A . Permeability using vibratome section showing cadaverine 555 dye ( red ) , CD31 ( green ) CD13 ( purple ) in brain vasculature of Glut1iECKO mice with ( bottom ) or without ( top ) lactate administration . Note 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 . of CD13 - positive pericytes coverage in brain vasculature of Glut1iECKO mice with ( orange ) or without ( blue ) lactate administration . ( n = 16 from 5 independent experiments . Error bars indicate standard error of mean ( ) from 1 - way ANOVA . ) C . of number of cadaverine 555 positive brain parenchyma in brain vasculature of Glut1iECKO mice with ( orange ) or without ( blue ) lactate administration . ( n = 24 from 5 independent experiments . Error bars indicate standard error of mean ( ) from 1 - way ANOVA . ) D . of CSF lactate level in WT Glut1iECKO mice with / without lactate supplementation . ( n = 3 from independent experiments . Error bars indicate standard error of mean ( ) from 1 - way ANOVA . )

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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 ) blunts inactivation of TORC1 upon acid removal . were treated with dsRNA targeting GFP as a negative control , or three independent , non - overlapping dsRNAs targeting eIF4A for 5 days then incubated with complete medium or medium lacking only 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 , caused by removal of acid subsets . After 5 days of knockdown , S2 were treated for 30 min with either complete Schneider ' s medium ( + aa ) , Schneider ' s medium lacking ( - aa ) or various subsets of , as indicated ( where EAA " essential " = 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 - acid removal reveals that eIF4A knockdown Kc167 maintain elevated S6K phosphorylation up to maximum possible timepoint of 60 minutes when start dying ( see drop in S6K tubulin ) . 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 . 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 immunoblot . 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 acid removal ( a ' ) . ( a ) S2 , treated with indicated dsRNAs for 5 days , on day 3 transfected with an inducible EGFP plasmid ( pMT - EGFP ) on day 4 induced for 18h . Non - targeting LacZ dsRNA used as a negative control . ( a ' ) S2 treated with indicated dsRNAs for 5 days , then incubated with medium lacking 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 acid withdrawal . Kc167 treated with indicated dsRNAs for 4 days then incubated with complete Schneider ' s medium or Schneider ' s medium lacking indicated 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 acid withdrawal in Kc167 . Kc167 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 ( - LIVASTQP ) . CHX treated still inactivate TORC1 ( compare lanes 8 to 7 ) whereas eIF4A knockdown do not ( lanes 14 vs 13 ) . Quantifications of two biological replicates are shown . CHX 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 does not prevent a drop in intracellular when are removed from medium for 30 min . of total intracellular , analyzed in a blinded fashion . of shown in Figure S2D . For CHX samples , cycloheximide ( 50µg / mL ) was added 5 minutes prior to , during treatment with medium containing or lacking . 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 eIF - 4G ) partially phenocopy eIF4A knockdown , leading to elevated TORC1 activity upon acid removal ( 30 min ) . Knockdown of other tested translation initiation factors do not cause elevated TORC1 upon acid removal ( main Figures 1c , 2a ' 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 ) of de novo synthesis rates by OPP incorporation reveals that eIF4E eIF4G knockdowns deplete eIF4F function less efficiently than eIF4A knockdown , explaining why effects of eIF4E eIF4G knockdowns on TORC1 activity ( panel a ) are a bit milder than eIF4A knockdown . Kc167 treated with dsRNA for 4 days then incubated with 20μM Click - it OPP reagent 30min before fixation labeling . of OPP per ( ) for two independent experiments is displayed ( 3 independent per condition ) , normalized to 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 Rag GTPases detected by co - immunoprecipitation from Kc167 in absence or presence of 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 Rag GTPase is mainly mediated by RagC , detected by expressing 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 Raptor detected via co - immunoprecipitation of epitope - tagged . were cross - linked with DSP prior to lysis 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 Raptor form a , detected by proximity ligation ( PLA ) . Specificity of PLA signal was controlled by knocking down either eIF4A or Raptor in Kc167 . ( d ) Representative . outlined in . ( d ' ) of number of PLA spots per . ( n > 200 . * * * p < 0 . 0001 , ANOVA1 , Dunnett ' s multiple comparisons , was performed on log - transformed data , box = + / - 1 quartile , whiskers = max / min ) . ( d ' ' ) Immunoblotting to detect efficiency of eIF4A 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 ) eIF4A - Raptor interaction does not take place on lysosomes ( e ) or endoplasmic reticulum ( ER ) ( f ) . Kc167 were either incubated with 100 µg / mL Dextran for 1 hour , washed incubated for 14 hours in normal growth medium to label late endosomes lysosomes ( e ) , or transfected to express an ER - resident GFP ( f ) . acid removal was for 45 min . Pearson ' s correlation coefficients for eIF4A - Raptor PLA Dextran in panel ( e ) are 0 . 09 0 . 08 for + aa - LIVASTQP respectively ( calculated for in 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 TSC2 do not elevate TORC1 activity in an additive manner , indicating they act in same , not parallel pathways . Whereas knockdown of eIF4A increases TORC1 activity in control Kc167 ( lanes 1 - 2 5 - 6 ) , eIF4A knockdown cannot further increase TORC1 activity in TSC2 knockdown ( lanes 3 - 4 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 p90RSK inhibitor BI - D1870 rescues elevated TORC1 activity caused by eIF4A knockdown in Kc167 , 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 TSC2 - V5 co - immunoprecipitate in Kc167 in absence of chemical cross - linker . were transfected with FLAG - eIF4A TSC2 - V5 expression vectors treated with media containing or lacking for indicated timepoints prior to lysis anti - FLAG immunoprecipitation . experiment was performed in absence of 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 acid removal . Kc167 were transfected to express either wild - type or mutant Rheb then incubated with Schneider ' s medium either containing or lacking 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 Rheb constructs to specifically transfected . 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 of co - immunoprecipitating with eIF4A reveals NAT1 as one of top interacting . eIF4A immunoprecipitations were performed in triplicate , from treated with medium either containing ( + aa ) or lacking ( - aa ) , average values are shown . Peptide for each in each replicate were normalized to eIF4A peptide . Raw peptide shown in parentheses . ( a ' ) Schematic diagram of eIF4G NAT1 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 NAT1 is regulated by acid availability in a TORC1 - independent fashion . Co - immunoprecipitation of tagged eIF4A NAT1 in control Kc167 , or treated with medium lacking 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 , treated with indicated dsRNAs for 4 days then incubated with medium containing or lacking for 30 min . Error bars : std . dev . n = 3 biological replicates .

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(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 absence of , eIF4A knockdown have elevated TORC1 activity , NAT1 knockdown have reduced TORC1 activity , eIF4A NAT1 double - knockdown have elevated TORC1 activity compared to control . were treated with indicated dsRNAs for 4 days then treated with medium containing or lacking indicated 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 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 ) of H9C2 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 . titin fragment 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 ) Venus - Nbr1 ( yellow ) or Cerulean - p62 ( cyan ) or respective truncated variants of latter lacking ubiquitin binding domain ( ∆ UBA ) . ( Throughout , nuclei are stained with DAPI ; blue ) . ( Scale bar is 10 μm in 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 + 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 of GFP + Tomato + Tomato + in large intestine of AhR wild type ( Lgr5 - GFPCreERT2 X Tomatof / f , GT ) AhR knock out ( AhRf / f X Lgr5 - GFPCreERT2 X Tomatof / f , HGT ) mice . Top left panel shows representative from WT negative control ( 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 ) terminated 1 d later . Bottom left panel is from a representative AhR WT mouse 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 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 ) of Lgr5 + ( 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 of GFP + Tomato + Tomato + in large intestine of AhR wild type AhR knock out mice . ( G ) Quantitative of of Lgr5 + 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 + 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 + per crypt , n = 60 ~ 70 crypts from n = 3 mice per group . Scale 50 µm . Solid indicates median , 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 microscopy of sorted GFPhigh GFPlow progenitor 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 of organoids generated from GFPhigh isolated from tamoxifen treated AhR WT mice ( Lgr5 - GFPCreERT2 X CDX2P - CreERT2 [ GC ] ) 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 ) organoid forming efficiency ( C ) diameter ( D ) from sorted GFPhigh 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 organoid grey 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 . ( E ) Representative brightfield of organoids .

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


( F ) Organoid forming efficiency ( G ) diameter . n = 4 - 8 mice per group in ( F ) . Each dot represents an organoid grey 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 ( I ) sorted in 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 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 ) comparison of top 25 upstream regulators between AhR KO ( KO / WT ) TCDD ( TCDD / vehicle in WT mice ) treated .

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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 , Cdc25B mRNA expression in progenitor with 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 , Cdc25B mRNA expression in WT progenitor with 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.


immunoprecipitation ( ChIP ) of AhR FoxM1 promoter interaction using WT colonic organoids treated with DMSO or TCDD , n = 3 or 4 per group . A 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.


immunoprecipitation ( ChIP ) of AhR FoxM1 promoter interaction using WT 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 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 PLK1 at 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 of mouse colonic organoids generated from WT Lgr5 + - GFPhi 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 ) of organoid forming efficiency ( J ) organoid ( K ) from WT colonic treated with DMSO or 5µM FDI - 6 . Data are represented as mean ± 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 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 B ) Proliferating Lgr5 + marked by EdU in distal colon from AhR WT ( GT ) KO ( HGT ) mice . Arrowhead star symbols denote proliferating ( GFP + EdU + ) static ( GFP + EdU - ) , respectively , during a 2 h EdU , 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 + per crypt in distal colon of AhR WT KO ( HGT ) mice , n = 3 mice per treatment . Dashed indicates median 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 proliferative zone in AhR WT KO ( HGT ) mice , presented as from crypt base to highest EdU + divided by total crypt . Red lines between crosses show measurement of . Each symbol represents one crypt , n = 3 mice per treatment , scale bar 20 µm . Grey 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 proliferation 5 d post DSS treatment . AhR WT ( AhRf / f [ H ] , n = 4 ) 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 proliferation 5 d post DSS treatment . Data represent mean ± , quantified as of EdU + relative to DAPI + 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 proliferation ( EdU + ) observed in AhR KO vs WT organoids derived from HGC 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 cycle S 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 number of colonic proliferation . Mice were intraperitoneally injected with FoxM1 inhibitor Thiostrepton ( 50 mg / kg ) for 2 consecutive days , n = 80 ~ 100 crypts from 4 mice per group . Scale bar , 50 µm . Dashed indicates median 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 from AhR WT 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 of representative colon sections from AhR WT 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 ( F ) adenocarcinoma multiplicity in AhR WT KO mice .

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


( G ) volume in AhR KO WT mice . Grey 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 . were quantified expressed as FoxM1 positive per crypt ( n = 5 mice per group ) . Scale 200 µm . Dashed indicates median 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 was averaged per on each slide ( n = 5 mice per group ) . Scale 200 µm . Dashed indicates median 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 markers LGR5 OLFM4 , n = 5 or 6 independent human samples . Paired student t - .

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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 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 - .

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


( H ) Representative immunoblots for FoxM1 PLK1 derived from organoids treated with DMSO or TCDD from n = 6 independent samples . Total 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 Caco2 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 CYP1A1 mRNA expression in different AhR WT CRISPR KO clones treated with TCDD for 1 d . C56 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 targeted AhR exon . Subsequent sequencing revealed indels at 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 ) association of FoxM1 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 indicates median 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 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 of human colonic organoids from sorted live 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 , ( P ) organoid diameter from sorted from human colonic organoids in presence of DMSO , TCDD CH223191 ( 10 μM ) for 6 d . n = 5 per group , 3 independent experiments . Grey 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 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 mobility plots for Armadillidium vulgare during 75 days post - injection . There were no significant differences between injected with wVulC , wDil or control treatment for these life history traits : Survival ( A ) , Gain of ( B ) 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).


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

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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 injected by wVulC compared to those from other treatments . On opposite , injection of wDil has no significant effect on 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 : level 0 corresponds to surface of substrate , negative values correspond to number of buried while positive values correspond to number of found at surface of substrate . For control treatment , not a single rose up at surface for individuals injected by wDil only few were spotted at surface . When inoculated by wVulC , rose up to 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 ( b ) data for : injection of wVulC kills individuals . However , die quicker than . 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 hemocytes of Porcellio d . dilatatus Armadillidium vulgare . ( A ) Comparison between ( i ) P . d . dilatatus in grey A . vulgare in injected with wVulC ( ii ) naturally infected A . vulgare in grey revealed no significant differences for Wolbachia loads in CNS , gonads hemocytes ( i . e immune ) , at both 30 60 days post - injection . ( B ) Comparison between ( i ) P . d . dilatatus in grey A . vulgare in injected with wDil ( ii ) naturally infected P . d . dilatatus in dim grey revealed no significant differences for Wolbachia loads in CNS at both 30 60 days PI . However , at 60 days post - injection , for gonads hemocytes wDil loads were significantly higher in native host P . d . dilatatus than in A . vulgare . A global comparison between wDil wVulC loads showed that wVulC ( A ) always exhibited higher 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 of Porcellio d . dilatatus injected by wVulC . These are localised in CNS ganglion . ( A ) At 30 days post - injection , some autolysosomes 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 ] . ( B ) At 60 days post - injection , nerve are severely damaged by accumulation of autophagic vesicles ( N : Nucleus , V : Vacuole , Ap : Autophagosomes ( Autophagic hallmark ) , Al : Autolysosomes , Arrow head : magnified 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 LC3B antibody labelling of adipocytes neighboring nerve of Porcellio d . dilatatus . These adipocytes are localised around nerve of CNS constitutes sheathing of this organ . ( A ) At 30 days post - injection , autolysosomes , autophagosomes but also were observed : autophagic process was already higher compared to control ( Ad : adipocyte , Ap : Autophagosomes ( Autophagic hallmark ) , Al : Autolysosomes , Wo : Wolbachia , Arrow head : magnified ) . ( B ) At 60 days post - injection , adipocytes were severely damaged by accumulation of autophagic vesicles ( Ad : adipocyte , Ap : Autophagosomes , Al : Autolysosomes , Wo : Wolbachia , Arrow head : magnified ) . ( C ) With antibody labelling anti - LC3B , LC3B implicated in autophagic processes was detected in adipocytes of P . d . dilatatus injected with wVulC at 45 days post - injection . highly labeled spherical structures would reflect incorporation of LC3B in 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 for on - target effects for Everolimus ( EV , P - RPS6 ) CX - 5461 ( p53 ) . Each lane represents equal amounts of 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 by MetaCore ® GeneGO of genes in " translation up " ( red ) " translation down " ( blue ) categories identified by anota2seq comparing lymph node isolated from mice in CX - 5461 + EV treatment group with V / V group ( n = 6 ) . " values were obtained by dividing number of significant genes in our data assigned to a molecular process by total number of genes in process in MetaCore ® ' s database . ( D ) Genes implicated in " Translation : initiation " " Translation : Elongation - Termination " processes 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 ) viability of indicated early - passage Eµ - Myc lymphoma 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 determined using liquid chromatography - mass spectrometry in CTRL , EV , CX CMB early - passage 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 ) oxygen consumption rate ( OCR ) in early - passage lines determined using Seahorse XF96 Extracellular Flux Analyzer ; graphs represent mean ± 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 of early - passage CX - 5461 - everolimus combination therapy - resistant ( CMB ) , CX - 5461 - resistant ( CX ) drug - naïve ( CTRL ) lymphoma treated with indicated concentrations of metformin for 48 hours .

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


( H ) CellTiterGLO ® - measuring ATP of CX - 5461 - resistant ( CX ) CX - 5461 - everolimus combination therapy - resistant ( CMB ) 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 of CTRL CMB treated with CX - 5461 everolimus in presence 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 of CTRL CX treated with CX - 5461 in presence absence of metformin for 48 hours as indicated .

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


( B ) Abundance of intracellular 3 ' 5 ' - cyclic AMP as measured by a liquid chromatography ( LC ) - mass spectrometry ( MS ) . Graphs represent mean ± 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 of EPAC1 EPAC2 abundance , as well as active GTP - bound RAP1 in indicated early - passage during their log - phase growth period ( n = 3 ) . Actin 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 treated with CX - 5461 everolimus as indicated in 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 of early - passage drug - naïve ( CTRL ) lymphoma treated with CX - 5461 everolimus in 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 of CMB treated with CX - 5461 everolimus as indicated in 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 of early - passage drug - naïve ( CTRL ) treated with CX - 5461 everolimus in presence or absence of selective EPAC activator 8 - pCPT - 2 - O - Me - cAMP 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.


( A ) A illustrating abundances of polysome - associated mRNAs that are associated with mRNA translation / synthesis on Gene Ontology Resource Database in drug - naïve ( CTRL ) or CX - 5461 - everolimus combination therapy - resistant ( CMB ) lines ( n = 3 ) . colours illustrate 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 as compared to CTRL CX - 5461 - resistant ( CX ) ( representatives of n = 3 ) . ( C ) Quantitation of polysome : monosome in indicated early - passage Eµ - Myc lymphoma as determined by measuring under curve using ImageJ software . Graph represents mean ± 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 on a nominal p - value < 0 . 01 cut - off in either CMB vs . CTRL or CX vs . CTRL as analysed by limma ) encoding components of mitochondrial oxidative phosphorylation 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 , 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 demonstrating effects of metformin treatment for 48 hours on of active GTP - bound RAP1 in CX CMB ( n = 3 ) 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 ( GFP ) - positive CMB ( clone # 8 ) in ( B ) lymph node ( 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 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 ) everolimus ( 5 mg / kg daily ) ( n = 8 ) , metformin ( 400 mg / kg twice daily ) ( n = 6 ) , or CX - 5461 , everolimus metformin ( 35 mg / kg twice weekly , 5 mg / kg daily 400 mg / kg twice daily , respectively ) ( n = 8 ) . grey : 5 - day metformin pre - treatment period , grey : treatment period . Data were analyzed by a log - rank ( Mantel - Cox ) . 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 of cilia ( a ) dot plots ( b ) showing cilia variation at different time points after serum starvation ( SS ) . Markers in ( a ) show ARL13B ( cilia ) PCNT ( Basal ) . graph shows of cilia from 3 biological replicates ( n > 150 cilia per sample experiment ) . Scale bar : 1µm . Data shown in include mean ± s . d . are calculated by two - unpaired student t - .

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

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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


d , FRAP experiment of SSTR3 - GFP in RPE1 WT SEPT2 KO . were starved for 48h before beginning of inspection imaged every 30sec for 10mins after bleaching . Scale bar : 5µm . e , 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 . P values are calculated by two - unpaired student t - .

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


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

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


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

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