B ) Caco - 2 cysts stably expressing empty vector ( pGIPZ ) or 2 distinct shFARP1 or shFARP2 were treated with Y27632 , allowed to invade for 2 days , fixed stained for F - actin ( Phalloidin ) . Boxed regions i , ii , iii iv are shown at high magnification . Arrowheads point to non - protruding , arrows point to protruding . stars show nuclei that engage in protrusions . Scale bars : 20μm . C ) Bar graphs representing of protrusive cysts formed from Caco - 2 transduced with lentiviruses encoding pGIPZ or shRNA against FARP1 FARP2 after treatment with Y27632 from at least 3 independent experiments ( Means ± , paired t - , ns : non significant , * * p < 0 . 01 ) .

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

B) Caco-2 cysts stably expressing empty vector (pGIPZ) or 2 distinct shFARP1 or shFARP2 were treated with Y27632, allowed to invade for 2 days, fixed and stained for F-actin (Phalloidin). Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells, arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. C) Bar graphs representing the percentage of protrusive cysts formed from Caco-2 cells transduced with lentiviruses encoding pGIPZ or shRNA against FARP1 and FARP2 after treatment with Y27632 from at least 3 independent experiments (Means ± SEM, paired t-test, ns: non significant, **p<0.01).


D ) Representative acquisition of Caco - 2 overexpressing GFP - FARP2 grown in 2D stained with ZO - 1 , in non - treated ( NT ) after 2h with Y27632 or H1152 . dashed lines crossing - junctions ( i , ii iii ) are representative of linescans measurements . Scale bars : 20μm . E ) Representative of ZO - 1 ( red ) GFP - FARP2 ( green ) along dashed i , ii iii represented corresponding to non - treated , Y27632 or H1152 treatments )

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

D) Representative acquisition of Caco-2 cells overexpressing GFP-FARP2 grown in 2D and stained with ZO-1, in non-treated (NT) and after 2h with Y27632 or H1152. The dashed lines crossing the cell-cell junctions (i, ii and iii) are representative of the linescans measurements. Scale bars: 20μm. E) Representative fluorescence intensity of ZO-1 (red) staining and GFP-FARP2 (green) along the dashed line i, ii and iii represented corresponding to non-treated, Y27632 or H1152 treatments)


F ) Bar graph representing average of GFP - FARP2 over ZO - 1 intensities measured with linescans crossing - junctions . was performed on 71 expressing GFP - FARP2 - FL from 3 independent experiments . For each , between 10 15 lines ( 12 pixels wide ) were drawn across random region of - junctions positive for ZO - 1 ( Mean ± Unpaired t - * * p < 0 . 01 , * p < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Bar graph representing the average ratio of GFP-FARP2 over ZO-1 fluorescence intensities measured with linescans crossing cell-cell junctions. The quantification was performed on 71 cells expressing GFP-FARP2-FL from 3 independent experiments. For each cell, between 10 and 15 lines (12 pixels wide) were drawn across random region of cell-cell junctions positive for ZO-1 staining (Mean ±SEM Unpaired t-test **p<0.01, *p<0.05).


A ) Caco - 2 cysts stably expressing GFP ( treated or not with Y27632 ) , or Cherry - FARP2 - full ( FARP2 - FL ) , FARP2 - 730 / 733 mutant ( FARP2 - Mut ) or FARP2 del PH 730 / 733 ( FARP2 - del / Mut ) were allowed to invade for 2 days , fixed stained for F - actin . Boxed regions i , ii , iii , iv v are shown at high magnification . Arrowheads point to non - protruding , arrow points to protruding , star shows nucleus that engages in protrusion . Scale bars : 20μm . B ) Bargraph representing of protrusive cysts expressing GFP ( treated or not with Y27632 ) or mentioned GFP - FARP2 constructs from 3 independent experiments ( Means ± , paired t - , * * p < 0 , 01 , n . s : non significant ) .

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

A) Caco-2 cysts stably expressing GFP (treated or not with Y27632), or Cherry-FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH 730/733 (FARP2-del/Mut) were allowed to invade for 2 days, fixed and stained for F-actin. Boxed regions i, ii, iii, iv and v are shown at high magnification. Arrowheads point to non-protruding cells, arrow points to protruding cells, white star shows nucleus that engages in protrusion. Scale bars: 20μm. B) Bargraph representing the percentage of protrusive cysts expressing GFP (treated or not with Y27632) or the mentioned GFP-FARP2 constructs from 3 independent experiments (Means ± SEM, paired t-test, **p<0,01, n.s: non significant).


C ) Representative of Caco - 2 cysts treated with Y27632 or Blebbistatin or transfected with siRNA targeting Myosin - IIA allowed to invade for 2 days . After fixation , cysts were stained for F - actin ( Phalloidin ) nuclei ( DAPI ) . Representative Y27632 - induced « invasive » phenotype Blebbistatin - induced « dendritic » phenotype are displayed . Arrows point to protruding , star shows nucleus that engages in protrusion arrowheads point to " dendritic " protrusions . D ) Bar graph representing of cysts with " invasive " or " dendritic " after treatment with Y27632 , Blebbistatin or siMyosin - IIA or siMyosin - IIB from at least 3 independent experiments ( Means ± , unpaired t - , * p < 0 , 05 , n . s : non significant ) .

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

C) Representative images of Caco-2 cysts treated with Y27632 or Blebbistatin or transfected with siRNA targeting Myosin-IIA and allowed to invade for 2 days. After fixation, the cysts were stained for F-actin (Phalloidin) and nuclei (DAPI). Representative Y27632-induced « invasive » phenotype and Blebbistatin-induced « dendritic » phenotype are displayed. Arrows point to protruding cells, white star shows nucleus that engages in protrusion and arrowheads point to "dendritic" protrusions. D) Bar graph representing the percentage of cysts with the "invasive" or "dendritic" phenotypes after treatment with Y27632, Blebbistatin or siMyosin-IIA or siMyosin-IIB from at least 3 independent experiments (Means ± SEM, unpaired t-test,*p<0,05, n.s: non significant).


E ) Mature Caco - 2 cysts were transduced with GFP or Cherry tagged FARP2 - full ( FARP2 - FL ) , FARP2 - 730 / 733 mutant ( FARP2 - Mut ) or FARP2 del PH / 730 / 733 ( FARP2 - del / Mut ) , treated ( or not , NT ) with Blebbistatin . 2 days after invasion , cysts were fixed stained for F - actin . Boxed regions i , ii , iii iv are shown at high magnification . Arrowheads point to non - protruding ; arrow points to protruding stars show nucleus that engage in protrusions . Scale bars : 20 μm . F ) Bargraph representing of protrusive Caco2 cysts expressing mentioned constructs , treated or not with blebbistatin , from at least 3 independent experiments ( Means ± , t - , * * p < 0 , 01 , n . s : non significant ) .

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

E) Mature Caco-2 cysts were transduced with GFP or Cherry tagged FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH/730/733 (FARP2-del/Mut), and treated (or not, NT) with Blebbistatin. 2 days after invasion,the cysts were fixed and stained for F-actin. Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells; arrow points to protruding cells and white stars show nucleus that engage in protrusions. Scale bars: 20 μm. F) Bargraph representing the percentage of protrusive Caco2 cysts expressing the mentioned constructs , treated or not with blebbistatin, from at least 3 independent experiments (Means ± SEM , t-test,**p<0,01, n.s: non significant).


Purified CUL3WT CUL3Δ403 - 459 analysed by SDS - PAGE Coomassie blue , following expression in SF21 , DAC - tag - affinity purification tag cleavage ( see Materials Methods ) . Bands migrating between 62 - 98 - kDa markers are CUL3WT CUL3Δ403 - 459 , respectively , while band below 17 kDa is Rbx1 . immunoblot for RBX1 is shown on right . band in CUL3WT denoted with * is co - purified insect CAND1 , which was later removed by SEC . identities were confirmed by mass spectrometry .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Purified CUL3WT and CUL3Δ403-459 analysed by SDS-PAGE and Coomassie blue staining, following expression in SF21 cells, and DAC-tag-affinity purification and tag cleavage (see Materials and Methods). Bands migrating between 62- and 98-kDa protein markers are CUL3WT and CUL3Δ403-459, respectively, while the band below 17 kDa is Rbx1. The immunoblot for RBX1 is shown on the right. The band in CUL3WT denoted with * is co-purified insect cell CAND1, which was later removed by SEC. All protein identities were confirmed by mass spectrometry.


KLHL3 binding to CUL3WT CUL3Δ403 - 459 is comparable as shown by pull - down assays with a KLHL3 resin generated from cross - linking anti - KLHL3 antibody to G sepharose , then binding saturating amounts of KLHL3 to anti - KLHL3 resin ( denoted KLHL3R ) , control resin ( ControlR ) is G sepharose ( see Materials Methods for further detail ) . CUL3WT or CUL3Δ403 - 459 was incubated with either KLHL3R or ControlR at 4 ° C for 1 h then washed thrice with 1 × PBS , resin volumes were minimised , SDS - Laemmli buffer was used to elute from resin prior to by SDS - PAGE with colloidal blue .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

KLHL3 binding to CUL3WT and CUL3Δ403-459 is comparable as shown by pull-down assays with a KLHL3 resin generated from cross-linking anti-KLHL3 antibody to Protein G sepharose, and then binding saturating amounts of KLHL3 to the anti-KLHL3 resin (denoted KLHL3R), the control resin (ControlR) is Protein G sepharose (see Materials and Methods for further detail). CUL3WT or CUL3Δ403-459 was incubated with either KLHL3R or ControlR at 4°C for 1 h and then washed thrice with 1× PBS, resin volumes were minimised, and SDS-Laemmli buffer was used to elute protein from resin prior to analysis by SDS-PAGE and staining with colloidal blue.


UBE2D3 ˜ ubiquitin - release assays analysed by non - reducing SDS - PAGE immunodetection . His - tagged UBE2D3 was charged with ubiquitin by incubation with ATP UAE , charging was stopped by ATP depletion with apyrase . No CUL3 ( negative control , lane 1 ) , CUL3WT or CUL3Δ403 - 459 was then added to charged E2 activity of RING domain , RBX1 , monitored by hydrolysis of ubiquitin from UBE2D3 , visualised by immunodetection with anti - His antibody ( upper panel , " ˜ " denotes thioester bond ) . lower panel is an immunoblot for CUL3 , showing equivalent amount of both CUL3WT CUL3Δ403 - 459 was added to reaction mix ; over time , appearance of higher molecular bands represents modification of CUL3 by covalent attachment of ubiquitin . right two lanes are control lanes incubated for 10 min with E2 ˜ Ubi but then treated with reducing agent prior to SDS - PAGE .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

UBE2D3˜ubiquitin-release assays analysed by non-reducing SDS-PAGE and immunodetection. His-tagged UBE2D3 was charged with ubiquitin by incubation with ATP and UAE, and charging was stopped by ATP depletion with apyrase. No CUL3 (negative control, lane 1), CUL3WT or CUL3Δ403-459 was then added to the charged E2 and the activity of the RING domain, RBX1, monitored by the hydrolysis of ubiquitin from UBE2D3, visualised by immunodetection with anti-His antibody (upper panel, "˜" denotes thioester bond). The lower panel is an immunoblot for CUL3, showing equivalent amount of both CUL3WT and CUL3Δ403-459 was added to the reaction mix; over time, the appearance of higher molecular weight bands represents the modification of CUL3 by covalent attachment of ubiquitin. The right two lanes are control lanes incubated for 10 min with E2˜Ubi but then treated with reducing agent prior to SDS-PAGE.


Structural model of CUL3WT on structure of CUL1 ( PDB : 1LDK ) , highlighting residues deleted in PHA2E mutant which are located close to N - terminal domain ( NTD ) C - terminal domain ( CTD ) border , using Phyre2 Chimera ( see Materials Methods ) . NTD is coloured purple , CTD cyan , residues 403 - 459 orange .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Structural model of CUL3WT based on the structure of CUL1 (PDB:1LDK), highlighting the residues deleted in the PHA2E mutant which are located close to the N-terminal domain (NTD) and C-terminal domain (CTD) border, using Phyre2 and Chimera (see Materials and Methods). The NTD is coloured purple, the CTD cyan, and residues 403-459 orange.


Time course of in vitro neddylation reactions . Purified NAE , UBE2M Nedd8 were mixed with either CUL3WT or CUL3Δ403 - 459 incubated at 30 ° C for time indicated . Due to covalent attachment of Nedd8 ( 8 . 6 kDa ) , modified have decreased mobility . band shift observed for CUL3WT ( lane 1 versus lane 2 ) is representative of a single Nedd8 modification , while multiple bands in CUL3Δ403 - 459 reaction reflect attachment of up to three Nedd8 molecules ( lane 5 versus lane 8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Time course of in vitro neddylation reactions. Purified NAE, UBE2M and Nedd8 were mixed with either CUL3WT or CUL3Δ403-459 and incubated at 30°C for the time indicated. Due to the covalent attachment of Nedd8 (8.6 kDa), modified proteins have decreased mobility. The band shift observed for CUL3WT (lane 1 versus lane 2) is representative of a single Nedd8 modification, while the multiple bands in the CUL3Δ403-459 reaction reflect the attachment of up to three Nedd8 molecules (lane 5 versus lane 8).


HEK - 293 over - expressing either FLAG - CUL3WT or FLAG - CUL3Δ403 - 459 were immunoprecipitated with M2 Flag - binding sepharose , in presence of a deneddylase inhibitor ( OPT ) . Extract , were lysed in presence of OPT clarified by centrifugation . IP : M2 , clarified supernatant samples were incubated for 1 h at 4 ° C with M2 ( anti - Flag ) sepharose , then washed with PBS eluted with SDS - Laemmli buffer , prior to SDS - PAGE immunodetection with anti - FLAG antibody . Bands are labelled as follows : 1 . CUL3WT , 2 . CUL3WT - N8 , 3 . CUL3Δ403 - 459 , 4 . CUL3Δ403 - 459 - N8 , 5 . CUL3Δ403 - 459 - 2 ( N8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HEK-293 cells over-expressing either FLAG-CUL3WT or FLAG-CUL3Δ403-459 were immunoprecipitated with M2 Flag-binding sepharose, in the presence of a deneddylase inhibitor (OPT). Extract, cells were lysed in the presence of OPT and clarified by centrifugation. IP:M2, clarified supernatant samples were incubated for 1 h at 4°C with M2 (anti-Flag) sepharose, then washed with PBS and eluted with SDS-Laemmli buffer, prior to SDS-PAGE and immunodetection with anti-FLAG antibody. Bands are labelled as follows: 1. CUL3WT, 2. CUL3WT-N8, 3. CUL3Δ403-459, 4. CUL3Δ403-459-N8, 5. CUL3Δ403-459-2(N8).


Neddylation in HEK - 293 over - expressing FLAG - CUL3WT or FLAG - CUL3Δ403 - 459 was blocked by treatment with a Nedd8 - E1 - enzyme inhibitor , 3 μM MLN4924 ( Millennium Pharmaceuticals ) , in over - expressing FLAG - CUL3WT FLAG - CUL3Δ403 - 459 respectively . extracts were sampled over time in presence of OPT to prevent de - neddylation subjected to SDS - PAGE immunodetection of FLAG tag .

O O O O O O O O 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

Neddylation in HEK-293 cells over-expressing FLAG-CUL3WT or FLAG-CUL3Δ403-459 was blocked by treatment with a Nedd8-E1-enzyme inhibitor, 3 μM MLN4924 (Millennium Pharmaceuticals), in cells over-expressing FLAG-CUL3WT and FLAG-CUL3Δ403-459 respectively. Cell extracts were sampled over time in the presence of OPT to prevent de-neddylation and subjected to SDS-PAGE and immunodetection of the FLAG tag.


FLAG - CUL3WT or FLAG - CUL3Δ403 - 459 was immunoprecipitated with M2 - sepharose as described in ( F ) . + MLN4924 indicates culture media supplementation with 3 μM MLN4924 for 5 h prior to lysis to achieve complete deneddylation of CUL3 . Following SDS - PAGE , immunoblotting with COP9 Signalosome ( CSN ) or CAND1 antibodies determined interaction between these Cullin regulators FLAG - CUL3WT FLAG - CUL3Δ403 - 459 , respectively .

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 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

FLAG-CUL3WT or FLAG-CUL3Δ403-459 was immunoprecipitated with M2-sepharose as described in (F). +MLN4924 indicates cell culture media supplementation with 3 μM MLN4924 for 5 h prior to cell lysis to achieve complete deneddylation of CUL3. Following SDS-PAGE, immunoblotting with COP9 Signalosome (CSN) or CAND1 antibodies determined the interaction between these Cullin regulators and FLAG-CUL3WT and FLAG-CUL3Δ403-459, respectively.


GST - CAND1 pull - down assays with CUL3 . Purified GST or GST - CAND1 immobilised on glutathione - sepharose was mixed with either purified CUL3WT or CUL3Δ403 - 459 incubated at 4 ° C for 1 h . sepharose was then washed thrice eluted from resin by incubation with SDS - Laemmli buffer prior to SDS - PAGE Coomassie . CUL3WT but not CUL3Δ403 - 459 interacts with CAND1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

GST-CAND1 pull-down assays with CUL3 proteins. Purified GST or GST-CAND1 immobilised on glutathione-sepharose was mixed with either purified CUL3WT or CUL3Δ403-459 and incubated at 4°C for 1 h. The sepharose was then washed thrice and eluted from the resin by incubation with SDS-Laemmli buffer prior to SDS-PAGE and Coomassie staining. CUL3WT but not CUL3Δ403-459 interacts with CAND1.


A - D In vitro ubiquitylation assays with purified . As ubiquitin is covalently attached to substrate lysine residue , appearance of higher molecular bands reflects modification of with ubiquitin . assays contain purified UBE1 , UBE2D3 , ubiquitin , 0 . 1 mM ATP , 1 mM MgCl2 were buffered in 50 mM HEPES , 150 mM NaCl incubated at 30 ° C for time indicated . Reactions were stopped by addition of SDS - Laemmli buffer to a of 1 × . SDS - PAGE , with Coomassie blue , or detection with indicated antibody following immunoblotting enabled visualisation of ubiquitylation . ( A ) to determine relative modification of KLHL3 by CUL3WT CUL3Δ403 - 459 , KLHL3 was included into reaction at 2 × molar over CUL3WT CUL3Δ403 - 459 . ( B , C ) Reactions serve to determine basal auto - ubiquitylation of CUL3WT or CUL3Δ403 - 459 do not contain KLHL3 or other potential substrate . Lysine residues on CUL3WT or CUL3Δ403 - 459 act as substrate . ( B ) High molecular bands reflect ubiquitin chain linkages on CUL3 or multiple mono - ubiquitylation of a number of CUL3 lysine residues . ( C ) Activity assays contain methylated ubiquitin , a form of ubiquitin incapable of forming ubiquitin chains ; as such , higher band shifts reflect attachment of mono - ubiquitin to one more lysine residue on CUL3WT or CUL3Δ403 - 459 , respectively . ( D ) Activity performed as in ( C ) with methyl - ubiquitin , boxes shown on gel are indicative of gel pieces excised for mass spectrometry in ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation. (A) to determine the relative modification of KLHL3 by CUL3WT and CUL3Δ403-459, KLHL3 was included into the reaction at 2× molar concentration over CUL3WT and CUL3Δ403-459. (B, C) Reactions serve to determine basal auto-ubiquitylation of CUL3WT or CUL3Δ403-459 and do not contain KLHL3 or other potential substrate proteins. Lysine residues on CUL3WT or CUL3Δ403-459 act as the substrate. (B) High molecular weight bands reflect ubiquitin chain linkages on CUL3 or the multiple mono-ubiquitylation of a number of CUL3 lysine residues. (C) Activity assays contain methylated ubiquitin, a form of ubiquitin incapable of forming ubiquitin chains; as such, higher band shifts reflect the attachment of mono-ubiquitin to one more lysine residue on CUL3WT or CUL3Δ403-459, respectively. (D) Activity assay performed as in (C) with methyl-ubiquitin, the boxes shown on the gel are indicative of the gel pieces excised for mass spectrometry analysis in (F).


F Structural docking model of CUL3Δ403 - 459 on structure of full - CUL1 ( 1LDK ) using Chimera ( see Materials Methods ) . NTD is coloured green , while CTD is red , four panels sample possible positioning of CTD relative to NTD in CUL3Δ403 - 459 , assuming full flexibility of linker after deletion of three helices encoded by exon 9 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Structural docking model of CUL3Δ403-459 based on the structure of full-length CUL1 (1LDK) using Chimera (see Materials and Methods). The NTD is coloured green, while the CTD is red, and the four panels sample possible positioning of the CTD relative to the NTD in CUL3Δ403-459, assuming full flexibility of the linker after deletion of three helices encoded by exon 9.


A - C In vitro ubiquitylation assays were performed as described in Figure 2 , but with addition of immunoprecipitatedWNK1 in ( A ) . In vitro ubiquitylation assays with purified . As ubiquitin is covalently attached to substrate lysine residue , appearance of higher molecular bands reflects modification of with ubiquitin . assays contain purified UBE1 , UBE2D3 , ubiquitin , 0 . 1 mM ATP , 1 mM MgCl2 were buffered in 50 mM HEPES , 150 mM NaCl incubated at 30 ° C for time indicated . Reactions were stopped by addition of SDS - Laemmli buffer to a of 1 × . SDS - PAGE , with Coomassie blue , or detection with indicated antibody following immunoblotting enabled visualisation of ubiquitylation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C In vitro ubiquitylation assays were performed as described in Figure 2, but with the addition of immunoprecipitatedWNK1 in (A). In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation.


A - C In vitro ubiquitylation assays were performed as described in Figure 2 , but with addition of immunoprecipitated over - expressed FLAG - WNK4 in ( B , C ) ( see Materials Methods ) . WNK kinases are modified by CUL3WT - KLHL3 , with higher molecular smear observed in anti - WNK1 anti - FLAG panels representative of multiple ubiquitin molecules being covalently attached to WNK . CUL3Δ403 - 459 is unable to modify WNKs . Samples from same reactions were divided to allow immunodetection of different components modified within same reaction . ( C ) CUL3WT , CUL3Δ403 - 459 an equimolar solution CUL3WT : CUL3Δ403 - 459 ( 1 : 1 Mix ) were incubated with KLHL3 immunoprecipitatedFLAG - WNK4 in ubiquitylation reactions to determine influence of CUL3Δ403 - 459 on ubiquitylation activity of CUL3WT . Notably , presence of CUL3Δ403 - 459 does not inhibit WNK ubiquitylation by CUL3WT . In vitro ubiquitylation assays with purified . As ubiquitin is covalently attached to substrate lysine residue , appearance of higher molecular bands reflects modification of with ubiquitin . assays contain purified UBE1 , UBE2D3 , ubiquitin , 0 . 1 mM ATP , 1 mM MgCl2 were buffered in 50 mM HEPES , 150 mM NaCl incubated at 30 ° C for time indicated . Reactions were stopped by addition of SDS - Laemmli buffer to a of 1 × . SDS - PAGE , with Coomassie blue , or detection with indicated antibody following immunoblotting enabled visualisation of ubiquitylation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C In vitro ubiquitylation assays were performed as described in Figure 2, but with the addition of immunoprecipitated over-expressed FLAG-WNK4 in (B, C) (see Materials and Methods). The WNK kinases are modified by CUL3WT-KLHL3, with the higher molecular weight smear observed in anti-WNK1 and anti-FLAG panels representative of multiple ubiquitin molecules being covalently attached to the WNK protein. CUL3Δ403-459 is unable to modify WNKs. Samples from the same assay reactions were divided to allow immunodetection of the different protein components modified within the same assay reaction. (C) CUL3WT, CUL3Δ403-459 and an equimolar solution CUL3WT:CUL3Δ403-459 (1:1 Mix) were incubated with KLHL3 and immunoprecipitatedFLAG-WNK4 in ubiquitylation reactions to determine the influence of CUL3Δ403-459 on the ubiquitylation activity of CUL3WT. Notably, the presence of CUL3Δ403-459 does not inhibit WNK ubiquitylation by CUL3WT.In vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS-Laemmli buffer to a concentration of 1×. SDS-PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation.


Western blot of whole kidney lysates from mice culled after a minimum 4 - h fast . Following exsanguination after surgery , mouse tissues were rapidly harvested stored , samples were then homogenised , clarified quantified prior to SDS - PAGE . Immunodetection with antibodies shown highlights elevated signalling through WNK kinase pathway in CUL3WT / Δ403 - 459 versus CUL3WT mice . lowest panel is an anti - CUL3 immunoprecipitation of kidney lysate samples , whereby CUL3 antibody was cross - linked to G sepharose used to affinity purify CUL3WT CUL3Δ403 - 459 from kidney lysates . samples were incubated together overnight to allow deneddylation of CUL3 to occur maximise CUL3 binding to anti - CUL3 resin . Samples were thoroughly washed eluted from resin prior to SDS - PAGE immunodetection for CUL3 . IP highlights that CUL3Δ403 - 459 is indeed present within kidney lysate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot of whole kidney lysates from mice culled after a minimum 4-h fast. Following exsanguination after surgery, mouse tissues were rapidly harvested and stored, samples were then homogenised, clarified and quantified prior to SDS-PAGE. Immunodetection with the antibodies shown highlights elevated signalling through the WNK kinase pathway in CUL3WT/Δ403-459 versus CUL3WT mice. The lowest panel is an anti-CUL3 immunoprecipitation of the kidney lysate samples, whereby the CUL3 antibody was cross-linked to Protein G sepharose and used to affinity purify CUL3WT and CUL3Δ403-459 from kidney lysates. The samples were incubated together overnight to allow deneddylation of CUL3 proteins to occur and maximise CUL3 binding to the anti-CUL3 resin. Samples were thoroughly washed and eluted from the resin prior to SDS-PAGE and immunodetection for CUL3. The IP highlights that CUL3Δ403-459 is indeed present within the kidney lysate.


Fresh frozen kidney tissues from transplant donor cadavers were homogenised , clarified quantified prior to SDS - PAGE . Western blot confirmed expression of KLHL3 CUL3 in normal healthy human kidneys .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fresh frozen kidney tissues from transplant patient and donor cadavers were homogenised, clarified and quantified prior to SDS-PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3 in normal healthy human kidneys.


Plasma aldosterone after a minimum 4 - h fast was calculated by HTRF ( homogeneous time - resolved ) aldosterone . average aldosterone level per mouse was calculated from duplicate samples run in parallel on . Blood was rapidly harvested in heparin - coated plasma extraction tubes following exsanguination after surgery , samples were snap - frozen for storage . A 58 % increase in aldosterone was detected in CUL3WT / Δ403 - 459 versus CUL3WT mice ( * P = 0 . 0245 ) . Two - unpaired Student ' s t - ; data are mean ± .

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

Plasma aldosterone after a minimum 4-h fast was calculated by HTRF (homogeneous time-resolved fluorescence) aldosterone assay. The average aldosterone level per mouse was calculated from duplicate samples run in parallel on the assay. Blood was rapidly harvested in heparin-coated plasma extraction tubes following exsanguination after surgery, and samples were snap-frozen for storage. A 58% increase in aldosterone was detected in CUL3WT/Δ403-459 versus CUL3WT mice (*P = 0.0245). Two-tailed unpaired Student's t-test; data are mean ± SEM.


Arterial blood biochemistries after a minimum 4 - h fast . Under anaesthesia , right carotid artery was cannulated to minimise atmospheric exposure of samples collected for iSTATbloodgas electrolyte measurements . CUL3WT / Δ403 - 459mice present with abnormal electrolyte homoeostasis compared to CUL3WTmice , exhibiting hyperkalaemia ( * * * P = 0 . 0004 ) hyperchloraemia ( * * * P = 9 . 5 × 10 − 5 ) with a compensated metabolic acidosis ( P = 0 . 7766 ) , marked by a decrease in bicarbonate ( ) ( * * * P = 3 . 4 × 10 − 5 ) , base excess ( BE ) ( * * * P = 9 . 1 × 10 − 5 ) partial pressure of carbon dioxide ( pCO2 ) ( * * * P = 0 . 0038 ) . Two - unpaired Student ' s t - ; data are mean ± .

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

Arterial blood biochemistries after a minimum 4-h fast. Under anaesthesia, the right carotid artery was cannulated to minimise atmospheric exposure of samples collected for iSTATbloodgas and electrolyte measurements. CUL3WT/Δ403-459mice present with abnormal electrolyte homoeostasis compared to CUL3WTmice, exhibiting hyperkalaemia (***P = 0.0004) and hyperchloraemia (***P = 9.5 × 10−5) with a compensated metabolic acidosis (P = 0.7766), marked by a decrease in bicarbonate () (***P = 3.4 × 10−5), base excess (BE) (***P = 9.1 × 10−5) and partial pressure of carbon dioxide (pCO2) (***P = 0.0038). Two-tailed unpaired Student's t-test; data are mean ± SEM.


Representative pseudocoloured maximum - z projections of immunofluorescence stained kidney sections showing distribution of WNK / SPAK pathway components between CUL3WT / Δ403 - 459 CUL3WTmice at a minimum 4 - h fasting baseline ( n = 4 per genotype ) . CUL3 KLHL3 are comparable between genotypes , with significantly higher of CUL3 in proximal convoluted tubule ( PCT ) compared to weak in distal convoluted tubule ( DCT ) thick ascending limb of loop of Henle ( TAL ) , while KLHL3 shows higher expression in DCT / TAL cytosol with of PCT confined to apical . Total ( t ) phospho ( p ) NCC Thr44 SPAK Thr243 show increased apical expression in parvalbumin ( PVALB ) - positive early late ( PVALB - negative ) DCT of CUL3WT / Δ403 - 459mice . Unexpectedly , increased of WNK4 SPAK resulted in formation of discrete specifically in DCT of CUL3WT / Δ403 - 459mice . It is possible that autophagy - lysosomal system may attempt to compensate degrade these excess although WNK4 do not colocalise with lysosomal marker LAMP1 . Scale bar , 20 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative pseudocoloured maximum-intensity z projections of immunofluorescence stained kidney sections showing the distribution of the WNK/SPAK pathway components between CUL3WT/Δ403-459 and CUL3WTmice at a minimum 4-h fasting baseline (n = 4 per genotype). CUL3 and KLHL3 are comparable between genotypes, with significantly higher levels of CUL3 in the proximal convoluted tubule (PCT) compared to the weak staining in distal convoluted tubule (DCT) and thick ascending limb of the loop of Henle (TAL), while KLHL3 shows higher expression in the DCT/TAL cytosol with staining of the PCT confined to the apical membrane. Total (t) and phospho (p) NCC Thr44 and SPAK Thr243 show increased apical membrane expression in the parvalbumin (PVALB)-positive early and late (PVALB-negative) DCT of CUL3WT/Δ403-459mice. Unexpectedly, the increased levels of WNK4 and SPAK resulted in the formation of discrete puncta specifically in the DCT of CUL3WT/Δ403-459mice. It is possible that the autophagy-lysosomal system may attempt to compensate and degrade these excess proteins although the WNK4 puncta do not colocalise with the lysosomal marker LAMP1. Scale bar, 20 μm.


Representative immunohistochemistry of KLHL3 CUL3 in humankidney sections ( n = 6 ) . KLHL3 shows preferential DCT / TAL similar to mouse despite no evidence of PCT apical as seen in ( A ) . CUL3 exhibits preferential basolateral of PCT with similarly low of diffuse in DCT / TAL to that of mouse . Scale bar , 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative immunohistochemistry staining of KLHL3 and CUL3 in humankidney sections (n = 6). KLHL3 shows preferential DCT/TAL cytosolic staining similar to the mouse despite no evidence of PCT apical staining as seen in (A). CUL3 exhibits preferential basolateral cytosolic staining of the PCT with similarly low levels of diffuse staining in the DCT/TAL to that of the mouse. Scale bar, 100 μm.


Continuous blood pressure measurements were taken in mice under general anaesthesia with thermostatically controlled , right carotid artery pressure transducer catheterisation ECG Lead II heart rate monitoring . CUL3WT / Δ403 - 459 mice have elevated systolic ( SBP ) ( * * * P = 1 × 10 − 12 ) diastolic ( DBP ) blood pressure ( * * * P = 8 . 5 × 10 − 8 ) , although they also present with 7 % lower heart rates ( * P = 0 . 0485 ) when compared to CUL3WT mice . Two - unpaired Student ' s t - ; data are mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Continuous blood pressure measurements were taken in mice under general anaesthesia with thermostatically controlled body temperature, right carotid artery pressure transducer catheterisation and ECG Lead II heart rate monitoring. CUL3WT/Δ403-459 mice have elevated systolic (SBP) (***P = 1 × 10−12) and diastolic (DBP) blood pressure (***P = 8.5 × 10−8), although they also present with 7% lower heart rates (*P = 0.0485) when compared to CUL3WT mice. Two-tailed unpaired Student's t-test; data are mean ± SEM.


waveform of blood pressure traces obtained in ( A ) reveals that CUL3WT / Δ403 - 459 mice have an increased pressure ( PP ) [ SBP - DBP ] ( * * * P = 2 . 1 × 10 − 10 ) , augmentation pressure ( AP ) [ SBP - anacrotic notch ( AN ) pressure ] ( * * * P = 3 . 5 × 10 − 6 ) , dicrotic notch ( DN ) pressure ( * * * P = 4 . 7 × 10 − 13 ) mean arterial pressure ( MAP ) [ 1 / 3 × SBP + 2 / 3 × DBP ] ( * * * P = 1 . 3 × 10 − 9 ) . This hypertensive phenotype is in part due to changes in vascular contractility in CUL3WT / Δ403 - 459 mice as evidenced by their higher augmentation index ( AIx ) [ AP / PP ] ( * * P = 0 . 0096 ) , a marker of arterial stiffness , is further supported by an increase in their diastolic pressure decay time constant ( τbourgeois ) [ 1 / slope of diastolic pressure decay ; measured 30 ms after DN 20 ms before end DBP ] ( * * P = 0 . 0083 ) , a surrogate marker of increased vascular resistance . Two - unpaired Student ' s t - ; data are mean ±

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Pulse waveform analysis of blood pressure traces obtained in (A) reveals that CUL3WT/Δ403-459 mice have an increased pulse pressure (PP) [SBP - DBP] (***P = 2.1 × 10−10), augmentation pressure (AP) [SBP - anacrotic notch (AN) pressure] (***P = 3.5 × 10−6), dicrotic notch (DN) pressure (***P = 4.7 × 10−13) and mean arterial pressure (MAP) [1/3 × SBP + 2/3 × DBP] (***P = 1.3 × 10−9). This hypertensive phenotype is in part due to changes in vascular contractility in CUL3WT/Δ403-459 mice as evidenced by their higher augmentation index (AIx) [AP/PP] (**P = 0.0096), a marker of arterial stiffness, and is further supported by an increase in their diastolic pressure decay time constant (τbourgeois) [1/slope of diastolic pressure decay; measured 30 ms after DN and 20 ms before end DBP] (**P = 0.0083), a surrogate marker of increased vascular resistance. Two-tailed unpaired Student's t-test; data are mean ± SEM


In vivo dose - responses to phenylephrine angiotensin II . After baseline measurements were obtained from mice in ( A ) , right external jugular vein was cannulated for administration of bolus doses of vasopressors in increasing half - log steps . Dose - response curves were generated data analysed using a logistical function to determine estimated dose producing half - maximal response ( ED50 ) maximum response ( Emax ) . fitted Emax for phenylephrine was increased in CUL3WT / Δ403 - 459 versus CUL3WT ( 183 . 9 ± 2 . 5 versus 164 . 9 ± 1 . 4 mmHg ) ( * * * P = 1 × 10 − 6 ) indicating an increased vasoconstrictor response to adrenergic stimulation elevating systolic blood pressure substantially above CUL3WT maximum . Similarly , fitted Emax for angiotensin II stimulation was higher in CUL3WT / Δ403 - 459 versus CUL3WT ( 155 . 5 ± 1 . 8 versus 138 . 3 ± 2 . 2 mmHg ) ( * * * P = 1 . 7 × 10 − 6 ) . However , there was no change between CUL3WT / Δ403 - 459 versus CUL3WT sensitivity ( as measured by ED50 ) to phenylephrine ( 36 . 6 ± 5 . 1 versus 31 . 4 ± 2 . 8 μg / kg bw ) ( P = 0 . 3778 ) or angiotensin II ( 0 . 77 ± 0 . 16 versus 0 . 93 ± 0 . 13 μg / kg bw ) ( P = 0 . 4401 ) . Two - unpaired Student ' s t - ; data are mean ± .

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

In vivo dose-responses to phenylephrine and angiotensin II. After baseline measurements were obtained from mice in (A), the right external jugular vein was cannulated for administration of bolus doses of vasopressors in increasing half-log steps. Dose-response curves were generated and data analysed using a logistical function to determine the estimated dose producing half-maximal response (ED50) and maximum response (Emax). The fitted Emax for phenylephrine was increased in CUL3WT/Δ403-459 versus CUL3WT (183.9 ± 2.5 versus 164.9 ± 1.4 mmHg) (***P = 1 × 10−6) indicating an increased vasoconstrictor response to adrenergic stimulation elevating systolic blood pressure substantially above the CUL3WT maximum. Similarly, the fitted Emax for angiotensin II stimulation was higher in CUL3WT/Δ403-459 versus CUL3WT (155.5 ± 1.8 versus 138.3 ± 2.2 mmHg) (***P = 1.7 × 10−6). However, there was no change between CUL3WT/Δ403-459 versus CUL3WT sensitivity (as measured by ED50) to phenylephrine (36.6 ± 5.1 versus 31.4 ± 2.8 μg/kg bw) (P = 0.3778) or angiotensin II (0.77 ± 0.16 versus 0.93 ± 0.13 μg/kg bw) (P = 0.4401). Two-tailed unpaired Student's t-test; data are mean ± SEM.


Western blot of tunica media - intimathoracic aorta lysates from mice culled after a minimum 4 - h fast . Following exsanguination after surgery , mouse tissues were rapidly harvested tunica adventitia removed before storage , later , samples were homogenised , clarified quantified prior to SDS - PAGE . Western blot confirmed expression of KLHL3 CUL3 . Similar to kidney , aorta of CUL3WT / Δ403 - 459 showed slightly lower of CUL3 compared to CUL3WT without any change in KLHL3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot of tunica media-intimathoracic aorta lysates from mice culled after a minimum 4-h fast. Following exsanguination after surgery, mouse tissues were rapidly harvested and the tunica adventitia removed before storage, and later, the samples were homogenised, clarified and quantified prior to SDS-PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3. Similar to the kidney, the aorta of CUL3WT/Δ403-459 showed slightly lower levels of CUL3 compared to CUL3WT without any change in KLHL3 levels.


Fresh frozen thoracic aorta tissues from donor cadavers were homogenised , clarified quantified prior to SDS - PAGE . Western blot confirmed expression of KLHL3 CUL3 in normal healthy human aorta .

Fresh frozen thoracic aorta tissues from donor cadavers were homogenised, clarified and quantified prior to SDS-PAGE. Western blot analysis confirmed the expression of KLHL3 and CUL3 in normal healthy human aorta.


Representative maximum - z projections of immunofluorescence stained thoracic aorta sections showing distribution of CUL3 KLHL3 between CUL3WT / Δ403 - 459 CUL3WT mice at a minimum 4 - h fasting baseline ( n = 4 per genotype ) . CUL3 KLHL3 localisation is comparable between genotypes . highest were detected in vascular smooth muscle endothelium , with a minimal expression in perivascular adipose tissue of adventitia . Scale bar , 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative maximum-intensity z projections of immunofluorescence stained thoracic aorta sections showing the distribution of CUL3 and KLHL3 between CUL3WT/Δ403-459 and CUL3WT mice at a minimum 4-h fasting baseline (n = 4 per genotype). CUL3 and KLHL3 localisation is comparable between genotypes. The highest levels were detected in the vascular smooth muscle cells and endothelium, with a minimal expression in the perivascular adipose tissue of the adventitia. Scale bar, 50 μm.


Representative immunohistochemistry of KLHL3 CUL3 in humanthoracic aorta sections ( n = 6 ) . Similar to mouse seen in ( C ) , vascular smooth muscle in tunica media of aortic wall are positive for KLHL3 CUL3 . Scale bar , 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative immunohistochemistry staining of KLHL3 and CUL3 in humanthoracic aorta sections (n = 6). Similar to the mouse staining seen in (C), vascular smooth muscle cells in the tunica media of the aortic wall are positive for KLHL3 and CUL3. Scale bar, 50 μm.


Morphometric of thoracic aortae reveals vascular remodelling in CUL3WT / Δ403 - 459 mice . There is an increase of ˜ 21 % in vessel wall intima - media thickness ( demarcated by arrows ) of CUL3WT / Δ403 - 459 compared to CUL3WT mice ( * * * P = 0 . 0003 ) . However , there is no change in number of elastin laminae ( P = 0 . 1458 ) therefore no increase in number of medial muscle layers between genotypes . Two - unpaired Student ' s t - ; data are mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Morphometric analysis of thoracic aortae reveals vascular remodelling in CUL3WT/Δ403-459 mice. There is an increase of ˜21% in the vessel wall intima-media thickness (demarcated by arrows) of CUL3WT/Δ403-459 compared to CUL3WT mice (***P = 0.0003). However, there is no change in the number of elastin laminae (P = 0.1458) and therefore no increase in the number of medial muscle layers between genotypes. Two-tailed unpaired Student's t-test; data are mean ± SEM.


A . Retinal whole mount immunostaining for Isolectin - B4 ( red ) Desmin ( green ) in WT ( top panel ) Glut1iECKO ( bottom panel ) mice showing pericyte coverage in vasculature . Left right panels show representative with 20x or 63x magnification , respectively . B . of pericytes coverage in retinal vasculature of WT ( blue ) Glut1iECKO ( orange ) mice . ( n = 9 from 3 independent experiments . Error bars indicate standard error of mean ( ) from unpaired Student ' s t . )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Retinal whole mount immunostaining for Isolectin-B4 (red) and Desmin (green) in WT (top panel) and Glut1iECKO (bottom panel) mice showing pericyte coverage in the vasculature. Left and right panels show representative images with 20x or 63x magnification, respectively. B. Quantification of the pericytes coverage in the retinal vasculature of 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.)


C . Immunostaining for CD31 ( green ) , CD13 ( ; pericyte marker ) αSMA ( cyan ) using paraffin section from WT ( top panel ) Glut1iECKO ( bottom panel ) mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Immunostaining for CD31 (green), CD13 (white; pericyte marker) and αSMA (cyan) using paraffin section from WT (top panel) and Glut1iECKO (bottom panel) mice.


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O 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 . of mTFP / Venus of laconic ( lactate sensor ) showing - dependent lactate uptake in HBVPs . ( n = 8 from 3 independent experiments . Error bars indicate standard error of mean ( ) ) B . Representative showing mTFP ( blue ) Venus ( yellow ) in control 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 ( high magnification ; 60x ) showing mTFP ( cyan ) , Venus ( yellow ) VECAD ( purple ) from BMECs laconic - infected HBVPs on a 2 well chamber . upper panel shows HBVPs with endothelial contact lower panel shows HBVPs without endothelial contact . G . of mTFP / Venus of laconic in HBVPs with endothelial contact ( EC - PC contact ) or HBVPs alone ( PC only ) . ( n = 16 from independent experiments . Error bars indicate standard error of mean ( ) from unpaired Student ' s t . )

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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 showing mTFP ( cyan ) Venus ( yellow ) in HBVPs which have direct contact with control ( upper panel ) or GLUT1 ( lower panel ) siRNA treated BMECs . VECAD immunostaining ( purple ) was used for endothelial . I . of mTFP / Venus 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 standard error of mean ( ) from unpaired Student ' s t . )

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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 for siRNA efficiency of MCT1 .

E. Western blot analysis for siRNA efficiency of MCT1.


G . Western blot for siRNA efficiency of MCT12 .

G. Western blot analysis for siRNA efficiency of MCT12.


I . Representative showing mTFP ( cyan ) Venus ( yellow ) in HBVPs which have direct contact with control ( upper panel ) or MCT1 ( lower panel ) siRNA - treated BMECs . VECAD immunostaining was used for endothelial . J . of mTFP / Venus 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 standard error of mean ( ) from unpaired Student ' s t . )

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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 showing mTFP ( cyan ) Venus ( yellow ) in HBVPs which have direct contact with control ( upper panel ) or MCT5 ( lower panel ) siRNA - treated BMECs . VECAD immunostaining was used for endothelial . L . of mTFP / Venus 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 standard error of mean ( ) from unpaired Student ' s t . )

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 showing expression level of MCT12 β - actin in HBVPs BMECs . E . Western blot 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 showing mTFP ( cyan ) Venus ( yellow ) in control ( upper panel ) or MCT12 ( lower panel ) siRNA treated HBVPs in a 2 well chamber system with BMECs . VECAD ( purple ) immunostaining was used for endothelial . H . of mTFP / Venus of Laconic in control ( blue ) or MCT12 ( orange ) siRNA treated HBVPs . ( n = 8 from independent experiments . Error bars indicate standard error of mean ( ) from unpaired Student ' s t .

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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 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 . )

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

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 .

(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 .

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 ) .

(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 .

(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.