted from top to bottom for each phosphopeptide for both cell lines with increases shown in green and decreases shown in red.


A , B EDN target phosphopeptides for ( A ) UACC257 ( B ) A2058 were segregated into six clusters each , using c - means clustering ( MFuzz ) . Each represents one phosphopeptide cluster membership is indicated by colour . Most likely kinases for each phosphosite were predicted using NetworKIN enrichment of kinase substrates was calculated for each cluster . Enriched kinases are indicated above each cluster . Note that substrates of these kinases only account for a fraction of phosphosites in a cluster .

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A,B EDN target phosphopeptides for (A) UACC257 and (B) A2058 were segregated into six clusters each, using c-means clustering (MFuzz). Each line represents one phosphopeptide and cluster membership is indicated by colour. Most likely kinases for each phosphosite were predicted using NetworKIN and enrichment of kinase substrates was calculated for each cluster. Enriched kinases are indicated above each cluster. Note that substrates of these kinases only account for a fraction of all phosphosites in a cluster.


C Average MS1 z - score kinetics for 217 predicted CaMKII - α sites that are shared between both lines . For comparison , average ( n = 6 ) calcium abundance kinetics are shown .

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C Average MS1 intensity z-score kinetics for 217 predicted CaMKII-α sites that are shared between both cell lines. For comparison, average (n = 6) calcium abundance kinetics are shown.