D . Quantification of the pericytes coverage using CD13 immunostaining in the brain vasculature of WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 7 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . ) E . Quantification of the smooth muscle cell coverage using αSMA immunostaining in the brain vasculature of WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 13 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

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D. Quantification of the pericytes coverage using CD13 immunostaining in the brain vasculature of WT (blue) and Glut1iECKO (orange) mice. (n=7 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) E. Quantification of the smooth muscle cell coverage using αSMA immunostaining in the brain vasculature of WT (blue) and Glut1iECKO (orange) mice. (n=13 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


J . Representative confocal images of fibrin ( green ) , CD31 ( red ) and DAPI ( blue ) immunostaining in the brain vasculature of WT ( top panel ) and Glut1iECKO ( bottom panel ) mice K . Quantification of the number of cadaverine 555 positive brain parenchyma cells in WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 13 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . ) L . Quantification of extravascular fibrin deposits in WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 9 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . ) M . Quantification of lactate in the cerebrospinal fluid of WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 3 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

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

J. Representative confocal images of fibrin (green), CD31 (red) and DAPI (blue) immunostaining in the brain vasculature of WT (top panel) and Glut1iECKO (bottom panel) mice K. Quantification of the number of cadaverine 555 positive brain parenchyma cells in WT (blue) and Glut1iECKO (orange) mice. (n=13 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) L. Quantification of extravascular fibrin deposits in WT (blue) and Glut1iECKO (orange) mice. (n=9 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) M. Quantification of lactate in the cerebrospinal fluid of WT (blue) and Glut1iECKO (orange) mice. (n=3 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


A . Quantification of the mTFP / Venus fluorescence ratio of laconic ( lactate sensor ) showing concentration - dependent lactate uptake in HBVPs . ( n = 8 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) ) B . Representative images showing mTFP ( blue ) and Venus ( yellow ) fluorescence in control and lactate ( 20mM ) treated HBVPs .

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A. Quantification of the mTFP/Venus fluorescence ratio of laconic (lactate sensor) showing concentration-dependent lactate uptake in HBVPs. (n=8 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM)) B. Representative images showing mTFP (blue) and Venus (yellow) fluorescence in control and lactate (20mM) treated HBVPs.


F . Representative images ( high magnification ; 60x ) showing mTFP ( cyan ) , Venus ( yellow ) and VECAD ( purple ) fluorescence from BMECs and laconic - infected HBVPs on a 2 well chamber . The upper panel shows HBVPs with endothelial contact and the lower panel shows HBVPs without endothelial contact . G . Quantification of the mTFP / Venus fluorescence ratio of laconic in HBVPs with endothelial contact ( EC - PC contact ) or HBVPs alone ( PC only ) . ( n = 16 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

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F. Representative images (high magnification; 60x) showing mTFP (cyan), Venus (yellow) and VECAD (purple) fluorescence from BMECs and laconic-infected HBVPs on a 2 well chamber. The upper panel shows HBVPs with endothelial contact and the lower panel shows HBVPs without endothelial contact. G. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs with endothelial contact (EC-PC contact) or HBVPs alone (PC only). (n=16 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


H . Representative images showing mTFP ( cyan ) and Venus ( yellow ) fluorescence in HBVPs which have direct contact with control ( upper panel ) or GLUT1 ( lower panel ) siRNA treated BMECs . VECAD immunostaining ( purple ) was used for endothelial staining . I . Quantification of the mTFP / Venus fluorescence ratio of laconic in HBVPs which have direct contact with control ( siCON ) or GLUT1 ( siGLUT1 ) siRNA - treated BMECs . ( n = 12 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

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H. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or GLUT1 (lower panel) siRNA treated BMECs. VECAD immunostaining (purple) was used for endothelial staining. I. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or GLUT1 (siGLUT1) siRNA-treated BMECs. (n=12 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


E . Western blot analysis for siRNA efficiency of MCT1 .

E. Western blot analysis for siRNA efficiency of MCT1.


G . Western blot analysis for siRNA efficiency of MCT12 .

G. Western blot analysis for siRNA efficiency of MCT12.


I . Representative images showing mTFP ( cyan ) and Venus ( yellow ) fluorescence in HBVPs which have direct contact with control ( upper panel ) or MCT1 ( lower panel ) siRNA - treated BMECs . VECAD immunostaining was used for endothelial staining . J . Quantification of the mTFP / Venus fluorescence ratio of laconic in HBVPs which have direct contact with control ( siCON ) or MCT1 ( siMCT1 ) siRNA - treated BMECs . ( n = 8 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

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I. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or MCT1 (lower panel) siRNA-treated BMECs. VECAD immunostaining was used for endothelial staining. J. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or MCT1 (siMCT1) siRNA-treated BMECs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


K . Representative images showing mTFP ( cyan ) and Venus ( yellow ) fluorescence in HBVPs which have direct contact with control ( upper panel ) or MCT5 ( lower panel ) siRNA - treated BMECs . VECAD immunostaining was used for endothelial staining . L . Quantification of the mTFP / Venus fluorescence ratio of laconic in HBVPs which have direct contact with control ( siCON ) or MCT5 ( siMCT5 ) siRNA - treated BMECs . ( n = 8 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

K. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or MCT5 (lower panel) siRNA-treated BMECs. VECAD immunostaining was used for endothelial staining. L. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or MCT5 (siMCT5) siRNA-treated BMECs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


D . Western blot analysis showing expression level of MCT12 and β - actin in HBVPs and BMECs . E . Western blot analysis for siRNA efficiency of MCT12 .

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D. Western blot analysis showing expression level of MCT12 and β-actin in HBVPs and BMECs. E. Western blot analysis for siRNA efficiency of MCT12.


G . Representative images showing mTFP ( cyan ) and Venus ( yellow ) fluorescence in control ( upper panel ) or MCT12 ( lower panel ) siRNA treated HBVPs in a 2 well chamber assay system with BMECs . VECAD ( purple ) immunostaining was used for endothelial staining . H . Quantification of the mTFP / Venus fluorescence ratio of Laconic in control ( blue ) or MCT12 ( orange ) siRNA treated HBVPs . ( n = 8 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test .

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

G. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in control (upper panel) or MCT12 (lower panel) siRNA treated HBVPs in a 2 well chamber assay system with BMECs. VECAD (purple) immunostaining was used for endothelial staining. H. Quantification of the mTFP/Venus fluorescence ratio of Laconic in control (blue) or MCT12 (orange) siRNA treated HBVPs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.


A . Permeability assay using vibratome section showing the cadaverine 555 dye ( red ) , CD31 ( green ) and CD13 ( purple ) in the brain vasculature of Glut1iECKO mice with ( bottom ) or without ( top ) lactate administration . Note the extravascular accumulation of cadaverine dye in control Glut1iECKO mice , but not in lactate supplemented Glut1iECKO mice .

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A. Permeability assay using vibratome section showing the cadaverine 555 dye (red), CD31 (green) and CD13 (purple) in the brain vasculature of Glut1iECKO mice with (bottom) or without (top) lactate administration. Note the extravascular accumulation of cadaverine dye in control Glut1iECKO mice, but not in lactate supplemented Glut1iECKO mice.


B . Quantification of the CD13 - positive pericytes coverage in the brain vasculature of Glut1iECKO mice with ( orange ) or without ( blue ) lactate administration . ( n = 16 from 5 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from 1 - way ANOVA . ) C . Quantification of the number of cadaverine 555 positive brain parenchyma cells in the brain vasculature of Glut1iECKO mice with ( orange ) or without ( blue ) lactate administration . ( n = 24 from 5 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from 1 - way ANOVA . ) D . Quantification of CSF lactate level in WT and Glut1iECKO mice with / without lactate supplementation . ( n = 3 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from 1 - way ANOVA . )

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

B. Quantification of the CD13-positive pericytes coverage in the brain vasculature of Glut1iECKO mice with (orange) or without (blue) lactate administration. (n=16 from 5 independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.) C. Quantification of the number of cadaverine 555 positive brain parenchyma cells in the brain vasculature of Glut1iECKO mice with (orange) or without (blue) lactate administration. (n=24 from 5 independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.) D. Quantification of CSF lactate level in WT and Glut1iECKO mice with/without lactate supplementation. (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.)


( a - b ) eIF4A knockdown in either S2 ( a ) or Kc167 ( b ) cells blunts the inactivation of TORC1 upon amino acid removal . Cells were treated with dsRNA targeting GFP as a negative control , or three independent , non - overlapping dsRNAs targeting eIF4A for 5 days and then incubated with complete medium or medium lacking only amino acids for 30 minutes . Representative of 3 biological replicates .

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(a-b) eIF4A knockdown in either S2 (a) or Kc167 (b) cells blunts the inactivation of TORC1 upon amino acid removal. Cells were treated with dsRNA targeting GFP as a negative control, or three independent, non-overlapping dsRNAs targeting eIF4A for 5 days and then incubated with complete medium or medium lacking only amino acids for 30 minutes. Representative of 3 biological replicates.


( d ) Impaired inactivation of TORC1 in response to eIF4A knockdown is most apparent upon partial depletion of amino acids , caused by removal of amino acid subsets . After 5 days of knockdown , S2 cells were treated for 30 min with either complete Schneider ' s medium ( + aa ) , Schneider ' s medium lacking all amino acids ( - aa ) or various subsets of amino acids , as indicated ( where EAA " essential amino acids " = H , I , L , K , M , T , W , V ) . Error bars : std dev . n = 3 biological replicates .

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(d) Impaired inactivation of TORC1 in response to eIF4A knockdown is most apparent upon partial depletion of amino acids, caused by removal of amino acid subsets. After 5 days of knockdown, S2 cells were treated for 30 min with either complete Schneider's medium (+aa), Schneider's medium lacking all amino acids (-aa) or various subsets of amino acids, as indicated (where EAA "essential amino acids" = H,I,L,K,M,T,W,V). Error bars: std dev. n=3 biological replicates.


( e ) Time course of amino - acid removal reveals that eIF4A knockdown Kc167 cells maintain elevated S6K phosphorylation up to the maximum possible timepoint of 60 minutes when the cells start dying ( see drop in S6K and tubulin levels ) . Representative of two biological replicates .

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(e) Time course of amino-acid removal reveals that eIF4A knockdown Kc167 cells maintain elevated S6K phosphorylation up to the maximum possible timepoint of 60 minutes when the cells start dying (see drop in S6K and tubulin levels). Representative of two biological replicates.


( f ) eIF4A mutant larvae have impaired TORC1 inactivation upon shifting to food lacking amino acids . Control ( w1118 ) or eIF4A1006 / 1013 1st instar larvae were transferred from standard food to plates containing either standard fly food or PBS / 1 % agarose + 2 % sucrose for 1h prior to lysis and immunoblot analysis . Two biological replicates are shown . Representative of 3 biological replicates .

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(f) eIF4A mutant larvae have impaired TORC1 inactivation upon shifting to food lacking amino acids. Control (w1118) or eIF4A1006/1013 1st instar larvae were transferred from standard food to plates containing either standard fly food or PBS/1% agarose+2%sucrose for 1h prior to lysis and immunoblot analysis. Two biological replicates are shown. Representative of 3 biological replicates.


( a - a ' ) Although knockdown of eIF4A or eIF3 - S2 equally blunt translation of EGFP from an inducible plasmid ( a ) , only knockdown of eIF4A but not eIF3 - S2 impairs TORC1 inactivation upon amino acid removal ( a ' ) . ( a ) S2 cells , treated with indicated dsRNAs for 5 days , on day 3 transfected with an inducible EGFP plasmid ( pMT - EGFP ) and on day 4 induced for 18h . Non - targeting LacZ dsRNA used as a negative control . ( a ' ) S2 cells treated with indicated dsRNAs for 5 days , then incubated with medium lacking amino acids for indicated time prior to lysis . Representative of two biological replicates .

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(a-a') Although knockdown of eIF4A or eIF3-S2 equally blunt translation of EGFP from an inducible plasmid (a), only knockdown of eIF4A but not eIF3-S2 impairs TORC1 inactivation upon amino acid removal (a'). (a) S2 cells, treated with indicated dsRNAs for 5 days, on day 3 transfected with an inducible EGFP plasmid (pMT-EGFP) and on day 4 induced for 18h. Non-targeting LacZ dsRNA used as a negative control. (a') S2 cells treated with indicated dsRNAs for 5 days, then incubated with medium lacking amino acids for indicated time prior to lysis. Representative of two biological replicates.


( b ) Knockdown of eIF4A , but not other translation initiation factors , blunts TORC1 inactivation upon amino acid withdrawal . Kc167 cells treated with indicated dsRNAs for 4 days and then incubated with complete Schneider ' s medium or Schneider ' s medium lacking the indicated amino acids for 30 minutes prior to lysis .

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(b) Knockdown of eIF4A, but not other translation initiation factors, blunts TORC1 inactivation upon amino acid withdrawal. Kc167 cells treated with indicated dsRNAs for 4 days and then incubated with complete Schneider's medium or Schneider's medium lacking the indicated amino acids for 30 minutes prior to lysis.


( c ) Inhibition of translation with cycloheximide does not block inactivation of TORC1 upon amino acid withdrawal in Kc167 cells . Kc167 cells treated with either eIF4A dsRNA for 4 days , or cycloheximide ( 50 µg / mL , " CHX " ) for 5 min prior to , as well as during removal of amino acids ( - LIVASTQP ) . CHX treated cells still inactivate TORC1 ( compare lanes 8 to 7 ) whereas eIF4A knockdown cells do not ( lanes 14 vs 13 ) . Quantifications of two biological replicates are shown . CHX and eIF4A samples were normalized to their respective control conditions .

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(c) Inhibition of translation with cycloheximide does not block inactivation of TORC1 upon amino acid withdrawal in Kc167 cells. Kc167 cells treated with either eIF4A dsRNA for 4 days, or cycloheximide (50 µg/mL, "CHX") for 5 min prior to, as well as during removal of amino acids (-LIVASTQP). CHX treated cells still inactivate TORC1 (compare lanes 8 to 7) whereas eIF4A knockdown cells do not (lanes 14 vs 13). Quantifications of two biological replicates are shown. CHX and eIF4A samples were normalized to their respective control conditions.


( d ) Knockdown of eIF4A in Kc167 cells does not prevent a drop in intracellular amino acids levels when amino acids are removed from the medium for 30 min . Quantification of total intracellular amino acids , analyzed in a blinded fashion . Levels of individual amino acids shown in Figure S2D . For CHX samples , cycloheximide ( 50µg / mL ) was added 5 minutes prior to , and during treatment with medium containing or lacking amino acids . Statistical significance tested by ANOVA2 using Scheffé ' s multiple comparisons method ( p < 0 . 05 ) . Error bars : std . dev . n = 5 .

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(d) Knockdown of eIF4A in Kc167 cells does not prevent a drop in intracellular amino acids levels when amino acids are removed from the medium for 30 min. Quantification of total intracellular amino acids, analyzed in a blinded fashion. Levels of individual amino acids shown in Figure S2D. For CHX samples, cycloheximide (50µg/mL) was added 5 minutes prior to, and during treatment with medium containing or lacking amino acids. Statistical significance tested by ANOVA2 using Scheffé's multiple comparisons method (p<0.05). Error bars: std. dev. n=5.


( a ) Knockdown of eIF4F components ( eIF - 4E and eIF - 4G ) partially phenocopy the eIF4A knockdown , leading to elevated TORC1 activity upon amino acid removal ( 30 min ) . Knockdown of all other tested translation initiation factors do not cause elevated TORC1 upon amino acid removal ( main Figures 1c , 2a ' and 2b ) . Error bars : std . dev . n = 3 biological replicates .

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(a) Knockdown of eIF4F components (eIF-4E and eIF-4G) partially phenocopy the eIF4A knockdown, leading to elevated TORC1 activity upon amino acid removal (30 min). Knockdown of all other tested translation initiation factors do not cause elevated TORC1 upon amino acid removal (main Figures 1c, 2a' and 2b). Error bars: std. dev. n=3 biological replicates.


( b ) Quantification of de novo protein synthesis rates by OPP incorporation reveals that eIF4E and eIF4G knockdowns deplete eIF4F function less efficiently than the eIF4A knockdown , explaining why the effects of eIF4E and eIF4G knockdowns on TORC1 activity ( panel a ) are a bit milder than the eIF4A knockdown . Kc167 cells treated with dsRNA for 4 days then incubated with 20μM Click - it OPP reagent 30min before fixation and fluorescent labeling . Quantification of OPP fluorescence per cell ( nuclear count ) for two independent experiments is displayed ( 3 independent images per condition ) , normalized to the no dsRNA condition . Scale bars : 25µm .

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(b) Quantification of de novo protein synthesis rates by OPP incorporation reveals that eIF4E and eIF4G knockdowns deplete eIF4F function less efficiently than the eIF4A knockdown, explaining why the effects of eIF4E and eIF4G knockdowns on TORC1 activity (panel a) are a bit milder than the eIF4A knockdown. Kc167 cells treated with dsRNA for 4 days then incubated with 20μM Click-it OPP reagent 30min before fixation and fluorescent labeling. Quantification of OPP fluorescence per cell (nuclear count) for two independent experiments is displayed (3 independent images per condition), normalized to the no dsRNA condition. Scale bars: 25µm.


( a ) Binding of eIF4G to the Rag GTPases detected by co - immunoprecipitation from Kc167 cells in absence or presence of the chemical cross - linker DSP . Representative of > 3 biological replicates .

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(a) Binding of eIF4G to the Rag GTPases detected by co-immunoprecipitation from Kc167 cells in absence or presence of the chemical cross-linker DSP. Representative of >3 biological replicates.


( b ) Binding between eIF4G and the Rag GTPase complex is mainly mediated by RagC , detected by expressing and immunoprecipitating either RagA or RagC alone . Representative of two biological replicates .

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(b) Binding between eIF4G and the Rag GTPase complex is mainly mediated by RagC, detected by expressing and immunoprecipitating either RagA or RagC alone. Representative of two biological replicates.


( c ) Interaction between eIF4A and Raptor detected via co - immunoprecipitation of epitope - tagged proteins . Proteins were cross - linked with DSP prior to lysis and immunoprecipitation . Representative of 3 biological replicates .

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(c) Interaction between eIF4A and Raptor detected via co-immunoprecipitation of epitope-tagged proteins. Proteins were cross-linked with DSP prior to lysis and immunoprecipitation. Representative of 3 biological replicates.


( d - d ' ' ) eIF4A and Raptor form a complex , detected by proximity ligation assay ( PLA ) . Specificity of the PLA signal was controlled by knocking down either eIF4A or Raptor in Kc167 . ( d ) Representative images . Cells outlined in white . ( d ' ) Quantification of number of PLA spots per cell . ( n > 200 . * * * p < 0 . 0001 , ANOVA1 , Dunnett ' s multiple comparisons test , was performed on log - transformed data , box = + / - 1 quartile , whiskers = max / min ) . ( d ' ' ) Immunoblotting to detect efficiency of eIF4A and Raptor knockdowns .

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(d-d'') eIF4A and Raptor form a complex, detected by proximity ligation assay (PLA). Specificity of the PLA signal was controlled by knocking down either eIF4A or Raptor in Kc167. (d) Representative images. Cells outlined in white. (d') Quantification of number of PLA spots per cell. (n>200. ***p<0.0001, ANOVA1, Dunnett's multiple comparisons test, was performed on log-transformed data, box = +/- 1 quartile, whiskers = max/min). (d'') Immunoblotting to detect efficiency of eIF4A and Raptor knockdowns.


( e - f ) The eIF4A - Raptor interaction does not take place on lysosomes ( e ) or the endoplasmic reticulum ( ER ) ( f ) . Kc167 cells were either incubated with 100 µg / mL Dextran for 1 hour , washed and incubated for 14 hours in normal growth medium to label late endosomes and lysosomes ( e ) , or transfected to express an ER - resident GFP ( f ) . Amino acid removal was for 45 min . Pearson ' s correlation coefficients for eIF4A - Raptor PLA and Dextran in panel ( e ) are 0 . 09 and 0 . 08 for + aa and - LIVASTQP respectively ( calculated for all cells in the acquired fields , which were chosen randomly , n = 40 ) . Scale bars : 5µm .

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(e-f) The eIF4A-Raptor interaction does not take place on lysosomes (e) or the endoplasmic reticulum (ER) (f). Kc167 cells were either incubated with 100 µg/mL Dextran for 1 hour, washed and incubated for 14 hours in normal growth medium to label late endosomes and lysosomes (e), or transfected to express an ER-resident GFP (f). Amino acid removal was for 45 min. Pearson's correlation coefficients for eIF4A-Raptor PLA and Dextran in panel (e) are 0.09 and 0.08 for +aa and -LIVASTQP respectively (calculated for all cells in the acquired fields, which were chosen randomly, n=40). Scale bars: 5µm.


( a ) Knockdown of eIF4A and TSC2 do not elevate TORC1 activity in an additive manner , indicating they act in the same , and not parallel pathways . Whereas knockdown of eIF4A increases TORC1 activity in control Kc167 cells ( lanes 1 - 2 and 5 - 6 ) , eIF4A knockdown cannot further increase TORC1 activity in TSC2 knockdown cells ( lanes 3 - 4 and 7 - 8 ) . Error bars : std dev . n = 3 biological replicates .

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(a) Knockdown of eIF4A and TSC2 do not elevate TORC1 activity in an additive manner, indicating they act in the same, and not parallel pathways. Whereas knockdown of eIF4A increases TORC1 activity in control Kc167 cells (lanes 1-2 and 5-6), eIF4A knockdown cannot further increase TORC1 activity in TSC2 knockdown cells (lanes 3-4 and 7-8). Error bars: std dev. n=3 biological replicates.


( b ) Activation of TSC2 with the p90RSK inhibitor BI - D1870 rescues the elevated TORC1 activity caused by eIF4A knockdown in Kc167 cells , consistent with TSC2 acting downstream of eIF4A . BI - D1870 leads to inhibition of TORC1 ( lanes 1 - 4 ) in a TSC2 - dependent manner ( see also Appendix Figure S5A ) . Representative of two biological replicates .

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(b) Activation of TSC2 with the p90RSK inhibitor BI-D1870 rescues the elevated TORC1 activity caused by eIF4A knockdown in Kc167 cells, consistent with TSC2 acting downstream of eIF4A. BI-D1870 leads to inhibition of TORC1 (lanes 1-4) in a TSC2-dependent manner (see also Appendix Figure S5A). Representative of two biological replicates.


( c ) FLAG - eIF4A and TSC2 - V5 co - immunoprecipitate in Kc167 cells in the absence of chemical cross - linker . Cells were transfected with FLAG - eIF4A and TSC2 - V5 expression vectors and treated with media containing or lacking amino acids for indicated timepoints prior to lysis and anti - FLAG immunoprecipitation . The experiment was performed in absence of the chemical cross - linker DSP . Representative of 3 biological replicates .

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(c) FLAG-eIF4A and TSC2-V5 co-immunoprecipitate in Kc167 cells in the absence of chemical cross-linker. Cells were transfected with FLAG-eIF4A and TSC2-V5 expression vectors and treated with media containing or lacking amino acids for indicated timepoints prior to lysis and anti-FLAG immunoprecipitation. The experiment was performed in absence of the chemical cross-linker DSP. Representative of 3 biological replicates.


( d ) Expression of TSC2 - insensitive ( S15H or Q63L ) , but not wild - type ( WT ) Rheb causes TORC1 activity to remain high upon amino acid removal . Kc167 cells were transfected to express either wild - type or mutant Rheb and then incubated with Schneider ' s medium either containing or lacking amino acids for 30 minutes . Elevated TORC1 activity can be observed either by looking at phosphorylation of endogenous S6K , or phosphorylation of an HA - tagged S6K that was co - transfected with the Rheb constructs to assay specifically the transfected cells . Representative of two biological replicates .

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(d) Expression of TSC2-insensitive (S15H or Q63L), but not wild-type (WT) Rheb causes TORC1 activity to remain high upon amino acid removal. Kc167 cells were transfected to express either wild-type or mutant Rheb and then incubated with Schneider's medium either containing or lacking amino acids for 30 minutes. Elevated TORC1 activity can be observed either by looking at phosphorylation of endogenous S6K, or phosphorylation of an HA-tagged S6K that was co-transfected with the Rheb constructs to assay specifically the transfected cells. Representative of two biological replicates.


( a ) Mass spectrometry analysis of proteins co - immunoprecipitating with eIF4A reveals NAT1 as one of the top interacting proteins . eIF4A immunoprecipitations were performed in triplicate , from cells treated with medium either containing ( + aa ) or lacking amino acids ( - aa ) , and average values are shown . Peptide counts for each protein in each replicate were normalized to eIF4A peptide counts . Raw peptide counts shown in parentheses . ( a ' ) Schematic diagram of eIF4G and NAT1 primary protein structures . Binding sites for other initiation factors are shown .

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(a) Mass spectrometry analysis of proteins co-immunoprecipitating with eIF4A reveals NAT1 as one of the top interacting proteins. eIF4A immunoprecipitations were performed in triplicate, from cells treated with medium either containing (+aa) or lacking amino acids (-aa), and average values are shown. Peptide counts for each protein in each replicate were normalized to eIF4A peptide counts. Raw peptide counts shown in parentheses.(a') Schematic diagram of eIF4G and NAT1 primary protein structures. Binding sites for other initiation factors are shown.


( b ) Binding between eIF4A and NAT1 is regulated by amino acid availability in a TORC1 - independent fashion . Co - immunoprecipitation of tagged eIF4A and NAT1 in control Kc167 cells , or cells treated with medium lacking amino acids or supplemented with 20 nM rapamycin for indicated times . Representative of 3 biological replicates .

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(b) Binding between eIF4A and NAT1 is regulated by amino acid availability in a TORC1-independent fashion. Co-immunoprecipitation of tagged eIF4A and NAT1 in control Kc167 cells, or cells treated with medium lacking amino acids or supplemented with 20 nM rapamycin for indicated times. Representative of 3 biological replicates.


( c ) Knockdown of NAT1 using 4 independent , non - overlapping dsRNAs leads to reduced TORC1 activity . Kc167 cells , treated with indicated dsRNAs for 4 days and then incubated with medium containing or lacking amino acids for 30 min . Error bars : std . dev . n = 3 biological replicates .

(c) Knockdown of NAT1 using 4 independent, non-overlapping dsRNAs leads to reduced TORC1 activity. Kc167 cells, treated with indicated dsRNAs for 4 days and then incubated with medium containing or lacking amino acids for 30 min. Error bars: std. dev. n=3 biological replicates.


( d ) eIF4A is epistatic to NAT1 for TORC1 regulation . In the absence of amino acids , eIF4A knockdown cells have elevated TORC1 activity , NAT1 knockdown cells have reduced TORC1 activity , and eIF4A NAT1 double - knockdown cells have elevated TORC1 activity compared to control cells . Cells were treated with indicated dsRNAs for 4 days and then treated with medium containing or lacking the indicated amino acids for 30 min . Error bars : std . dev . n = 3 biological replicates .

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(d) eIF4A is epistatic to NAT1 for TORC1 regulation. In the absence of amino acids, eIF4A knockdown cells have elevated TORC1 activity, NAT1 knockdown cells have reduced TORC1 activity, and eIF4A NAT1 double-knockdown cells have elevated TORC1 activity compared to control cells. Cells were treated with indicated dsRNAs for 4 days and then treated with medium containing or lacking the indicated amino acids for 30 min. Error bars: std. dev. n=3 biological replicates.


( e ) TSC2 is epistatic to NAT1 for TORC1 regulation . As in ( d ) , knockdown of TSC2 rescues the reduced TORC1 activity caused by NAT1 knockdown .

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(e) TSC2 is epistatic to NAT1 for TORC1 regulation. As in (d), knockdown of TSC2 rescues the reduced TORC1 activity caused by NAT1 knockdown.


A Anti - TK Western blot of ubiquitinated titin samples .

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A Anti-TK Western blot of ubiquitinated titin samples.


( B - D ) Micrographs of H9C2 cells corresponding to ( B ) expressions of single species : MuRF1 - mCherry ( red ) , titin EGFP - A168 - TK ( green ) , Venus - Nbr1 ( yellow ) or Cerulean - p62 ( cyan ) ; ( C ) Co - expression of titin EGFP - A168 - TK with mCherry - MuRF1 . Neither Nbr1 nor p62 were co - transfected . The titin fragment and MuRF1 co - localize forming filamentous formations , which is in agreement with their known complexation ; ( D ) Co - expression of MuRF1 - mCherry ( red ) , titin EGFP - A168 - TK ( green ) and Venus - Nbr1 ( yellow ) or Cerulean - p62 ( cyan ) or respective truncated variants of the latter lacking the ubiquitin binding domain ( ∆ UBA ) . ( Throughout , cell nuclei are stained with DAPI ; blue ) . ( Scale bar is 10 μm in all cases ) .

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(B-D) Micrographs of H9C2 cells corresponding to (B) expressions of single species: MuRF1-mCherry (red), titin EGFP-A168-TK (green), Venus-Nbr1 (yellow) or Cerulean-p62 (cyan); (C) Co-expression of titin EGFP-A168-TK with mCherry-MuRF1. Neither Nbr1 nor p62 were co-transfected. The titin fragment and MuRF1 co-localize forming filamentous formations, which is in agreement with their known complexation; (D) Co-expression of MuRF1-mCherry (red), titin EGFP-A168-TK (green) and Venus-Nbr1 (yellow) or Cerulean-p62 (cyan) or respective truncated variants of the latter lacking the ubiquitin binding domain (∆UBA). (Throughout, cell nuclei are stained with DAPI; blue). (Scale bar is 10 μm in all cases).


Colonic organoids grown from Lgr5 - EGFP - IRES - CreERT2 mice were treated with an AhR agonist ( 10 nM TCDD ) or antagonist ( 10 µM CH223191 ) for 3 d . ( A ) Induced Cyp1a1 mRNA expression in response to different treatments ( n = 3 per group ) .

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Colonic organoids grown from Lgr5-EGFP-IRES-CreERT2 mice were treated with an AhR agonist (10 nM TCDD) or antagonist (10 µM CH223191) for 3 d. (A) Induced Cyp1a1 mRNA expression in response to different treatments (n=3 per group).


Colonic organoids grown from Lgr5 - EGFP - IRES - CreERT2 mice were treated with an AhR agonist ( 10 nM TCDD ) or antagonist ( 10 µM CH223191 ) for 3 d . ( B ) Assessment of GFP + stem cell percentages ( n = 3 replicates per treatment ) . Bars with different letters are significantly different ( p < 0 . 05 ) .

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Colonic organoids grown from Lgr5-EGFP-IRES-CreERT2 mice were treated with an AhR agonist (10 nM TCDD) or antagonist (10 µM CH223191) for 3 d. (B) Assessment of GFP+ stem cell percentages (n=3 replicates per treatment). Bars with different letters are significantly different (p<0.05).


( D ) Flow cytometry analysis of GFP + Tomato + and Tomato + cells in the large intestine of AhR wild type ( Lgr5 - GFPCreERT2 X Tomatof / f , GT ) and AhR knock out ( AhRf / f X Lgr5 - GFPCreERT2 X Tomatof / f , HGT ) mice . Top left panel shows representative cells from WT negative control animal ( no GFP or Tomato expression ) . Top right panel shows Tomato only control ( CDX2P - CreERT2 X Tomatof / f ) . Middle panels represent DMSO or TCDD treatment in AhR WT mice , respectively . Mice were gavaged every other day , 4 times total , with vehicle or TCDD ( 25 µg / kg bw ) and terminated 1 d later . Bottom left panel is from a representative AhR WT mouse and bottom right panel is from a representative AhR KO mouse .

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(D) Flow cytometry analysis of GFP+Tomato+ and Tomato+ cells in the large intestine of AhR wild type (Lgr5-GFPCreERT2 X Tomatof/f, GT) and AhR knock out (AhRf/f X Lgr5-GFPCreERT2 X Tomatof/f, HGT) mice. Top left panel shows representative cells from WT negative control animal (no GFP or Tomato expression). Top right panel shows Tomato only control (CDX2P-CreERT2 X Tomatof/f). Middle panels represent DMSO or TCDD treatment in AhR WT mice, respectively. Mice were gavaged every other day, 4 times total, with vehicle or TCDD (25 µg/kg bw) and terminated 1 d later. Bottom left panel is from a representative AhR WT mouse and bottom right panel is from a representative AhR KO mouse.


( E ) Induced Cyp1a1 mRNA expression in stem cells after oral gavage with TCDD ( n = 4 ) .

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(E) Induced Cyp1a1 mRNA expression in stem cells after oral gavage with TCDD (n=4).


( F ) Percentage of Lgr5 + stem cells ( n = 4 mice per group ) in ( D ) .

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(F) Percentage of Lgr5+ stem cells (n=4 mice per group) in (D).


Flow cytometry analysis of GFP + Tomato + and Tomato + cells in the large intestine of AhR wild type and AhR knock out mice . ( G ) Quantitative analysis of the percentage of Lgr5 + stem cells in ( D ) , n = 7 - 10 mice per group .

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Flow cytometry analysis of GFP+Tomato+ and Tomato+ cells in the large intestine of AhR wild type and AhR knock out mice. (G) Quantitative analysis of the percentage of Lgr5+ stem cells in (D), n=7-10 mice per group.


Representative immunohistochemistry of GFP + stem cells per crypt , n = 60 ~ 70 crypts from n = 3 mice per group . Scale 50 µm .

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Representative immunohistochemistry of GFP+ stem cells per crypt, n=60~70 crypts from n=3 mice per group. Scale 50 µm.


Representative violin plots of GFP + stem cells per crypt , n = 60 ~ 70 crypts from n = 3 mice per group . Scale 50 µm . Solid line indicates median , and dotted lines indicate 25th / 75th quartiles .

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Representative violin plots of GFP+ stem cells per crypt, n=60~70 crypts from n=3 mice per group. Scale 50 µm. Solid line indicates median, and dotted lines indicate 25th/75th quartiles.


( A ) Representative flow cytometry gating and fluorescence microscopy images of sorted GFPhigh stem cells and GFPlow progenitor cells from Lgr5 - EGFP - IRES - CreERT2 mice .

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(A) Representative flow cytometry gating and fluorescence microscopy images of sorted GFPhigh stem cells and GFPlow progenitor cells from Lgr5-EGFP-IRES-CreERT2 mice.


( B ) Representative brightfield images of organoids generated from GFPhigh stem cells isolated from tamoxifen treated AhR WT mice ( Lgr5 - GFPCreERT2 X CDX2P - CreERT2 [ GC ] ) and KO mice ( AhRf / f X Lgr5 - GFPCreERT2 X CDX2P - CreERT2 [ HGC ] ) , treated with DMSO ( control ) or TCDD for 5 d . Data are from ≥ 4 mice , scale bar 200 µm .

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(B) Representative brightfield images of organoids generated from GFPhigh stem cells isolated from tamoxifen treated AhR WT mice (Lgr5-GFPCreERT2 X CDX2P-CreERT2 [GC]) and KO mice (AhRf/f X Lgr5-GFPCreERT2 X CDX2P-CreERT2 [HGC]), treated with DMSO (control) or TCDD for 5 d. Data are from ≥4 mice, scale bar 200 µm.


( C , D ) The organoid forming efficiency ( C ) and diameter ( D ) from sorted GFPhigh stem cells in AhR WT or KO mice treated for 5 d with DMSO or TCDD ( 10 nM ) . n = 4 - 5 mice per group in ( C ) . Each dot represents an individual organoid and grey line indicates mean in ( D ) . Bars with different letters are significantly different ( p < 0 . 05 ) .

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(C, D) The organoid forming efficiency (C) and diameter (D) from sorted GFPhigh stem cells in AhR WT or KO mice treated for 5 d with DMSO or TCDD (10 nM). n=4-5 mice per group in (C). Each dot represents an individual organoid and grey line indicates mean in (D). Bars with different letters are significantly different (p<0.05).


Organoid growth from GFPlow progenitor cells . ( E ) Representative brightfield images of organoids .

Organoid growth from GFPlow progenitor cells. (E) Representative brightfield images of organoids.


( F ) Organoid forming efficiency and ( G ) diameter . n = 4 - 8 mice per group in ( F ) . Each dot represents an individual organoid and grey line indicates mean in ( G ) . Scale bar 200 μm . Bars with different letters are significantly different ( p < 0 . 05 ) .

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(F) Organoid forming efficiency and (G) diameter. n=4-8 mice per group in (F). Each dot represents an individual organoid and grey line indicates mean in (G). Scale bar 200 μm. Bars with different letters are significantly different (p<0.05).


( H , I ) ( H ) Lgr5 expression in organoids and ( I ) sorted stem cells in the absence or presence of TCDD treatment ( n ≥ 3 independent samples per group ) .

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(H, I) (H) Lgr5 expression in organoids and (I) sorted stem cells in the absence or presence of TCDD treatment (n≥3 independent samples per group).


( J ) Representative brightfield images for secondary organoid formation pretreated with DMSO or 10 nM TCDD for 2d . Scale 200 µm .

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(J) Representative brightfield images for secondary organoid formation pretreated with DMSO or 10 nM TCDD for 2d. Scale 200 µm.


( C ) Heatmap comparison of the top 25 upstream regulators between AhR KO ( KO / WT ) and TCDD ( TCDD / vehicle in WT mice ) treated cells .

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(C) Heatmap comparison of the top 25 upstream regulators between AhR KO (KO/WT) and TCDD (TCDD/vehicle in WT mice) treated cells.


( A ) FoxM1 , Plk1 , and Cdc25B mRNA expression in stem and progenitor cells with and without AhR , n = 4 mice per group .

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(A) FoxM1, Plk1, and Cdc25B mRNA expression in stem and progenitor cells with and without AhR, n=4 mice per group.


( B ) FoxM1 , Plk1 , and Cdc25B mRNA expression in WT stem and progenitor cells with and without TCDD , n = 3 - 4 mice per group .

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(B) FoxM1, Plk1, and Cdc25B mRNA expression in WT stem and progenitor cells with and without TCDD, n=3-4 mice per group.


Chromatin immunoprecipitation ( ChIP ) analysis of AhR and FoxM1 promoter interaction using WT colonic organoids treated with DMSO or TCDD , n = 3 or 4 per group . A DNA fragment amplified in a gene desert on mouse chromosome 6 was used as a negative control .

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Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT colonic organoids treated with DMSO or TCDD, n=3 or 4 per group. A DNA fragment amplified in a gene desert on mouse chromosome 6 was used as a negative control.


Chromatin immunoprecipitation ( ChIP ) analysis of AhR and FoxM1 promoter interaction using WT and KO crypts ( E ) treated with DMSO or TCDD , n = 3 or 4 per group .

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Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT and KO crypts (E) treated with DMSO or TCDD, n=3 or 4 per group.


( F ) FoxM1 luciferase activity determination for young adult mouse colonocytes ( YAMCs ) treated with DMSO or 10 nM TCDD for 1 d , n = 3 per group .

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(F) FoxM1 luciferase activity determination for young adult mouse colonocytes (YAMCs) treated with DMSO or 10 nM TCDD for 1 d, n=3 per group.


( G Expression of FoxM1 and PLK1 at mRNA level , , n > = 3 independent organoids per treatment .

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(G Expression of FoxM1 and PLK1 at mRNA level, , n>=3 independent organoids per treatment.


H ) Expression of FoxM1 and PLK1 at protein level , n > = 3 independent organoids per treatment .

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H) Expression of FoxM1 and PLK1 at protein level, n>=3 independent organoids per treatment.


( I ) Representative brightfield images of mouse colonic organoids generated from WT Lgr5 + - GFPhi stem cells treated with DMSO or 5µM FDI - 6 . Scale bar 200 µm .

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(I) Representative brightfield images of mouse colonic organoids generated from WT Lgr5+-GFPhi stem cells treated with DMSO or 5µM FDI-6. Scale bar 200 µm.


( J , K ) Quantification of organoid forming efficiency ( J ) and organoid area ( K ) from WT colonic stem cells treated with DMSO or 5µM FDI - 6 . Data are represented as mean ± SEM in ( K ) .

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(J, K) Quantification of organoid forming efficiency (J) and organoid area (K) from WT colonic stem cells treated with DMSO or 5µM FDI-6. Data are represented as mean ± SEM in (K).


( L ) Organoid expression of Lgr5 in response to FDI - 6 . Lgr5 expression level was dramatically suppressed by FDI - 6 in a dose - dependent manner , n > = 3 per treatment .

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(L) Organoid expression of Lgr5 in response to FDI-6. Lgr5 expression level was dramatically suppressed by FDI-6 in a dose-dependent manner, n>=3 per treatment.


( M ) Representative brightfield images for secondary organoid formation following treatment with DMSO or 5 µM FDI - 6 for 2 d . Scale 200 µm .

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(M) Representative brightfield images for secondary organoid formation following treatment with DMSO or 5 µM FDI-6 for 2 d. Scale 200 µm.


( A and B ) Proliferating Lgr5 + stem cells marked by EdU in the distal colon from AhR WT ( GT ) and KO ( HGT ) mice . Arrowhead and star symbols denote proliferating stem cells ( GFP + EdU + ) and static stem cells ( GFP + EdU - ) , respectively , during a 2 h EdU pulse , n = 3 per treatment .

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(A and B) Proliferating Lgr5+ stem cells marked by EdU in the distal colon from AhR WT (GT) and KO (HGT) mice. Arrowhead and star symbols denote proliferating stem cells (GFP+EdU+) and static stem cells (GFP+EdU-), respectively, during a 2 h EdU pulse, n=3 per treatment.


( C ) Total number of EdU + cells per crypt in the distal colon of AhR WT and KO ( HGT ) mice , n = 3 mice per treatment . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles .

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(C) Total number of EdU+ cells per crypt in the distal colon of AhR WT and KO (HGT) mice, n=3 mice per treatment. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.


( D & E ) Crypt cell proliferative zone analysis in AhR WT and KO ( HGT ) mice , presented as the distance from the crypt base to the highest EdU + cell divided by total crypt length . Red lines between crosses show the measurement of distance . Each symbol represents one crypt , n = 3 mice per treatment , scale bar 20 µm . Grey line represents mean .

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(D&E) Crypt cell proliferative zone analysis in AhR WT and KO (HGT) mice, presented as the distance from the crypt base to the highest EdU+ cell divided by total crypt length. Red lines between crosses show the measurement of distance. Each symbol represents one crypt, n=3 mice per treatment, scale bar 20 µm. Grey line represents mean.


( F & AhR KO promoted cell proliferation 5 d post DSS treatment . AhR WT ( AhRf / f [ H ] , n = 4 ) and AhR KO ( AhRf / f X Villin - Cre [ HV ] , n = 4 ) mice were used .

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(F& AhR KO promoted cell proliferation 5 d post DSS treatment. AhR WT (AhRf/f [H], n=4) and AhR KO (AhRf/f X Villin-Cre [HV], n=4) mice were used.


G ) AhR KO promoted cell proliferation 5 d post DSS treatment . Data represent mean ± SEM , quantified as the ratio of the EdU + area relative to the DAPI + area using KeyenceTM software .

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G) AhR KO promoted cell proliferation 5 d post DSS treatment. Data represent mean ± SEM, quantified as the ratio of the EdU+ area relative to the DAPI+ area using KeyenceTM software.


( H ) Increased cell proliferation ( EdU + cells ) observed in AhR KO vs WT organoids derived from HGC and GC mice , respectively ( n = 6 from 3 mice per group ) .

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(H) Increased cell proliferation (EdU+ cells) observed in AhR KO vs WT organoids derived from HGC and GC mice, respectively (n=6 from 3 mice per group).


( I ) Effect of AhR KO on cell cycle S and G2 - M phases ( n = 3 mice per group ) .

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(I) Effect of AhR KO on cell cycle S and G2-M phases (n=3 mice per group).


( J ) . FoxM1 inhibition reduced the number of colonic stem cells and cell proliferation . Mice were intraperitoneally injected with the FoxM1 inhibitor Thiostrepton ( 50 mg / kg ) for 2 consecutive days , n = 80 ~ 100 crypts from 4 mice per group . Scale bar , 50 µm . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles . Bars with different letters are significantly different ( p < 0 . 05 ) .

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(J). FoxM1 inhibition reduced the number of colonic stem cells and cell proliferation. Mice were intraperitoneally injected with the FoxM1 inhibitor Thiostrepton (50 mg/kg) for 2 consecutive days, n=80~100 crypts from 4 mice per group. Scale bar, 50 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles. Bars with different letters are significantly different (p<0.05).


( B ) Representative colon images from AhR WT and AhR KO mice following AOM / DSS treatment . Scale bar , 9 mm .

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(B) Representative colon images from AhR WT and AhR KO mice following AOM/DSS treatment. Scale bar, 9 mm.


( C ) H & E staining of representative colon tumor sections from AhR WT and KO mice , scale bar 200 μm .

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(C) H&E staining of representative colon tumor sections from AhR WT and KO mice, scale bar 200 μm.


( E , F ) ( E ) Adenoma and ( F ) adenocarcinoma multiplicity in AhR WT and KO mice .

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(E, F) (E) Adenoma and (F) adenocarcinoma multiplicity in AhR WT and KO mice.


( G ) Tumor volume in AhR KO and WT mice . Grey line indicates mean .

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(G) Tumor volume in AhR KO and WT mice. Grey line indicates mean.


( H ) FoxM1 mRNA expression in normal ( uninvolved ) crypts adjacent to colonic tumors . Images were quantified and expressed as FoxM1 positive area per crypt ( n = 5 mice per group ) . Scale 200 µm . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles .

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(H) FoxM1 mRNA expression in normal (uninvolved) crypts adjacent to colonic tumors. Images were quantified and expressed as FoxM1 positive area per crypt (n=5 mice per group). Scale 200 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.


( I ) FoxM1 mRNA expression in colon tumors . FoxM1 mRNA ISH area was averaged per tumor on each slide ( n = 5 mice per group ) . Scale 200 µm . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles .

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(I) FoxM1 mRNA expression in colon tumors. FoxM1 mRNA ISH area was averaged per tumor on each slide (n=5 mice per group). Scale 200 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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