( A - B ) mRNA expression of human colonic stem cell markers LGR5 and OLFM4 , n = 5 or 6 independent human samples . Paired student t - test .

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(A-B) mRNA expression of human colonic stem cell markers LGR5 and OLFM4, n=5 or 6 independent human samples. Paired student t-test.


( C ) Representative images of LGR5 ISH in human organoids treated with DMSO or TCDD . Scale bar 50 µm .

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(C) Representative images of LGR5 ISH in human organoids treated with DMSO or TCDD. Scale bar 50 µm.


mRNA expression of target genes , n = 5 or 6 independent human samples . Paired student t - test .

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mRNA expression of target genes, n=5 or 6 independent human samples. Paired student t-test.


( H ) Representative immunoblots for FoxM1 and PLK1 protein derived from organoids treated with DMSO or TCDD from n = 6 independent samples . Total protein was used as a loading control .

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(H) Representative immunoblots for FoxM1 and PLK1 protein derived from organoids treated with DMSO or TCDD from n=6 independent samples. Total protein was used as a loading control.


( I ) Determination of luciferase reporter activity of human FoxM1 in SW48 and Caco2 cell lines treated with DMSO or TCDD . n = 3 - 4 per group .

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(I) Determination of luciferase reporter activity of human FoxM1 in SW48 and Caco2 cell lines treated with DMSO or TCDD. n=3-4 per group.


( J ) FOXM1 and CYP1A1 mRNA expression in different AhR WT and CRISPR KO clones treated with TCDD for 1 d . C56 and C61 designate different human subjects .

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(J) FOXM1 and CYP1A1 mRNA expression in different AhR WT and CRISPR KO clones treated with TCDD for 1 d. C56 and C61 designate different human subjects.


( K ) Validation of CRISPR KO clones was assessed by PCR amplification using primers flanking the targeted AhR exon . Subsequent sequencing revealed indels at the expected locations .

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(K) Validation of CRISPR KO clones was assessed by PCR amplification using primers flanking the targeted AhR exon. Subsequent sequencing revealed indels at the expected locations.


( L ) The association of FoxM1 and AhR mRNA expression in human normal colon biopsies ( n = 304 ) . AhR low ( n = 152 ) is defined as below its median expression ; AhR high ( n = 152 ) is defined as above its median expression . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles .

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(L) The association of FoxM1 and AhR mRNA expression in human normal colon biopsies (n=304). AhR low (n=152) is defined as below its median expression; AhR high (n=152) is defined as above its median expression. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.


( M ) Correlation between FoxM1 and Cyp1b1 mRNA expression in human normal colon biopsies ( n = 303 ) analyzed by Spearman rank correlation .

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(M) Correlation between FoxM1 and Cyp1b1 mRNA expression in human normal colon biopsies (n=303) analyzed by Spearman rank correlation.


( N ) Representative brightfield images of human colonic organoids from sorted live cells at day 6 , scale bar 200 μm .

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(N) Representative brightfield images of human colonic organoids from sorted live cells at day 6, scale bar 200 μm.


( O , P ) ( O ) Human organoid forming efficiency , and ( P ) organoid diameter from sorted individual cells from human colonic organoids in the presence of DMSO , TCDD and CH223191 ( 10 μM ) for 6 d . n = 5 per group , 3 independent experiments . Grey line indicates mean in ( P ) . Treatments not sharing a common letter are significantly different , p < 0 . 05 .

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(O, P) (O) Human organoid forming efficiency, and (P) organoid diameter from sorted individual cells from human colonic organoids in the presence of DMSO, TCDD and CH223191 (10 μM) for 6 d. n=5 per group, 3 independent experiments. Grey line indicates mean in (P). Treatments not sharing a common letter are significantly different, p<0.05.


( Q ) Representative brightfield images of human colonic organoids treated with DMSO or FDI - 6 . FoxM1 inhibition suppressed human colonic organoid growth in a dose - dependent manner , scale bar 200 µm .

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(Q) Representative brightfield images of human colonic organoids treated with DMSO or FDI-6. FoxM1 inhibition suppressed human colonic organoid growth in a dose-dependent manner, scale bar 200 µm.


Survival , gain of weight and mobility plots for Armadillidium vulgare during 75 days post - injection . There were no significant differences between animals injected with wVulC , wDil or control treatment for all these life history traits : Survival ( A ) , Gain of weight ( B ) and Mobility ( C ) .

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Survival, gain of weight and mobility plots for Armadillidium vulgare during 75 days post-injection.There were no significant differences between animals injected with wVulC, wDil or control treatment for all these life history traits: Survival (A), Gain of weight (B) and Mobility (C).


Animals were injected by pure suspension of wVulC , diluted wVulC inoculum ( only for females ) , wDil or control treatment and monitored during 75 days post - injection . ( A ) Gain of weight : Both Wolbachia strains negatively impact this parameter compared to the control treatment . However , wVulC ( i . e pure or 1 / 100 diluted inoculum ) induces a lower growth rate than the native strain

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Animals were injected by pure suspension of wVulC, diluted wVulC inoculum (only for females), wDil or control treatment and monitored during 75 days post-injection. (A) Gain of weight: Both Wolbachia strains negatively impact this parameter compared to the control treatment. However, wVulC (i.e pure or 1/100 diluted inoculum) induces a lower growth rate than the native strain wDil


( B ) Mobility : this parameter is dramatically reduced for animals injected by wVulC compared to those from other treatments . On the opposite , injection of wDil has no significant effect on the host ' s mobility .

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(B) Mobility: this parameter is dramatically reduced for animals injected by wVulC compared to those from other treatments. On the opposite, injection of wDil has no significant effect on the host's mobility.


( C ) Burying behaviour : The level 0 corresponds to the surface of the substrate , negative values correspond to the number of buried animals while positive values correspond to the number of animals found at the surface of the substrate . For control treatment animals , not a single individual rose up at the surface and for individuals injected by wDil only few animals were spotted at the surface . When inoculated by wVulC , all animals rose up to the substrate after 60 days .

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(C) Burying behaviour: The level 0 corresponds to the surface of the substrate, negative values correspond to the number of buried animals while positive values correspond to the number of animals found at the surface of the substrate. For control treatment animals, not a single individual rose up at the surface and for individuals injected by wDil only few animals were spotted at the surface. When inoculated by wVulC, all animals rose up to the substrate after 60 days.


( D ) Survival ( a ) data for females and ( b ) data for males : The injection of wVulC kills all the individuals . However , the males die quicker than the females . No extra - mortalities were reported for injection with wDil compared to control treatment .

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(D) Survival (a) data for females and (b) data for males: The injection of wVulC kills all the individuals. However, the males die quicker than the females. No extra-mortalities were reported for injection with wDil compared to control treatment.


Wolbachia loads in CNS , gonads and hemocytes of Porcellio d . dilatatus and Armadillidium vulgare . ( A ) Comparison between ( i ) P . d . dilatatus in dark grey and A . vulgare in white injected with wVulC and ( ii ) naturally infected A . vulgare in light grey revealed no significant differences for the Wolbachia loads in CNS cells , gonads and hemocytes ( i . e immune cells ) , at both 30 and 60 days post - injection . ( B ) Comparison between ( i ) P . d . dilatatus in dark grey and A . vulgare in white injected with wDil and ( ii ) naturally infected P . d . dilatatus in dim grey revealed no significant differences for the Wolbachia loads in CNS cells at both 30 and 60 days PI . However , at 60 days post - injection , for gonads and hemocytes wDil loads were significantly higher in the native host P . d . dilatatus than in A . vulgare . A global comparison between wDil and wVulC loads showed that wVulC ( A ) always exhibited higher bacterial loads than wDil ( B ) in both host species .

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Wolbachia loads in CNS, gonads and hemocytes of Porcellio d. dilatatus and Armadillidium vulgare.(A) Comparison between (i) P. d. dilatatus in dark grey and A. vulgare in white injected with wVulC and (ii) naturally infected A. vulgare in light grey revealed no significant differences for the Wolbachia loads in CNS cells, gonads and hemocytes (i.e immune cells), at both 30 and 60 days post-injection. (B) Comparison between (i) P. d. dilatatus in dark grey and A. vulgare in white injected with wDil and (ii) naturally infected P. d. dilatatus in dim grey revealed no significant differences for the Wolbachia loads in CNS cells at both 30 and 60 days PI. However, at 60 days post-injection, for gonads and hemocytes wDil loads were significantly higher in the native host P. d. dilatatus than in A. vulgare. A global comparison between wDil and wVulC loads showed that wVulC (A) always exhibited higher bacterial loads than wDil (B) in both host species.


Micrograph of nerve cells of Porcellio d . dilatatus injected by wVulC . These cells are localised in the CNS ganglion . ( A ) At 30 days post - injection , some autolysosomes and bacteria are visible : autophagic process is already quite high compared to control [ N : Nucleus , V : Vacuole , Ap : Autophagosomes ( Autophagic hallmark ) , Al : Autolysosomes , Wo : Wolbachia , * : a Wolbachia which seems to be surrounded by an autolysosome , Arrow head : magnified area ] . ( B ) At 60 days post - injection , the nerve cells are severely damaged by the accumulation of autophagic vesicles ( N : Nucleus , V : Vacuole , Ap : Autophagosomes ( Autophagic hallmark ) , Al : Autolysosomes , Arrow head : magnified area and probably a Wolbachia in autophagic degradation ) .

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Micrograph of nerve cells of Porcellio d. dilatatus injected by wVulC.These cells are localised in the CNS ganglion. (A) At 30 days post-injection, some autolysosomes and bacteria are visible: autophagic process is already quite high compared to control [N: Nucleus, V: Vacuole, Ap: Autophagosomes (Autophagic hallmark), Al: Autolysosomes, Wo: Wolbachia, *: a Wolbachia which seems to be surrounded by an autolysosome, Arrow head: magnified area]. (B) At 60 days post-injection, the nerve cells are severely damaged by the accumulation of autophagic vesicles (N: Nucleus, V: Vacuole, Ap: Autophagosomes (Autophagic hallmark), Al: Autolysosomes, Arrow head: magnified area and probably a Wolbachia in autophagic degradation).


Micrograph and LC3B antibody labelling of adipocytes neighboring the nerve cells of Porcellio d . dilatatus . These adipocytes are localised around the nerve cells of the CNS and constitutes the sheathing of this organ . ( A ) At 30 days post - injection , autolysosomes , autophagosomes but also bacteria were observed : autophagic process was already higher compared to the control ( Ad : adipocyte , Ap : Autophagosomes ( Autophagic hallmark ) , Al : Autolysosomes , Wo : Wolbachia , Arrow head : magnified area ) . ( B ) At 60 days post - injection , the adipocytes were severely damaged by the accumulation of autophagic vesicles ( Ad : adipocyte , Ap : Autophagosomes , Al : Autolysosomes , Wo : Wolbachia , Arrow head : magnified area ) . ( C ) With the antibody labelling anti - LC3B , the protein LC3B implicated in autophagic processes was detected in adipocytes of P . d . dilatatus injected with wVulC at 45 days post - injection . The highly labeled spherical structures would reflect the incorporation of the LC3B protein in the phagophores . ( D ) With adipocytes of P . d . dilatatus injected by wDil but also in other tested situations , only really few of these structures were detected by epiflorescent microscopy .

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Micrograph and LC3B antibody labelling of adipocytes neighboring the nerve cells of Porcellio d. dilatatus.These adipocytes are localised around the nerve cells of the CNS and constitutes the sheathing of this organ. (A) At 30 days post-injection, autolysosomes, autophagosomes but also bacteria were observed: autophagic process was already higher compared to the control (Ad: adipocyte, Ap: Autophagosomes (Autophagic hallmark), Al: Autolysosomes, Wo: Wolbachia, Arrow head: magnified area). (B) At 60 days post-injection, the adipocytes were severely damaged by the accumulation of autophagic vesicles (Ad: adipocyte, Ap: Autophagosomes, Al: Autolysosomes, Wo: Wolbachia, Arrow head: magnified area). (C) With the antibody labelling anti-LC3B, the protein LC3B implicated in autophagic processes was detected in adipocytes of P. d. dilatatus injected with wVulC at 45 days post-injection. The highly labeled spherical structures would reflect the incorporation of the LC3B protein in the phagophores. (D) With adipocytes of P. d. dilatatus injected by wDil but also in other tested situations, only really few of these structures were detected by epiflorescent microscopy.


( A ) Western analysis for on - target effects for Everolimus ( EV , P - RPS6 ) and CX - 5461 ( p53 ) . Each lane represents equal amounts of protein from lymph node tissue isolated from a single mouse that received drug vehicles ( everolimus vehicle : 1 % methylcellulose ; CX - 5461 vehicle : 25 mM NaH2PO 4 ) ( V / V ; mouse # 1 - 6 ) , 5 mg / kg everolimus ( EV ; mouse # 7 - 12 ) , 35 mg / kg CX - 5461 ( mouse # 13 - 18 ) or both drugs ( CX - 5461 + EV ; mouse # 19 - 24 ) for 2 hours ( n = 6 per treatment group ) . Actin was used as a loading control .

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(A) Western analysis for on-target effects for Everolimus (EV, P-RPS6) and CX-5461 (p53). Each lane represents equal amounts of protein from lymph node tissue isolated from a single mouse that received drug vehicles (everolimus vehicle: 1% methylcellulose; CX-5461 vehicle: 25 mM NaH2PO4) (V/V; mouse #1-6), 5 mg/kg everolimus (EV; mouse #7-12), 35 mg/kg CX-5461 (mouse #13-18) or both drugs (CX-5461+EV; mouse #19-24) for 2 hours (n=6 per treatment group). Actin was used as a loading control.


( C ) Enrichment analysis by MetaCore ® GeneGO of genes in " translation up " ( red ) and " translation down " ( blue ) categories identified by anota2seq analysis comparing lymph node cells isolated from mice in CX - 5461 + EV treatment group with the V / V group ( n = 6 ) . Ratio " values were obtained by dividing the number of significant genes in our data assigned to a molecular process by the total number of genes in the process in MetaCore ® ' s database . ( D ) Genes implicated in " Translation : initiation " and " Translation : Elongation - Termination " processes based on Fig

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(C) Enrichment analysis by MetaCore® GeneGO of genes in "translation up" (red) and "translation down" (blue) categories identified by anota2seq analysis comparing lymph node cells isolated from mice in CX-5461+EV treatment group with the V/V group (n=6). Ratio" values were obtained by dividing the number of significant genes in our data assigned to a molecular process by the total number of genes in the process in MetaCore®'s database. (D) Genes implicated in "Translation: initiation" and "Translation: Elongation-Termination" processes based on Fig 1C


( B ) Cell viability analysis of the indicated early - passage Eµ - Myc lymphoma cells treated with indicated compound ( s ) for 48 hours as determined by Beckman ® Coulter counter .

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(B) Cell viability analysis of the indicated early-passage Eµ-Myc lymphoma cells treated with indicated compound(s) for 48 hours as determined by Beckman® Coulter counter.


( E ) Intracellular ATP levels determined using the liquid chromatography - mass spectrometry in CTRL , EV , CX and CMB early - passage cell lines .

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(E) Intracellular ATP levels determined using the liquid chromatography-mass spectrometry in CTRL, EV, CX and CMB early-passage cell lines.


( F ) Basal extracellular acidification rate ( ECAR ) and oxygen consumption rate ( OCR ) in the early - passage cell lines determined using the Seahorse XF96 Extracellular Flux Analyzer ; graphs represent mean ± SEM of 6 - 8 technical replicates for each biological replicate ( n = 3 ) .

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(F) Basal extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in the early-passage cell lines determined using the Seahorse XF96 Extracellular Flux Analyzer; graphs represent mean ± SEM of 6-8 technical replicates for each biological replicate (n=3).


( G ) Propidium iodide ( PI ) exclusion analysis of early - passage CX - 5461 - everolimus combination therapy - resistant ( CMB ) , CX - 5461 - resistant ( CX ) and drug - naïve ( CTRL ) lymphoma cells treated with indicated concentrations of metformin for 48 hours .

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(G) Propidium iodide (PI) exclusion analysis of early-passage CX-5461-everolimus combination therapy-resistant (CMB), CX-5461-resistant (CX) and drug-naïve (CTRL) lymphoma cells treated with indicated concentrations of metformin for 48 hours.


( H ) CellTiterGLO ® - based assay measuring cellular ATP levels of the CX - 5461 - resistant ( CX ) and CX - 5461 - everolimus combination therapy - resistant ( CMB ) cells treated with metformin as indicated for 48 hours .

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(H) CellTiterGLO®-based assay measuring cellular ATP levels of the CX-5461-resistant (CX) and CX-5461-everolimus combination therapy-resistant (CMB) cells treated with metformin as indicated for 48 hours.


( I ) PI analysis of CTRL and CMB cells treated with CX - 5461 and everolimus in the presence and absence of metformin for 48 hours as indicated .

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(I) PI analysis of CTRL and CMB cells treated with CX-5461 and everolimus in the presence and absence of metformin for 48 hours as indicated.


( J ) PI analysis of CTRL and CX cells treated with CX - 5461 in the presence and absence of metformin for 48 hours as indicated .

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(J) PI analysis of CTRL and CX cells treated with CX-5461 in the presence and absence of metformin for 48 hours as indicated.


( B ) Abundance of intracellular 3 ' 5 ' - cyclic AMP as measured by a liquid chromatography ( LC ) - mass spectrometry ( MS ) analysis . Graphs represent mean ± SEM of n = 3 ( 5 - 6 technical replicates each ) .

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(B) Abundance of intracellular 3'5'-cyclic AMP as measured by a liquid chromatography (LC)-mass spectrometry (MS) analysis. Graphs represent mean ± SEM of n=3 (5-6 technical replicates each).


( C ) Western analysis of EPAC1 and EPAC2 abundance , as well as active GTP - bound RAP1 levels in the indicated early - passage cells during their log - phase growth period ( n = 3 ) . Actin and total RAP1 were used as loading controls .

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(C) Western analysis of EPAC1 and EPAC2 abundance, as well as active GTP-bound RAP1 levels in the indicated early-passage cells during their log-phase growth period (n=3). Actin and total RAP1 were used as loading controls.


( D ) Propidium iodide ( PI ) exclusion assays of CMB cells treated with CX - 5461 and everolimus as indicated in the presence or absence of a selective PKA inhibitor H89 for 48 hours .

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(D) Propidium iodide (PI) exclusion assays of CMB cells treated with CX-5461 and everolimus as indicated in the presence or absence of a selective PKA inhibitor H89 for 48 hours.


( E ) PI exclusion analysis of early - passage drug - naïve ( CTRL ) lymphoma cells treated with CX - 5461 and everolimus in the presence or absence of selective PKA activator 6 - Bnz - cAMP for 48 hours .

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(E) PI exclusion analysis of early-passage drug-naïve (CTRL) lymphoma cells treated with CX-5461 and everolimus in the presence or absence of selective PKA activator 6-Bnz-cAMP for 48 hours.


( F ) PI exclusion analysis of the CMB cells treated with CX - 5461 and everolimus as indicated in the presence or absence of EPAC1 inhibitor CE3F4 or EPAC2 inhibitor ESI - 05 for 48 hours .

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(F) PI exclusion analysis of the CMB cells treated with CX-5461 and everolimus as indicated in the presence or absence of EPAC1 inhibitor CE3F4 or EPAC2 inhibitor ESI-05 for 48 hours.


( G ) PI exclusion analysis of early - passage drug - naïve ( CTRL ) cells treated with CX - 5461 and everolimus in the presence or absence of the selective EPAC activator 8 - pCPT - 2 - O - Me - cAMP for 48 hours .

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(G) PI exclusion analysis of early-passage drug-naïve (CTRL) cells treated with CX-5461 and everolimus in the presence or absence of the selective EPAC activator 8-pCPT-2-O-Me-cAMP for 48 hours.


( A ) A heatmap illustrating the abundances of polysome - associated mRNAs that are associated with the mRNA translation / protein synthesis based on the Gene Ontology Resource Database in drug - naïve ( CTRL ) or CX - 5461 - everolimus combination therapy - resistant ( CMB ) cell lines ( n = 3 ) . The colours illustrate the expression values of indicated mRNAs that were normalized using voom ( red : high expression ; blue : low expression ) .

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(A) A heatmap illustrating the abundances of polysome-associated mRNAs that are associated with the mRNA translation/protein synthesis based on the Gene Ontology Resource Database in drug-naïve (CTRL) or CX-5461-everolimus combination therapy-resistant (CMB) cell lines (n=3). The colours illustrate the expression values of indicated mRNAs that were normalized using voom (red: high expression; blue: low expression).


( B ) Polysome profiles demonstrating increased ribosome abundance in CMB cells as compared to CTRL and CX - 5461 - resistant ( CX ) cells ( representatives of n = 3 ) . ( C ) Quantitation of polysome : monosome ratio in the indicated early - passage Eµ - Myc lymphoma cells as determined by measuring the area under the curve using ImageJ software . Graph represents mean ± SEM of n = 3 .

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(B) Polysome profiles demonstrating increased ribosome abundance in CMB cells as compared to CTRL and CX-5461-resistant (CX) cells (representatives of n=3). (C) Quantitation of polysome:monosome ratio in the indicated early-passage Eµ-Myc lymphoma cells as determined by measuring the area under the curve using ImageJ software. Graph represents mean ± SEM of n=3.


( F ) Polysomal abundance of mRNAs ( selected based on a nominal p - value < 0 . 01 cut - off in either the CMB vs . CTRL or CX vs . CTRL as analysed by limma ) encoding components of mitochondrial oxidative phosphorylation based on polysome profiling data . Red denotes upregulation , blue denotes downregulation ( n = 3 ; genes with an adjusted P value ( false discovery rate ; FDR ) ≤ 0 . 05 were considered to be significant , and are denoted with a black border ) .

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(F) Polysomal abundance of mRNAs (selected based on a nominal p-value < 0.01 cut-off in either the CMB vs. CTRL or CX vs. CTRL as analysed by limma) encoding components of mitochondrial oxidative phosphorylation based on polysome profiling data. Red denotes upregulation, blue denotes downregulation (n=3; genes with an adjusted P value (false discovery rate; FDR) ≤ 0.05 were considered to be significant, and are denoted with a black border).


( A ) Western analysis demonstrating the effects of metformin treatment for 48 hours on the levels of active GTP - bound RAP1 in CX and CMB cells ( n = 3 ) and its quantitation .

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(A) Western analysis demonstrating the effects of metformin treatment for 48 hours on the levels of active GTP-bound RAP1 in CX and CMB cells (n=3) and its quantitation.


( B , C ) Proportion of green fluorescent protein ( GFP ) - positive CMB ( clone # 8 ) cells in ( B ) lymph node and ( C ) spleen of transplanted C57BL / 6 mice treated as indicated for 6 hours on day 12 post - transplant .

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(B, C) Proportion of green fluorescent protein (GFP)-positive CMB (clone #8) cells in (B) lymph node and (C) spleen of transplanted C57BL/6 mice treated as indicated for 6 hours on day 12 post-transplant.


( D ) Kaplan - Meier curve of C57BL / 6 mice transplanted with CX - 5461 - everolimus - resistant ( CMB # 8 ) early - passage Eµ - Myc lymphoma cells treated with vehicles ( everolimus vehicle : 1 % methylcellulose ; CX - 5461 / metformin vehicle : 25 mM NaH2PO 4 ) ( n = 6 ) ; CX - 5461 ( 35 mg / kg every twice weekly ) and everolimus ( 5 mg / kg daily ) ( n = 8 ) , metformin ( 400 mg / kg twice daily ) ( n = 6 ) , or CX - 5461 , everolimus and metformin ( 35 mg / kg twice weekly , 5 mg / kg daily and 400 mg / kg twice daily , respectively ) ( n = 8 ) . Light grey : 5 - day metformin pre - treatment period , dark grey : treatment period . Data were analyzed by a log - rank ( Mantel - Cox ) test . Vehicle vs . CX - 5461 - everolimus : P = 0 . 0006 , Vehicle vs . CX - 5461 - everolimus - metformin : P = 0 . 0001 . CX - 5461 - everolimus vs . CX - 5461 - everolimus - metformin : P = 0 . 0003 .

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

(D) Kaplan-Meier curve of C57BL/6 mice transplanted with CX-5461-everolimus-resistant (CMB #8) early-passage Eµ-Myc lymphoma cells treated with vehicles (everolimus vehicle: 1% methylcellulose; CX-5461/metformin vehicle: 25 mM NaH2PO4) (n=6); CX-5461 (35 mg/kg every twice weekly) and everolimus (5 mg/kg daily) (n=8), metformin (400 mg/kg twice daily) (n=6), or CX-5461, everolimus and metformin (35 mg/kg twice weekly, 5 mg/kg daily and 400 mg/kg twice daily, respectively) (n=8). Light grey: 5-day metformin pre-treatment period, dark grey: treatment period. Data were analyzed by a log-rank (Mantel-Cox) test. Vehicle vs. CX-5461-everolimus: P = 0.0006, Vehicle vs. CX-5461-everolimus-metformin: P = 0.0001. CX-5461-everolimus vs. CX-5461-everolimus-metformin: P = 0.0003.


a - b , Representative images of cilia ( a ) and dot plots ( b ) showing primary cilia length variation at different time points after serum starvation ( SS ) . Markers in ( a ) show ARL13B ( cilia ) and PCNT ( Basal body ) . The graph shows individual length of cilia from 3 biological replicates ( n > 150 cilia per sample and experiment ) . Scale bar : 1µm . Data shown in include mean ± s . d . are calculated by two - tailed unpaired student t - test .

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a-b, Representative images of cilia (a) and dot plots (b) showing primary cilia length variation at different time points after serum starvation (SS). Markers in (a) show ARL13B (cilia) and PCNT (Basal body). The graph shows individual length of cilia from 3 biological replicates (n>150 cilia per sample and experiment). Scale bar: 1µm. Data shown in include mean ±s.d. are calculated by two-tailed unpaired student t-test.


d , Representative images show cilia segmentation based on acetylated tubulin ( Ac tub ) , polyglutamylated tubulin ( Glu tub ) , ARL13B and PCNT . The cartoon on the left indicates proximal segments ( PS , Glu tub rich - region ) and distal segments ( DS ) . DS were determined by calculating the difference between PS and total cilia length based on ARL13B staining as depicted by the dashed lines on the images . Scale bar : 1 . 5µm . e , Quantification of d . Comparative analysis of average cilia length based on Ac tubulin and ARL13B ( cilia ) staining shown for specified cilia length groups ( 2 < , 2 - 3 , 3 - 4 and > 4µm ) . Three biological replicates , n = 400 cilia per sample and repetition . The numbers above the cartoons show the ratio of the calculated Ac tub / whole cilia length . Data shown include mean ± s . d . are calculated by two - tailed unpaired student t - test .

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d, Representative images show cilia segmentation based on acetylated tubulin (Ac tub), polyglutamylated tubulin (Glu tub), ARL13B and PCNT. The cartoon on the left indicates proximal segments (PS, Glu tub rich-region) and distal segments (DS). DS were determined by calculating the difference between PS and total cilia length based on ARL13B staining as depicted by the dashed lines on the images. Scale bar: 1.5µm. e, Quantification of d. Comparative analysis of average cilia length based on Ac tubulin and ARL13B (cilia) staining shown for specified cilia length groups (2<, 2-3, 3-4 and >4µm). Three biological replicates, n=400 cilia per sample and repetition. The numbers above the cartoons show the ratio of the calculated Ac tub/whole cilia length. Data shown include mean ±s.d. are calculated by two-tailed unpaired student t-test.


a , Representative images of 48h serum starved RPE1 cells treated with solvent control ( DMSO ) , cytochalasin D ( CytoD ) and / or nocodazole ( Noco ) for 3h before immunostaining with the indicated antibodies . Scale bar : 3µm . b , Quantification of ( a ) showing average length of proximal segment ( PS , Glu tub ) and whole cilia ( ARL13B ) . The numbers above the cartoons show the ratio of the calculated distal segment ( DS ) / whole cilia length . Three biological replicates , n = 100 cilia per sample and repetition . Data shown include mean ± s . d . ; P values are calculated by two - tailed unpaired student t - test .

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a, Representative images of 48h serum starved RPE1 cells treated with solvent control (DMSO), cytochalasin D (CytoD) and/or nocodazole (Noco) for 3h before immunostaining with the indicated antibodies. Scale bar: 3µm. b, Quantification of (a) showing average length of proximal segment (PS, Glu tub) and whole cilia (ARL13B). The numbers above the cartoons show the ratio of the calculated distal segment (DS)/whole cilia length. Three biological replicates, n=100 cilia per sample and repetition. Data shown include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test.


RPE1 cells were treated with the indicated siRNAs before serum starvation for 48h . Representative immunostained images with indicated antibodies Scale bar : 2µm .

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RPE1 cells were treated with the indicated siRNAs before serum starvation for 48h. Representative immunostained images with indicated antibodies Scale bar: 2µm.


e - f , Cells were treated with the indicated siRNAs before serum starvation for 48h and treatment with DMSO or Noco for 3h before immunostaining with the indicated antibodies . Representative images ( e ) and quantification of cilia length based on ARL13B ( f ) are shown . Scale bar : 2µm . Three biological replicates , n = 100 cilia per sample and repetition . Data shown include mean ± s . d . ; P values are calculated by two - tailed unpaired student t - test .

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e-f, Cells were treated with the indicated siRNAs before serum starvation for 48h and treatment with DMSO or Noco for 3h before immunostaining with the indicated antibodies. Representative images (e) and quantification of cilia length based on ARL13B (f) are shown. Scale bar: 2µm. Three biological replicates, n=100 cilia per sample and repetition. Data shown include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test.


a , Images show the localisation of endogeneous SEPT2 and SEPT7 in RPE1 ARLB13B - mRuby2 cells with basal body staining . The insets to the right show enlargements of the cilium . Scale bar : 4µm ( large ) and 2µm ( small ) . b , STED microscopy images showing ciliated RPE1 cells expressing EGFP - SEPT2 or SEPT7 - EGFP and stained for ARL13B . Scale bar : 1µm .

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a, Images show the localisation of endogeneous SEPT2 and SEPT7 in RPE1 ARLB13B-mRuby2 cells with basal body staining. The insets to the right show enlargements of the cilium. Scale bar: 4µm (large) and 2µm (small). b, STED microscopy images showing ciliated RPE1 cells expressing EGFP-SEPT2 or SEPT7-EGFP and stained for ARL13B. Scale bar: 1µm.


d , Representative images showing co - localisation of MKS3 or CEP290 in ciliated RPE1 ARLB13B - mRuby2 and GFP - SEPT2 cells . The white arrows point to the specific MKS3 or CEP290 signals . Scale bar : 1µm . e , Line scan measurements show the normalised signal intensities along the cilium for the indicated proteins as specified in ( d ) ( dashed lines ) .

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d, Representative images showing co-localisation of MKS3 or CEP290 in ciliated RPE1 ARLB13B-mRuby2 and GFP-SEPT2 cells. The white arrows point to the specific MKS3 or CEP290 signals. Scale bar: 1µm. e, Line scan measurements show the normalised signal intensities along the cilium for the indicated proteins as specified in (d) (dashed lines).


f , Effect of nocodazole ( Noco ) treatment ( 3h ) on the localisation of endogenous SEPT2 and SEPT7 at cilia in RPE1 cells . The insets show magnifications of the cilium . Scale bar : 5µm ( large ) and 2 . 5µm ( small ) . g , Relative signal intensity of endogenous SEPT2 or SEPT7 along the cilium from ( f ) . SEPT2 or SEPT7 signals at cilia were normalized to the total cilia length . Three biological replicates , n > 90 cilia per sample and repetition . Data show in ( g ) includes mean ± s . d . ; P values are calculated by two - tailed unpaired student t - test . See source data .

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f, Effect of nocodazole (Noco) treatment (3h) on the localisation of endogenous SEPT2 and SEPT7 at cilia in RPE1 cells. The insets show magnifications of the cilium. Scale bar: 5µm (large) and 2.5µm (small). g, Relative signal intensity of endogenous SEPT2 or SEPT7 along the cilium from (f). SEPT2 or SEPT7 signals at cilia were normalized to the total cilia length. Three biological replicates, n>90 cilia per sample and repetition. Data show in (g) includes mean ±s.d.; P values are calculated by two-tailed unpaired student t-test. See source data.


g , Electron microscopy of RPE1 WT and SEPT2 - KO cilia . Black arrows point to the axoneme and basal body ( BB ) . Scale bar : 400nm . h , Quantification of ( g ) . Only cilia detected from bottom ( BB ) to tip in serial sections were quantified . N > 15 cilia from two independent experiments . Data include mean ± s . d . ; P values are calculated by unpaired Wilcoxon - Mann - Whitney Rank Sum

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g, Electron microscopy of RPE1 WT and SEPT2-KO cilia. Black arrows point to the axoneme and basal body (BB). Scale bar: 400nm. h, Quantification of (g). Only cilia detected from bottom (BB) to tip in serial sections were quantified. N>15 cilia from two independent experiments. Data include mean ±s.d.; P values are calculated by unpaired Wilcoxon-Mann-Whitney Rank Sum Test


i - j , RPE1 WT and SEPT2 - KO cells after 48h SS were treated with solvent control ( DMSO ) , cytochalasin D ( CytoD ) and / or nocodazole ( Noco ) for 3h before immunostaining with the indicated antibodies . Representative images ( i ) and quantifications of PS ( Glu tub ) and whole cilia ( ARL13B ) length ( j ) are shown . The numbers above the cartoons in ( j ) show the ratio of the calculated DS / whole cilia length . Scale bar : 3µm . Three biological replicates , n = 100 cilia per sample and repetition . Data show include mean ± s . d . ; P values are calculated by two - tailed unpaired student t -

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i-j, RPE1 WT and SEPT2-KO cells after 48h SS were treated with solvent control (DMSO), cytochalasin D (CytoD) and/or nocodazole (Noco) for 3h before immunostaining with the indicated antibodies. Representative images (i) and quantifications of PS (Glu tub) and whole cilia (ARL13B) length (j) are shown. The numbers above the cartoons in (j) show the ratio of the calculated DS/whole cilia length. Scale bar: 3µm. Three biological replicates, n=100 cilia per sample and repetition. Data show include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test


quantification of the relative signal intensities for the indicated proteins ( b ) . IFT88 and KIF17 signal intensities were normalized to the whole cilia length . KIF7 signal intensity was quantified at ciliary tip or base Three biological replicates , n > 80 cilia per sample and repetition . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test .

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quantification of the relative signal intensities for the indicated proteins (b). IFT88 and KIF17 signal intensities were normalized to the whole cilia length. KIF7 signal intensity was quantified at ciliary tip or base Three biological replicates, n>80 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


c - d , KIF7 localisation at the tip and base of cilia ( indicated by arrows ) in RPE1 cells after 48h SS treated for 3h with solvent control ( DMSO ) or nocodazole ( Noco ) and quantification of the relative signal intensities for the ciliary base and tip of KIF7 ( d ) . Scale bar : 2µm . KIF7 signal intensity was quantified at the ciliary tip or base as indicated in ( c ) ( white arrows ) . Three biological replicates , at least 80 cilia per sample and repetition . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test .

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c-d, KIF7 localisation at the tip and base of cilia (indicated by arrows) in RPE1 cells after 48h SS treated for 3h with solvent control (DMSO) or nocodazole (Noco) and quantification of the relative signal intensities for the ciliary base and tip of KIF7 (d). Scale bar: 2µm. KIF7 signal intensity was quantified at the ciliary tip or base as indicated in (c) (white arrows). Three biological replicates, at least 80 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


e - f , RPE1 WT and SEPT2 - KO cells were treated with control or KIF7 siRNA after 48h SS and immunostained with the indicated antibodies . Representative images ( e ) and quantifications of proximal segment ( Glu tub ) and whole cilia ( ARL13B ) length ( f ) are shown . The numbers above the cartoons in ( f ) show the ratio of the calculated distal segments / whole cilia length . Three biological replicates , n = 100 cilia per sample and repetition . Scale bar : 2µm . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test .

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e-f, RPE1 WT and SEPT2-KO cells were treated with control or KIF7 siRNA after 48h SS and immunostained with the indicated antibodies. Representative images (e) and quantifications of proximal segment (Glu tub) and whole cilia (ARL13B) length (f) are shown. The numbers above the cartoons in (f) show the ratio of the calculated distal segments /whole cilia length. Three biological replicates, n=100 cilia per sample and repetition. Scale bar: 2µm. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


d , FRAP experiment of SSTR3 - GFP in RPE1 WT and SEPT2 KO cells . Cells were starved for 48h before the beginning of inspection and imaged every 30sec for 10mins after bleaching . Scale bar : 5µm . e , Percentage of normalised SSTR3 - GFP signal recovery at whole cilia after photobleching from ( d ) . n = 20 cilia are quantified from three biological replicates Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test .

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d, FRAP experiment of SSTR3-GFP in RPE1 WT and SEPT2 KO cells. Cells were starved for 48h before the beginning of inspection and imaged every 30sec for 10mins after bleaching. Scale bar: 5µm. e, Percentage of normalised SSTR3-GFP signal recovery at whole cilia after photobleching from (d). n=20 cilia are quantified from three biological replicates Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


f - g , Influence of MKS3 depletion on cilia length of RPE WT and SEPT2 - KO cells . Cells are treated with the indicated siRNA and serum starved ( SS ) for 48h before immunostaining with the indicated antibodies . Representative images ( f ) and quantifications of proximal segment ( Glu tub ) and whole cilia ( ARL13B ) length ( g ) are shown . The numbers above the cartoons in ( g ) show the ratio of the calculated distal segments / whole cilia length . Scale bar : 2µm . Three 3 biological replicates , n = 100 cilia per sample and repetition . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test .

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f-g, Influence of MKS3 depletion on cilia length of RPE WT and SEPT2-KO cells. Cells are treated with the indicated siRNA and serum starved (SS) for 48h before immunostaining with the indicated antibodies. Representative images (f) and quantifications of proximal segment (Glu tub) and whole cilia (ARL13B) length (g) are shown. The numbers above the cartoons in (g) show the ratio of the calculated distal segments/whole cilia length. Scale bar: 2µm. Three 3 biological replicates, n=100 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


h - i , A functional TZ is required for KIF7 cilia tip localisation in RPE1 cells . KIF7 signal intensities are measured in ciliated RPE1 cells treated with control or MKS3 siRNA and 48h SS . Representative images ( h ) and quantification of KIF7 signal intensity at the base and tip of cilia ( h ) are shown . Scale bar : 2µm . Three biological replicates , at least 60 cilia per sample and repetition . Data include mean ± s . d . and P values are calculated by two - tailed unpaired student t - test .

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h-i, A functional TZ is required for KIF7 cilia tip localisation in RPE1 cells. KIF7 signal intensities are measured in ciliated RPE1 cells treated with control or MKS3 siRNA and 48h SS. Representative images (h) and quantification of KIF7 signal intensity at the base and tip of cilia (h) are shown. Scale bar: 2µm. Three biological replicates, at least 60 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


a , Time - lapse images of RPE1 WT and SEPT2 - KO cells stably expressing ARL13B - GFP and γ - tubulin - mRuby2 . Cells are starved for 32h before the beginning of inspection and imaged every 15min for 9 . 5h . Scale bar : 5µm . b , Quantification of ( a ) showing changes in cilia length during time - lapse imaging . Three biological replicates , 10 cilia per sample and repetition . c , Time - lapse images show ectocytosis events observed in RPE1 SEPT2 - KO cells . Experiments are done as in ( a ) with exception that cells are imaged every 15min . Scale bar : 2 . 5µm .

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a, Time-lapse images of RPE1 WT and SEPT2-KO cells stably expressing ARL13B-GFP and γ-tubulin-mRuby2. Cells are starved for 32h before the beginning of inspection and imaged every 15min for 9.5h. Scale bar: 5µm. b, Quantification of (a) showing changes in cilia length during time-lapse imaging. Three biological replicates, 10 cilia per sample and repetition. c, Time-lapse images show ectocytosis events observed in RPE1 SEPT2-KO cells. Experiments are done as in (a) with exception that cells are imaged every 15min. Scale bar: 2.5µm.


g - h , RPE1 SEPT2 - KO cells expressing ARL13B - mRuby2 and Neongreen - EFHC1 after 48h SS and immunostained with Glu - tub antibody . Representative images ( g ) and quantifications of proximal segment ( Glu tub ) , EFHC1 and whole cilia ( ARL13B ) length ( h ) are shown . Three biological replicates , n = 100 cilia per sample and repetition . Scale bar : 2µm .

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g-h, RPE1 SEPT2-KO cells expressing ARL13B-mRuby2 and Neongreen-EFHC1 after 48h SS and immunostained with Glu-tub antibody. Representative images (g) and quantifications of proximal segment (Glu tub), EFHC1 and whole cilia (ARL13B) length (h) are shown. Three biological replicates, n=100 cilia per sample and repetition. Scale bar: 2µm.


Male mice , housed at 30 ° C or 6 ° C for 10 days , were intraperitoneally ( IP ) administered with [ U - 13C ] glucose ( 2 g / kg ) . 15 minutes after injection , BAT was harvested for metabolic enrichment assay . ( A ) m + 6 glucose enrichment in serum and BAT . ( B ) relative glucose abundance in serum and BAT .

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Male mice, housed at 30°C or 6°C for 10 days, were intraperitoneally (IP) administered with [U-13C]glucose (2 g/kg). 15 minutes after injection, BAT was harvested for metabolic enrichment assay. (A) m+6 glucose enrichment in serum and BAT. (B) relative glucose abundance in serum and BAT.


( D , E ) Metabolic 13C enrichments in BAT of male mice are shown as m + 3 glycolytic intermediates ( D ) , m + 2 TCA cycle intermediates ( E ) .

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(D,E) Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates (D), m+2 TCA cycle intermediates (E).


( A ) Representative immunofluorescence staining shows UCP1 ( green ) , perilipin ( red ) and DAPI ( blue ) in sWAT of mice housed at 6 ° C .

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(A) Representative immunofluorescence staining shows UCP1 (green), perilipin (red) and DAPI (blue) in sWAT of mice housed at 6°C.


( B - G ) Male mice , housed at 30 ° C or 6 ° C for 10 days , were administered with [ U - 13C ] glucose ( 2 g / kg , IP ) . 15 minutes after injection , sWAT and gWAT were harvested for metabolic enrichment assay . Metabolic 13C enrichments in sWAT of male mice are shown as m + 6 glucose ( B ) , m + 3 glycolytic intermediates ( C ) , and m + 2 TCA cycle intermediates ( D ) . Metabolic 13C enrichments in gWAT of male mice are shown as m + 6 glucose ( E ) , m + 3 glycolysis intermediates ( F ) , and m + 2 TCA cycle intermediates ( G ) .

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(B-G) Male mice, housed at 30°C or 6°C for 10 days, were administered with [U-13C]glucose (2 g/kg, IP). 15 minutes after injection, sWAT and gWAT were harvested for metabolic enrichment assay. Metabolic 13C enrichments in sWAT of male mice are shown as m+6 glucose (B), m+3 glycolytic intermediates (C), and m+2 TCA cycle intermediates (D). Metabolic 13C enrichments in gWAT of male mice are shown as m+6 glucose (E), m+3 glycolysis intermediates (F), and m+2 TCA cycle intermediates (G).


( H ) Plasma 13CO2 enrichment , after normalizing to the [ U - 13C ] glucose tracer enrichment .

(H) Plasma 13CO2 enrichment, after normalizing to the [U-13C]glucose tracer enrichment.


Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions ( SVF ) of the interscapular BAT . ( A ) Upper : Oil red staining of the cells during differentiation . Lower : Relative mRNA levels of brown adipocyte markers were measured by qPCR .

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Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (A) Upper: Oil red staining of the cells during differentiation. Lower: Relative mRNA levels of brown adipocyte markers were measured by qPCR.


Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions ( SVF ) of the interscapular BAT . ( B ) Glycolytic intermediates were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours .

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Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (B) Glycolytic intermediates were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose for 2 hours.


Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions ( SVF ) of the interscapular BAT . ( C ) TCA cycle intermediates were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours .

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Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (C) TCA cycle intermediates were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose for 2 hours.


( A ) Fully differentiated brown adipocytes were treated with β3 - AR agonist CL316 , 243 ( 10 µM ) . Relative mRNA levels of thermogenic markers were measured by qPCR .

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(A) Fully differentiated brown adipocytes were treated with β3-AR agonist CL316,243 (10 µM). Relative mRNA levels of thermogenic markers were measured by qPCR.


( B ) Pyruvate and lactate enrichment were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose and 10 µM CL316 , 243 for 2 hours .

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(B) Pyruvate and lactate enrichment were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose and 10 µM CL316,243 for 2 hours.


( C ) Citrate and malate enrichment were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose and 10 µM CL316 , 243 for 2 hours .

(C) Citrate and malate enrichment were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose and 10 µM CL316,243 for 2 hours.


( A ) oxygen consumption rate ( OCR ) of mouse brown adipocytes treated with MPC inhibitor CHC ( 2 mM ) or UK5099 ( 2 µM ) , n = 6 - 7 biological repeats .

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(A) oxygen consumption rate (OCR) of mouse brown adipocytes treated with MPC inhibitor CHC (2 mM) or UK5099 (2 µM), n=6-7 biological repeats.


( B ) Differentiated mouse brown adipocytes were pre - treated with β3 - AR agonist CL316 , 243 for 4 hours , before cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours with CHC or UK5099 . Metabolic 13C enrichments in brown adipocytes were shown as m + 3 glycolytic intermediates , m + 2 and m + 3 TCA cycle intermediates , n = 3 biological replicates .

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(B) Differentiated mouse brown adipocytes were pre-treated with β3-AR agonist CL316,243 for 4 hours, before cultured with medium containing 10 mM [U-13C]glucose for 2 hours with CHC or UK5099. Metabolic 13C enrichments in brown adipocytes were shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=3 biological replicates.


( C ) After housed at 6 ° C for 10 days , mice were IP injected with PBS or CHC ( 500 mg / kg ) . 30 minutes after CHC treatment , mice were IP administered with [ U - 13C ] glucose ( 2 g / kg ) . Metabolic 13C enrichments in BAT of male mice are shown as m + 3 glycolytic intermediates , m + 2 and m + 3 TCA cycle intermediates , n = 7 .

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(C) After housed at 6°C for 10 days, mice were IP injected with PBS or CHC (500 mg/kg). 30 minutes after CHC treatment, mice were IP administered with [U-13C]glucose (2 g/kg). Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=7.


( D ) Body temperature of the CHC ( left : 400 mg / kg , n = 5 ) and ( right : 500 mg / kg , n = 7 ) treated mice .

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(D) Body temperature of the CHC (left: 400 mg/kg, n=5) and (right: 500 mg/kg, n=7) treated mice.


B Elution volumes ( peak positions ) of the Munc18 - 1 mutants in the absence ( top ) and presence ( bottom ) of Syx1 using size - exclusion chromatography on Superdex - 200 column .

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B Elution volumes (peak positions) of the Munc18-1 mutants in the absence (top) and presence (bottom) of Syx1 using size-exclusion chromatography on Superdex-200 column.


C Effects of the domain 3a mutations on MUN - catalyzed transition detected by native - PAGE assay . Components addition was indicated on the top of every lane . D Quantitative analysis of the integrated densities of Munc18 - 1 / Syx1 bands in C .

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C Effects of the domain 3a mutations on MUN-catalyzed transition detected by native-PAGE assay. Components addition was indicated on the top of every lane. D Quantitative analysis of the integrated densities of Munc18-1/Syx1 bands in C.


Effects of the QKAA , KKEE and L348R mutations on MUN - catalyzed transition detected by FRET assay . Schematic diagram is shown on the top of the chart . FRET between BODIPY FL - labeled Syb2 ( donor ) and TMR - labeled SN25 ( acceptor ) was monitored .

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Effects of the QKAA, KKEE and L348R mutations on MUN-catalyzed transition detected by FRET assay. Schematic diagram is shown on the top of the chart. FRET between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored.


F Effects of the QKAA , KKEE and L348R mutations on lipid mixing between liposomes with Munc18 - 1 / Syx1 and liposomes containing Syb2 in the presence of Munc13 ( M13 ) , SN25 and Syt1 / Ca2 + . Illustration of the reactions is shown on the top of the chart .

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F Effects of the QKAA, KKEE and L348R mutations on lipid mixing between liposomes with Munc18-1/Syx1 and liposomes containing Syb2 in the presence of Munc13 (M13), SN25 and Syt1/Ca2+. Illustration of the reactions is shown on the top of the chart.


A Representative traces ( left ) , summary graphs of normalized frequency ( middle ) and normalized amplitude ( right ) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18 - 1 WT or mutants , with or without Munc18 - 1 shRNAs ( Control , n = 25 ; None , n = 17 ; WT , n = 17 ; QKAA , n = 18 ; KKEE , n = 16 ; P335A , n = 23 ; L348R , n = 21 ) . B Sample traces ( left ) , summary graphs of normalized amplitude ( middle ) and normalized charge transfer ( right ) of evoked IPSCs recorded from neurons as described in panel A ( Control , n = 19 ; None , n = 19 ; WT , n = 19 ; QKAA , n = 13 ; KKEE , n = 18 ; P335A , n = 17 ; L348R , n = 21 ) . C Sample traces ( left ) and quantification of the normalized charge transfer evoked by hypertonic sucrose ( right ) recorded from the neurons described in panel A ( Control , n = 17 ; None , n = 13 ; WT , n = 17 ; QKAA , n = 17 ; KKEE , n = 18 ; P335A , n = 15 ; L348R , n = 14 ) .

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A Representative traces (left), summary graphs of normalized frequency (middle) and normalized amplitude (right) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18-1 WT or mutants, with or without Munc18-1 shRNAs (Control, n = 25; None, n = 17; WT, n = 17; QKAA, n = 18; KKEE, n = 16; P335A, n = 23; L348R, n = 21). B Sample traces (left), summary graphs of normalized amplitude (middle) and normalized charge transfer (right) of evoked IPSCs recorded from neurons as described in panel A (Control, n = 19; None, n = 19; WT, n = 19; QKAA, n = 13; KKEE, n = 18; P335A, n = 17; L348R, n = 21). C Sample traces (left) and quantification of the normalized charge transfer evoked by hypertonic sucrose (right) recorded from the neurons described in panel A (Control, n = 17; None, n = 13; WT, n = 17; QKAA, n = 17; KKEE, n = 18; P335A, n = 15; L348R, n = 14).


A Representative traces ( left ) , summary graphs of normalized frequency ( middle ) and normalized amplitude ( right ) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18 - 1 WT or mutants , with or without Munc18 - 1 shRNAs ( Control , n = 25 ; None , n = 17 ; WT , n = 17 ; QKAA , n = 18 ; KKEE , n = 16 ; P335A , n = 23 ; L348R , n = 21 ) .

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A Representative traces (left), summary graphs of normalized frequency (middle) and normalized amplitude (right) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18-1 WT or mutants, with or without Munc18-1 shRNAs (Control, n = 25; None, n = 17; WT, n = 17; QKAA, n = 18; KKEE, n = 16; P335A, n = 23; L348R, n = 21).


A Representative images of HEK293T cells transfected with mCherry2 - Syx1 or EGFP - Munc18 - 1 . Lipophilic dye DiD ( 1 , 1 - Dioctadecyl - 3 , 3 , 3 , 3 - tetramethylindodicarbocyanine ) was used as the plasma membrane marker . B Representative images of HEK293T cells co - transfected with mCherry2 - Syx1 and EGFP labeled WT or mutants of Munc18 - 1 .

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A Representative images of HEK293T cells transfected with mCherry2-Syx1 or EGFP-Munc18-1. Lipophilic dye DiD (1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine) was used as the plasma membrane marker. B Representative images of HEK293T cells co-transfected with mCherry2-Syx1 and EGFP labeled WT or mutants of Munc18-1.


C , D Summary graphs of the membrane distribution of Syx1 ( C ) and Munc18 - 1 WT or mutants ( D ) quantified by the ratio of fluorescence at the plasma membrane ( PM ) to the total fluorescence in the whole cell ( total ) . DiD is the PM marker . Numbers of pictures analyzed are shown as follows : no Syx1 , n = 14 ; no M18 , n = 15 ; WT , n = 24 ; QKAA , n = 23 ; KKEE , n = 12 ; L348R , n = 22 .

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C,D Summary graphs of the membrane distribution of Syx1 (C) and Munc18-1 WT or mutants (D) quantified by the ratio of fluorescence at the plasma membrane (PM) to the total fluorescence in the whole cell (total). DiD is the PM marker. Numbers of pictures analyzed are shown as follows: no Syx1, n = 14; no M18, n = 15; WT, n = 24; QKAA, n = 23; KKEE, n = 12; L348R, n = 22.


E Munc18 - 1 mutants retain interaction with Syx1 shown by GST pull - down experiment . A representative gel displayed is from one of three independent replicates .

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E Munc18-1 mutants retain interaction with Syx1 shown by GST pull-down experiment. A representative gel displayed is from one of three independent replicates.


B Example histograms of smFRET efficiency of Syx1 in complex with Munc18 - 1 WT in the absence and presence of the MUN domain , and within the SNARE complex . C − E Example histograms of smFRET efficiency of Syx1 bound to Munc18 - 1 QKAA ( C ) , KKEE ( D ) , and L348R ( E ) in the absence or presence of the MUN domain . The QKAA mutation strongly impaired the shift of smFRET efficiency in the presence of the MUN domain .

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B Example histograms of smFRET efficiency of Syx1 in complex with Munc18-1 WT in the absence and presence of the MUN domain, and within the SNARE complex. C−E Example histograms of smFRET efficiency of Syx1 bound to Munc18-1 QKAA (C), KKEE (D), and L348R (E) in the absence or presence of the MUN domain. The QKAA mutation strongly impaired the shift of smFRET efficiency in the presence of the MUN domain.


F Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C − E .

F Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C−E.


G Effects of QKAA , KKEE , and L348R mutations on the interaction between the Munc18 - 1 / Syx1 complex and the MUN domain by GST pull - down assay combined with immunoblotting . Results were shown with 6 × His - tag antibody and GST - tag antibody .

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G Effects of QKAA, KKEE, and L348R mutations on the interaction between the Munc18-1/Syx1 complex and the MUN domain by GST pull-down assay combined with immunoblotting. Results were shown with 6×His-tag antibody and GST-tag antibody.


B Effects of the QKAA , KKEE , and L348R mutations on binding of Syb2 to Munc18 - 1 / Syx1 complex in the presence and absence of MUN domain detected by GST pull - down assay . Bands of Munc18 - 1 are indicated with red asterisks . C Quantification of the normalized binding of Syb2 to Munc18 - 1 / Syx1 complex . Integrated intensities of Munc18 - 1 bands were quantified . QKAA and L348R , but not KKEE , disrupted Syb2 binding to the Munc18 - 1 / Syx1 complex .

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B Effects of the QKAA, KKEE, and L348R mutations on binding of Syb2 to Munc18-1/Syx1 complex in the presence and absence of MUN domain detected by GST pull-down assay. Bands of Munc18-1 are indicated with red asterisks. C Quantification of the normalized binding of Syb2 to Munc18-1/Syx1 complex. Integrated intensities of Munc18-1 bands were quantified. QKAA and L348R, but not KKEE, disrupted Syb2 binding to the Munc18-1/Syx1 complex.


B Effects of the QKAA , KKEE , and L348R mutations on binding of Syb2 to Munc18 - 1 / Syx1 complex in the presence and absence of MUN domain detected by GST pull - down assay . Bands of Munc18 - 1 are indicated with red asterisks .

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B Effects of the QKAA, KKEE, and L348R mutations on binding of Syb2 to Munc18-1/Syx1 complex in the presence and absence of MUN domain detected by GST pull-down assay. Bands of Munc18-1 are indicated with red asterisks.


A Ensemble FRET assay showing the different effects of the Munc18 - 1 P335A mutation and the Syx1 LEAA mutation in the transition from the Munc18 - 1 / Syx1 complex to the SNARE complex with and without the MUN domain . FRET signal between BODIPY FL - labeled Syb2 ( donor ) and TMR - labeled SN25 ( acceptor ) was monitored . B Quantification of the FRET results in A .

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A Ensemble FRET assay showing the different effects of the Munc18-1 P335A mutation and the Syx1 LEAA mutation in the transition from the Munc18-1/Syx1 complex to the SNARE complex with and without the MUN domain. FRET signal between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored. B Quantification of the FRET results in A.


A Ensemble FRET assay showing the different effects of the Munc18 - 1 P335A mutation and the Syx1 LEAA mutation in the transition from the Munc18 - 1 / Syx1 complex to the SNARE complex with and without the MUN domain . FRET signal between BODIPY FL - labeled Syb2 ( donor ) and TMR - labeled SN25 ( acceptor ) was monitored .

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A Ensemble FRET assay showing the different effects of the Munc18-1 P335A mutation and the Syx1 LEAA mutation in the transition from the Munc18-1/Syx1 complex to the SNARE complex with and without the MUN domain. FRET signal between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored.


C Single - molecule FRET assay showing the MUN domain drives the open of the Syx1 linker region independent of the conformational change of domain 3a . Example histograms of smFRET efficiency of Syx1 bound to Munc18 - 1 P335A mutant in the absence and presence of the MUN domain . D Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C .

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C Single-molecule FRET assay showing the MUN domain drives the open of the Syx1 linker region independent of the conformational change of domain 3a. Example histograms of smFRET efficiency of Syx1 bound to Munc18-1 P335A mutant in the absence and presence of the MUN domain. D Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C.


C Single - molecule FRET assay showing the MUN domain drives the open of the Syx1 linker region independent of the conformational change of domain 3a . Example histograms of smFRET efficiency of Syx1 bound to Munc18 - 1 P335A mutant in the absence and presence of the MUN domain .

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C Single-molecule FRET assay showing the MUN domain drives the open of the Syx1 linker region independent of the conformational change of domain 3a. Example histograms of smFRET efficiency of Syx1 bound to Munc18-1 P335A mutant in the absence and presence of the MUN domain.


B Munc18 - 1 , Habc and H3 construct a tertiary complex .

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B Munc18-1, Habc and H3 construct a tertiary complex.


C The tertiary complex globally resembles the Munc18 - 1 / Syx1 complex .

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C The tertiary complex globally resembles the Munc18-1/Syx1 complex.


D Formation of the tertiary complex with Munc18 - 1 QKAA , KKEE , and L348R mutants . KKEE strongly impaired formation of the tertiary complex .

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D Formation of the tertiary complex with Munc18-1 QKAA, KKEE, and L348R mutants. KKEE strongly impaired formation of the tertiary complex.


E Munc18 - 1 displays interaction with Syx1 - Habc .

E Munc18-1 displays interaction with Syx1-Habc.


F QKAA , KKEE , and L348R all retained interaction with Syx1 - Habc .

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F QKAA, KKEE, and L348R all retained interaction with Syx1-Habc.


( B ) NSC - 34 and SH - SY5Y cells were transfected with various concentrations of siPRPH or siNTC . The efficiency of siRNA knockdown at 48h . p . t . was validated by Western blot while cytotoxicity was determined by AlamarBlue assay . siPRPH - or siNTC - transfected cells were infected with EV - A71 S41 , and the viral titers were determined at 48h . p . i .

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(B) NSC-34 and SH-SY5Y cells were transfected with various concentrations of siPRPH or siNTC. The efficiency of siRNA knockdown at 48h.p.t. was validated by Western blot while cytotoxicity was determined by AlamarBlue assay. siPRPH- or siNTC-transfected cells were infected with EV-A71 S41, and the viral titers were determined at 48h.p.i.


Two - week old AG129 mice ( n = 2 - 3 / group / time point ) were infected with EV - A71 S41 . ( A ) At various time points post - infection , mice were euthanized and the relative expression of PRPH was detected by Western blot in tissue homogenates ( brainstem , spinal cord and limb muscle ) . Actin and GAPDH serve as loading controls , and protein expression was compared to age - matched non - infected mice .

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Two-week old AG129 mice (n=2-3 /group/time point) were infected with EV-A71 S41. (A) At various time points post-infection, mice were euthanized and the relative expression of PRPH was detected by Western blot in tissue homogenates (brainstem, spinal cord and limb muscle). Actin and GAPDH serve as loading controls, and protein expression was compared to age-matched non-infected mice.


Two - week old AG129 mice ( n = 2 - 3 / group / time point ) were infected with EV - A71 S41 . ( B ) Immunostaining of PRPH and EV - A71 particles on brainstem and spinal cord sections harvested at 6 d . p . i .

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Two-week old AG129 mice (n=2-3 /group/time point) were infected with EV-A71 S41. (B) Immunostaining of PRPH and EV-A71 particles on brainstem and spinal cord sections harvested at 6 d.p.i.


( C ) Proximity ligation assay . NSC - 34 and SH - SY5Y cells were infected with EV - A71 S41 at MOI 10 and 0 . 01 , respectively . Cells were fixed at 48h . p . i . prior to PLA staining using anti - PRPH and anti - VP0 antibodies .

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(C) Proximity ligation assay. NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 10 and 0.01, respectively. Cells were fixed at 48h.p.i. prior to PLA staining using anti-PRPH and anti-VP0 antibodies.


( A ) NSC - 34 and SH - SY5Y cells were reverse - transfected with siPRPH or siNTC for 48 hrs prior to transfection with EV - A71 RNA . The culture supernatants were harvested for viral titer determination at 24h . p . t .

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(A) NSC-34 and SH-SY5Y cells were reverse-transfected with siPRPH or siNTC for 48 hrs prior to transfection with EV-A71 RNA. The culture supernatants were harvested for viral titer determination at 24h.p.t.


( A ) NSC - 34 and SH - SY5Y cells were infected with EV - A71 S41 at MOI 30 and 0 . 01 , respectively . At 48 h . p . i . , cells were fixed and permeabilized prior to immunostaining .

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(A) NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 30 and 0.01, respectively. At 48 h.p.i., cells were fixed and permeabilized prior to immunostaining.


( B ) Total infected cell lysates were pulled down with anti - PRPH or IgG isotype control antibody prior to immunoblotting . Uninfected cells were included as negative control . IP : immunoprecipitation ; IB : immunoblotting .

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(B) Total infected cell lysates were pulled down with anti-PRPH or IgG isotype control antibody prior to immunoblotting. Uninfected cells were included as negative control. IP: immunoprecipitation; IB: immunoblotting.


C ) Proximity ligation assay . NSC - 34 and SH - SY5Y cells were infected with EV - A71 S41 at MOI 10 and 0 . 01 , respectively . At 48 h . p . i . , the cells were fixed and permeabilized prior to PLA staining using antibodies specific to PPRH , VP0 , 3C and 3D .

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C) Proximity ligation assay. NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 10 and 0.01, respectively. At 48 h.p.i., the cells were fixed and permeabilized prior to PLA staining using antibodies specific to PPRH, VP0, 3C and 3D.


( C ) Viral titers in siRNA - treated NSC - 34 cells . Cells were transfected with various concentrations of siRNA specific to the indicated target genes . The efficiency of siRNA - mediated knockdown at 48h . p . t . was validated by Western blot while cytotoxicity was determined by AlamarBlue assay siRNA - or siNTC - transfected cells were infected with EV - A71 S41 at MOI 10 , and the viral titers were determined at 48h . p . i .

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(C) Viral titers in siRNA-treated NSC-34 cells. Cells were transfected with various concentrations of siRNA specific to the indicated target genes. The efficiency of siRNA-mediated knockdown at 48h.p.t. was validated by Western blot while cytotoxicity was determined by AlamarBlue assay siRNA- or siNTC-transfected cells were infected with EV-A71 S41 at MOI 10, and the viral titers were determined at 48h.p.i.


( D ) Drug treatment . EV - A71 - infected NSC - 34 cells were treated with the indicated drugs , and the viral titers were determined at 48h . p . i .

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(D) Drug treatment. EV-A71-infected NSC-34 cells were treated with the indicated drugs, and the viral titers were determined at 48h.p.i.


( A ) NSC - 34 and SH - SY5Y cells were infected with EV - A71 S41 at MOI 30 and 0 . 01 , respectively . At 48h . p . i . , the cells were fixed and permeabilized prior to immunostaining .

(A) NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 30 and 0.01, respectively. At 48h.p.i., the cells were fixed and permeabilized prior to immunostaining.


B ) Proximity ligation assay . NSC - 34 and SH - SY5Y cells were transfected with LucEV - A71 replicon . Cells were fixed and permeabilized at 24h . p . t . prior to PLA staining using antibodies specific to Rac1 , 3C and 3D .

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B) Proximity ligation assay. NSC-34 and SH-SY5Y cells were transfected with LucEV-A71 replicon. Cells were fixed and permeabilized at 24h.p.t. prior to PLA staining using antibodies specific to Rac1, 3C and 3D.


Concentration of total cell - free DNA ( cfDNA ) in 13 CSF samples . Samples in which SCNAs were detected are shown in dark blue , and tended to have higher levels of total

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Concentration of total cell-free DNA (cfDNA) in 13 CSF samples. Samples in which SCNAs were detected are shown in dark blue, and tended to have higher levels of total cfDNA


Relative copy number estimation in 20 genes of interest , and 18 large genomic regions , determined by sWGS of CSF from 13 glioma patients . Genes are ordered by genomic position , and their chromosomal locations are indicated in parentheses . Amplifications are shown in dark blue , deletions in orange , and copy number neutral regions are in grey . The top green bar indicates the tumor largest length dimension ( < 42 mm vs > 42 mm ) , and the top violet bar indicates the glioma subtype . SCNAs were more frequently detected in CSF from patients with large glioblastoma

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Relative copy number estimation in 20 genes of interest, and 18 large genomic regions, determined by sWGS of CSF from 13 glioma patients. Genes are ordered by genomic position, and their chromosomal locations are indicated in parentheses. Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in grey. The top green bar indicates the tumor largest length dimension (<42 mm vs >42 mm), and the top violet bar indicates the glioma subtype. SCNAs were more frequently detected in CSF from patients with large glioblastoma tumors


SCNAs determined by sWGS in 4 tumor subparts ( T1 - T4 ) and the CSF sample , collected from patient G1 . Amplifications are shown in dark blue , deletions in orange , and copy number neutral regions are in dark grey . sWGS from plasma and urine samples collected at the same time as the CSF sample showed no

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SCNAs determined by sWGS in 4 tumor subparts (T1 - T4) and the CSF sample, collected from patient G1. Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in dark grey. sWGS from plasma and urine samples collected at the same time as the CSF sample showed no SCNAs


Heat map summarizing the SCNAs detected by sWGS of 28 genes of interest in tumor biopsies and CSF from patient G1 ( 4 tumor subparts and 1 CSF sample ) . Amplifications are shown in dark blue , deletions in orange , and copy number neutral regions are in light

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Heat map summarizing the SCNAs detected by sWGS of 28 genes of interest in tumor biopsies and CSF from patient G1 (4 tumor subparts and 1 CSF sample). Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in light grey


Heat map summarizing detection of EGFR and PTEN alterations in tumor tissue and in CSF samples . Shared detection in tissue and CSF is indicated in green , detection of the alteration only in tissue in orange , and non - detection in blue . The top bars indicate the cfDNA concentration ( copies / mL ; in a range of purples ) , the size of the tumors ( in a range of browns ) , the type of glioma ( in a range of blues ) , and whether the tumor was in direct contact with the CSF or not ( based on MRI , green or red , respectively ) . Samples are ranked from the left to right by decreasing concentration of cfDNA ( copies / mL )

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Heat map summarizing detection of EGFR and PTEN alterations in tumor tissue and in CSF samples. Shared detection in tissue and CSF is indicated in green, detection of the alteration only in tissue in orange, and non-detection in blue. The top bars indicate the cfDNA concentration (copies/mL; in a range of purples), the size of the tumors (in a range of browns), the type of glioma (in a range of blues), and whether the tumor was in direct contact with the CSF or not (based on MRI, green or red, respectively). Samples are ranked from the left to right by decreasing concentration of cfDNA (copies/mL)


A ) Fragment size distribution , determined by sWGS , in CSF ( blue line ) , plasma ( red dashed line ) and urine ( black dashed line ) from patient G1 . The 3 samples were collected simultaneously before initiation of treatment . B ) Fragment size distribution of cfDNA , determined by sWGS , in CSF from 13 glioma patients . The samples contained a high fraction of reads from DNA fragments with lengths corresponding to ~ 145 bp and ~ 167 bp . The fragment size profile of cfDNA from the plasma sample from patient G1 is shown in red . All samples were collected simultaneously before initiation of treatment C ) Cumulative frequency analysis of the average density of fragment size in CSF ( blue ) and plasma ( red ) . The vertical dashed line represents 167

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

A) Fragment size distribution, determined by sWGS, in CSF (blue line), plasma (red dashed line) and urine (black dashed line) from patient G1. The 3 samples were collected simultaneously before initiation of treatment. B) Fragment size distribution of cfDNA, determined by sWGS, in CSF from 13 glioma patients. The samples contained a high fraction of reads from DNA fragments with lengths corresponding to ~145 bp and ~167 bp. The fragment size profile of cfDNA from the plasma sample from patient G1 is shown in red. All samples were collected simultaneously before initiation of treatment C) Cumulative frequency analysis of the average density of fragment size in CSF (blue) and plasma (red). The vertical dashed line represents 167 bp


Mean fragment size density in patients with detected SCNAs in the CSF ( yellow ) , and those with no detected SCNAs ( dark blue )

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Mean fragment size density in patients with detected SCNAs in the CSF (yellow), and those with no detected SCNAs (dark blue)


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Log2 ratio of the difference in cfDNA fragment sizes between CSF samples with detected SCNAs and those samples in which no SCNAs were detected


The amplitude of the 10 bp periodic peaks in fragment size is a signature for non - tumor DNA in the CSF . The amplitude of the 10 bp periodic peaks in fragment length distribution ( in the range between 75 and 150 bp , see Methods ) when compared to the mean copy number alteration calculated from sWGS revealed a significant negative correlation ( Pearson - 0 . 85 ; p = 0 . 0002 )

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The amplitude of the 10 bp periodic peaks in fragment size is a signature for non-tumor DNA in the CSF. The amplitude of the 10 bp periodic peaks in fragment length distribution (in the range between 75 and 150 bp, see Methods) when compared to the mean copy number alteration calculated from sWGS revealed a significant negative correlation (Pearson -0.85; p=0.0002)