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 .

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

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 .

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

(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

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 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 .

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

(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 .

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

(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 ) .

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

(F) 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 .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-DISEASE O O O O O O B-SMALL_MOLECULE O O O O

(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 .

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

(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 .

O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O

(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 .

O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O

(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 ) .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 ) .

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

(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 .

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

(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 ) .

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

(F) 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 .

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

(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 .

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

(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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD B-DISEASE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 .

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

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 .

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

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 .

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

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 .

B-CELL_LINE O O O O O O O O B-TISSUE O O O O O B-EXP_ASSAY O O O O O O O O O

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 .

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

e-f, 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 .

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

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 ) .

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

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 .

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

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

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

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 -

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

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 .

O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O B-TISSUE O B-TISSUE O O O O O B-SMALL_MOLECULE O O B-TISSUE O B-TISSUE O

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 ) .

O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O

(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 .

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

(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 ) .

O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O B-TISSUE O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-TISSUE O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O B-TISSUE O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O

(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 .

B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY O

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 .

B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O

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 .

B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O

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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O 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) 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 .

O O O O O O O O O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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

(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 .

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

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 .

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

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 .

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

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 .

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

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 ) .

O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O

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

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O B-DISEASE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O B-TISSUE O O O O B-DISEASE

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 B-EXP_ASSAY O O B-TISSUE O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O B-TISSUE O O B-DISEASE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-TISSUE O O O O B-TISSUE 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)


U2OS cells were transfected , synchronized at the G1 / S - border by thymidine for 24 hours and then either released into nocodazole for 16 hours ( unperturbed mitotic entry ) or released for 7 hours , treated with 0 . 5 μM Adriamycin for one hour and after 16 hour G2 arrest were induced to recover by addition of caffeine ( checkpoint recovery ) . Mitotic index was scored based on the percentage of Histone H3 - pS10 positive DAPI - nuclei and normalized to the untransfected controls . Black diamonds indicate individual siRNA - targeted genes from the library , light grey diamonds indicate positive ( Wip1 - depleted ) and negative ( untransfected and GAPDH - depleted ) controls and dark grey diamonds indicate the hits based on the two screens . Dotted lines indicate selection criteria for recovery - specific genes .

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

U2OS cells were transfected, synchronized at the G1/S-border by thymidine for 24 hours and then either released into nocodazole for 16 hours (unperturbed mitotic entry) or released for 7 hours, treated with 0.5 μM Adriamycin for one hour and after 16 hour G2 arrest were induced to recover by addition of caffeine (checkpoint recovery). Mitotic index was scored based on the percentage of Histone H3-pS10 positive DAPI-nuclei and normalized to the untransfected controls. Black diamonds indicate individual siRNA-targeted genes from the library, light grey diamonds indicate positive (Wip1-depleted) and negative (untransfected and GAPDH-depleted) controls and dark grey diamonds indicate the hits based on the two screens. Dotted lines indicate selection criteria for recovery-specific genes.


A ) U2OS cells were transfected with four independent siRNA ' s from the pools used in the screen , treated as in figure 1 and analyzed for mitotic index . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * for P ≤ 0 . 01 )

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A) U2OS cells were transfected with four independent siRNA's from the pools used in the screen, treated as in figure 1 and analyzed for mitotic index. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01)


B ) U2OS cells were synchronized with a single thymidine block , released into G2 and damaged with 0 . 5 μM Adriamycin for one hour . After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for Tlk2 protein levels .

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B) U2OS cells were synchronized with a single thymidine block, released into G2 and damaged with 0.5 μM Adriamycin for one hour. After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for Tlk2 protein levels.


C ) Live cell imaging of thymidine synchronized unperturbed or damaged G2 cells . Cumulative percentage of cells entering mitosis were scored and plotted .

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C) Live cell imaging of thymidine synchronized unperturbed or damaged G2 cells. Cumulative percentage of cells entering mitosis were scored and plotted.


D ) U2OS cells were transfected with either a control siRNA or Tlk2 siRNA # 3 , synchronized and damaged in G2 . Cells were either harvested or treated with caffeine for 8 hours before harvest and cell cycle distribution was analyzed by FACS . Percentages of cells in each quadrant are indicated .

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

D) U2OS cells were transfected with either a control siRNA or Tlk2 siRNA #3, synchronized and damaged in G2. Cells were either harvested or treated with caffeine for 8 hours before harvest and cell cycle distribution was analyzed by FACS. Percentages of cells in each quadrant are indicated.


E ) Tlk2Δ cells were generated using CRISPR / Cas9 genome editing . Cells were synchronized in G2 by thymidine release , damaged with 0 . 5 μM Adriamycin for one hour . After a 16 - hour G2 arrest cells were induced to recover by addition of caffeine for 8 hours and analyzed by FACS . Error bars represent SD , n = 4 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * * for P ≤ 0 . 001 ) .

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

E) Tlk2Δ cells were generated using CRISPR/Cas9 genome editing. Cells were synchronized in G2 by thymidine release, damaged with 0.5 μM Adriamycin for one hour. After a 16-hour G2 arrest cells were induced to recover by addition of caffeine for 8 hours and analyzed by FACS. Error bars represent SD, n=4. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, *** for P≤0.001).


F ) U2TR cells stably expressing Tlk2 siRNA # 3 - insensitive tetracycline - inducible FLAG - Tlk2 - wt or FLAG - Tlk2 - D613A were thymidine synchronized and damaged in G2 . Tetracycline was present form the start of the experiment where indicated .

O O B-CELL_LINE O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

F) U2TR cells stably expressing Tlk2 siRNA #3-insensitive tetracycline-inducible FLAG-Tlk2-wt or FLAG-Tlk2-D613A were thymidine synchronized and damaged in G2. Tetracycline was present form the start of the experiment where indicated.


G ) Caffeine induced recovery assay of cell lines shown in 2F . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * for P ≤ 0 . 01 , * * * for P ≤ 0 . 001 ) .

O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G) Caffeine induced recovery assay of cell lines shown in 2F. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01, *** for P≤0.001).


A ) U2OS cells were grown on glass coverslips and synchronized in G2 after thymidine release . Cells were fixed at the indicated times after adriamycin - induced DNA damage and stained for DAPI and γH2AX . Scale bar indicates 10 μm . B ) Quantification of cells in 3A . Each dot represents the total signal of γH2AX in the nucleus of a single cell . Bars indicate the mean and error bars indicate the SD of the data points in each condition . Whiskers represent 5 - 95 % of data points . Statistical significance was tested using an unpaired two - tailed t - test ( NS for P > 0 . 05 , * * * * for P ≤ 0 . 0001 ) .

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

A) U2OS cells were grown on glass coverslips and synchronized in G2 after thymidine release. Cells were fixed at the indicated times after adriamycin-induced DNA damage and stained for DAPI and γH2AX. Scale bar indicates 10 μm. B) Quantification of cells in 3A. Each dot represents the total signal of γH2AX in the nucleus of a single cell. Bars indicate the mean and error bars indicate the SD of the data points in each condition. Whiskers represent 5-95% of data points. Statistical significance was tested using an unpaired two-tailed t-test (NS for P>0.05, **** for P≤0.0001).