E ) Images of an invading gland of a colorectal NOS adenocarcinoma , at the light microscopic level ( i , semithin section , 100x objective ) , and as seen by electron microscopy ( ii , 1000x magnification ; and iii , 4000x magnification ) . Note that the bulk of the invading neoplastic gland is in a state of glandular organization ( regions denoted by arrowheads in the left panel ) : epithelia ( E , middle panel ) are polarized to form a lumen ( L ) , and are attached to a basal membrane ( asterisk ) by hemidesmosomes ; the gland is surrounded by fibroblasts ( F ) . However , this organization is focally disrupted at the tubular invasion pole ( arrows in the left panel ) where neoplastic epithelial cells have lost polarization and directly contact the surrounding stroma as the basal membrane is missing ( right panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Images of an invading gland of a colorectal NOS adenocarcinoma, at the light microscopic level (i, semithin section, 100x objective), and as seen by electron microscopy (ii, 1000x magnification; and iii, 4000x magnification). Note that the bulk of the invading neoplastic gland is in a state of glandular organization (regions denoted by arrowheads in the left panel): epithelia (E, middle panel) are polarized to form a lumen (L), and are attached to a basal membrane (asterisk) by hemidesmosomes; the gland is surrounded by fibroblasts (F). However, this organization is focally disrupted at the tubular invasion pole (arrows in the left panel) where neoplastic epithelial cells have lost polarization and directly contact the surrounding stroma as the basal membrane is missing (right panel).


A ) Representative images of Caco - 2 cysts transfected with control siRNA ( siGlo ) or siRNAs targeting ROCK1 and ROCK2 , fixed and stained for prominin - 1 , F - actin ( Phalloidin ) and Nuclei ( Hoechst ) . Confocal Z - sections of cysts are displayed . Arrowheads show non - protruding cells . Arrows point to protruding cells . White stars mark nuclei that are off - centered relative to the cyst ' s monolayer . Scale bars : 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

A) Representative images of Caco-2 cysts transfected with control siRNA (siGlo) or siRNAs targeting ROCK1 and ROCK2, fixed and stained for prominin-1, F-actin (Phalloidin) and Nuclei (Hoechst). Confocal Z-sections of cysts are displayed. Arrowheads show non-protruding cells. Arrows point to protruding cells. White stars mark nuclei that are off-centered relative to the cyst's monolayer. Scale bars: 20 μm.


B ) Representative confocal images of Caco - 2 cysts treated with ROCK inhibitors Y27632 and H1152 , non - treated ( NT ) or transduced with ROCK1 and ROCK2 shRNAs ( shROCK1 + 2 ) . The cysts were fixed 2 days after invasion and stained for E - cadherin , F - actin ( Phalloidin ) and nuclei ( DAPI ) . Boxed regions i and ii are displayed at high magnification . White arrowheads point to non - protruding cells , green arrowheads point to the apical pole , arrows point to protruding cells and white stars show off - centered nuclei . Scale bar : 20 μm .

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 O O O O O O O O O O O O O O O O O O O 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) Representative confocal images of Caco-2 cysts treated with ROCK inhibitors Y27632 and H1152, non-treated (NT) or transduced with ROCK1 and ROCK2 shRNAs (shROCK1+2). The cysts were fixed 2 days after invasion and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i and ii are displayed at high magnification. White arrowheads point to non-protruding cells, green arrowheads point to the apical pole, arrows point to protruding cells and white stars show off-centered nuclei. Scale bar: 20 μm.


C ) Bar graph representing the percentage of protrusive Caco - 2 cysts from three independent experiments . Caco - 2 cysts non - treated ( NT ) , incubated with ROCK inhibitors Y27632 and H1152 or infected with lentiviruses encoding shRNA control and targeting ROCK1 and ROCK2 . The cysts were fixed 2 days after invasion , over 100 cysts were counted for each condition . Error bar represents the Standard Error of the Mean ( Means ± SEM ) . P values were calculated using unpaired t - test ( * * * p < 0 . 001 , * * 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 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 O O O O O O O O O O O O O O O O O O O O O O

C) Bar graph representing the percentage of protrusive Caco-2 cysts from three independent experiments. Caco-2 cysts non-treated (NT), incubated with ROCK inhibitors Y27632 and H1152 or infected with lentiviruses encoding shRNA control and targeting ROCK1 and ROCK2. The cysts were fixed 2 days after invasion, over 100 cysts were counted for each condition. Error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (***p<0.001, **p<0.01).


E ) The number of cells that have invaded from the top to the bottom chamber of the Boyden chamber was quantified by automated segmentation in non - treated ( NT ) , in ROCK inhibited conditions ( Y27632 , H1152 ) or in cells transfected with siRNA against ROCK1 and ROCK2 ( siROCK1 + 2 ) . The number of cells that have invaded was normalized to control conditions and reported as the average invasion rate from at least three independent experiments ( Means ± SEM ) . P values were calculated using unpaired t - test ( * * * * p < 0 . 0001 , * * 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 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 O 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) The number of cells that have invaded from the top to the bottom chamber of the Boyden chamber was quantified by automated segmentation in non-treated (NT), in ROCK inhibited conditions (Y27632, H1152) or in cells transfected with siRNA against ROCK1 and ROCK2 (siROCK1+2). The number of cells that have invaded was normalized to control conditions and reported as the average invasion rate from at least three independent experiments (Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001, **p<0.01).


F ) High magnification of boxed region from ( D ) . The top ( i ) and the bottom ( ii ) side of the Boyden chamber membrane were stained for F - actin , E - cadherin , ZO - 1 , Ezrin and Hoechst . Arrow points to the protrusive cells of an invasive cyst and the white stars show the nuclei that engage in the protrusion . Scale bars : 40 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

F) High magnification of boxed region from (D). The top (i) and the bottom (ii) side of the Boyden chamber membrane were stained for F-actin, E-cadherin, ZO-1, Ezrin and Hoechst. Arrow points to the protrusive cells of an invasive cyst and the white stars show the nuclei that engage in the protrusion. Scale bars: 40 μm.


A ) Representative images of organoids formed from patient derived xenografts ( PDXs ) that have been embedded in collagen - I gels during 3 days , in non - treated ( NT ) or ROCK inhibitors treated - conditions ( + Y27632 ) and ( + H1152 ) . At end - point , organoids were stained for E - cadherin , F - actin ( Phalloidin ) and nuclei ( DAPI ) . i and ii are high magnification of boxed regions of + Y27632 and + H1152 organoids respectively . Arrows point to protruding cells . White stars show nuclei that engage in protrusions . Scale bars : 20μm . B ) The bar graph represents the percentage of protrusive organoids resulting from three independent experiments . Over 50 organoids were counted per condition ( Means ± SEM ) . P values were calculated using paired t - test ( * * * p < 0 . 001 ) .

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

A) Representative images of organoids formed from patient derived xenografts (PDXs) that have been embedded in collagen-I gels during 3 days, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152). At end-point, organoids were stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). i and ii are high magnification of boxed regions of +Y27632 and +H1152 organoids respectively. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. B) The bar graph represents the percentage of protrusive organoids resulting from three independent experiments. Over 50 organoids were counted per condition (Means ± SEM). P values were calculated using paired t-test (***p<0.001).


C ) Representative images of human adenocarcinoma explants after 2 days in 3D collagen - I gel , in non - treated ( NT ) or ROCK inhibitors treated - conditions ( + Y27632 ) and ( + H1152 ) and stained for the apical pole ( Ezrin ) , F - actin ( Phalloidin ) and nuclei ( DAPI ) . Boxed regions of NT , + Y27632 , and + H1152 explants 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 . D Explants displaying ( D ) lumens from 10 patients were quantified and results presented as percentage of total explants . Over 50 explants were counted per condition ( duplicate ) and per patient ( Means ± SEM ) . P values were calculated using unpaired t - test ( * * * * p < 0 . 0001 , 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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

C) Representative images of human adenocarcinoma explants after 2 days in 3D collagen-I gel, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152) and stained for the apical pole (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions of NT, +Y27632, and +H1152 explants 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. D Explants displaying (D) lumens from 10 patients were quantified and results presented as percentage of total explants. Over 50 explants were counted per condition (duplicate) and per patient(Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001 , n.s : non significant).


Explants displaying ( E ) protrusive cells from 10 patients were quantified and results presented as percentage of total explants . Over 50 explants were counted per condition ( duplicate ) and per patient ( Means ± SEM ) . P values were calculated using unpaired t - test ( * * * * p < 0 . 0001 , 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 O O O O O O O O O O

Explants displaying (E) protrusive cells from 10 patients were quantified and results presented as percentage of total explants. Over 50 explants were counted per condition (duplicate) and per patient(Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001 , n.s : non significant).


F ) Time - lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen - I gel in the presence of Y27632 monitored by DIC microscopy for 2 days Arrowheads point to non - protruding cells , arrows point to protruding cells and white stars point to lumens . Scale bars : 50 μm .

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

F) Time-lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen-I gel in the presence of Y27632 monitored by DIC microscopy for 2 days Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars point to lumens. Scale bars: 50 μm.


A ) Caco - 2 cysts expressing an empty vector ( pLKO ) or shROCK1 ( # 02 ) or shROCK2 ( # 36 ) after 2 days of invasion were fixed and stained for E - cadherin , F - actin ( Phalloidin ) and nuclei ( DAPI ) . Boxed regions i , ii and iii are shown at high magnifications . Arrowheads point to non - protruding cells . Arrow points to protruding cells . White stars show nuclei that engage in protrusions . Scale bars : 20μm . B ) The number of protrusive Caco - 2 cyst was quantified in each of the mentioned conditions and represented in a bar graph as percentage of total cysts ( Means ± SEM of at least 3 independent experiments , paired t - test , * * * p < 0 . 001 , * p < 0 . 05 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 expressing an empty vector (pLKO) or shROCK1 (#02) or shROCK2 (#36) after 2 days of invasion were fixed and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii and iii are shown at high magnifications. Arrowheads point to non-protruding cells. Arrow points to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. B) The number of protrusive Caco-2 cyst was quantified in each of the mentioned conditions and represented in a bar graph as percentage of total cysts (Means ± SEM of at least 3 independent experiments, paired t-test, ***p<0.001, *p<0.05 n.s: non significant).


C ) Caco - 2 cysts stably expressing GFP alone or GFP - ROCK2DN # 2 , were submitted to a 2 days period invasion assay , then fixed and stained for F - actin ( Phalloidin ) and nuclei ( DAPI ) . Boxed regions i , ii and iii are shown at high magnification . Arrowheads point to non - protruding cells . Arrows point to protruding cells . White and black stars show nuclei that engage in protrusions . Scale bars : 20μm . D ) Bargraph representing the percentage of protrusive cysts transduced with GFP ( CTRL , with or without Y27632 ) or with ROCK2DN # 2 ( Means ± SEM of at least 3 independent experiments , paired t - test , * * 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 O O O O O O O O O O O O O O O O O O O O O O O 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

C) Caco-2 cysts stably expressing GFP alone or GFP-ROCK2DN#2, were submitted to a 2 days period invasion assay, then fixed and stained for F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii and iii are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. White and black stars show nuclei that engage in protrusions. Scale bars: 20μm. D) Bargraph representing the percentage of protrusive cysts transduced with GFP (CTRL, with or without Y27632) or with ROCK2DN#2 (Means ± SEM of at least 3 independent experiments, paired t-test, **p<0.01 n.s: non significant).


E ) T84 cells grown in 3D organotypic culture form polarized cysts with a central lumen that display protruding leader cells at the basal pole ( arrows ) reminiscent of collective invasion . T84 cysts were transduced with lentiviruses encoding inducible GFP or Venus - ROCK2 - DA and treated with doxycycline 1μg / ml . Scale bars : 20μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) T84 cells grown in 3D organotypic culture form polarized cysts with a central lumen that display protruding leader cells at the basal pole (arrows) reminiscent of collective invasion. T84 cysts were transduced with lentiviruses encoding inducible GFP or Venus-ROCK2-DA and treated with doxycycline 1μg/ml. Scale bars: 20μm.


F ) Bar graph representing the percentage of protrusive T84 cysts in control ( WT ) , treated with ROCK inhibitor ( Y27632 ) or transduced with lentiviruses expressing GFP or GFP - ROCK2 - DA after induction by Doxycycline ( Means ± SEM of at least 3 independent experiments ) ( paired t - test , * * * p < 0 . 001 , * 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 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

F) Bar graph representing the percentage of protrusive T84 cysts in control (WT), treated with ROCK inhibitor (Y27632) or transduced with lentiviruses expressing GFP or GFP-ROCK2-DA after induction by Doxycycline (Means ± SEM of at least 3 independent experiments) (paired t-test, ***p<0.001, *p<0.05).


G ) Overall Survival Prognostic value of ROCK2 expression in colon cancer patients of the GSE33582 data set ( n = 477 patients with survival annotation ) using Kaplan - Meier survival analysis . The patient cohort was divided into tumours with or without a down - regulation of ROCK2 relatively to non - tumoural tissues ( expression cut - off value corresponds to the 1st decile of ROCK2 expression in non - tumoural sample , i . e . 7 . 6 ) . Log - rank test p - values for survival distribution difference are indicated at 3 and 5 years .

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

G) Overall Survival Prognostic value of ROCK2 expression in colon cancer patients of the GSE33582 data set (n=477 patients with survival annotation) using Kaplan-Meier survival analysis. The patient cohort was divided into tumours with or without a down-regulation of ROCK2 relatively to non-tumoural tissues (expression cut-off value corresponds to the 1st decile of ROCK2 expression in non-tumoural sample, i.e. 7.6). Log-rank test p-values for survival distribution difference are indicated at 3 and 5 years.


B ) DIC acquisition of fixed mosaic Caco - 2 cysts expressing GFP alone , GFP - ROCK2DN # 1 or GFP - ROCK2DN # 2 . Boxed regions i and ii are shown at high magnification . Arrowheads point to non - protruding cells . Arrows point to protruding cells . Scale bars : 20μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DIC acquisition of fixed mosaic Caco-2 cysts expressing GFP alone, GFP-ROCK2DN#1 or GFP-ROCK2DN#2. Boxed regions i and ii are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. Scale bars: 20μm.


C ) Bar graph representing the percentage of mosaic Caco - 2 cysts transduced with GFP or GFP - ROCK2DN # , with or without Y27632 , harbouring a protruding morphology ( Means ± SEM of at least 3 independent experiments , unpaired t - test , * * * p < 0 . 001 , * 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 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

C) Bar graph representing the percentage of mosaic Caco-2 cysts transduced with GFP or GFP-ROCK2DN#, with or without Y27632, harbouring a protruding morphology (Means ± SEM of at least 3 independent experiments, unpaired t-test, ***p<0.001, *p<0.05).


D ) Quantification of the fate of the GFP positive cells , leader or follower , in mosaic cysts transduced with GFP - alone and treated with Y27632 ( GFP + Y27632 ) or GFP - ROCK2DN # 2 from 3 independent experiments ( Means ± SEM , unpaired t - test , * * * p < 0 . 001 ) .

O O O 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 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

D) Quantification of the fate of the GFP positive cells, leader or follower, in mosaic cysts transduced with GFP-alone and treated with Y27632 (GFP+Y27632) or GFP-ROCK2DN#2 from 3 independent experiments (Means ± SEM, unpaired t-test, ***p<0.001).


E ) Representative pull - down of RAC1 - GTP by PAK - Crib , in Caco - 2 cells treated with ROCK inhibitor Y27632 , H1152 or blebbistatin and lysed at 12 or 16 hours . Immunoblot analyses were performed with anti - Rac1 and anti - GAPDH antibody from three independent experiments .

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

E) Representative pull-down of RAC1-GTP by PAK-Crib, in Caco-2 cells treated with ROCK inhibitor Y27632, H1152 or blebbistatin and lysed at 12 or 16 hours. Immunoblot analyses were performed with anti-Rac1 and anti-GAPDH antibody from three independent experiments.


F ) DIC acquisition of fixed Caco - 2 cysts treated with ROCK inhibitors alone ( Y27632 or H1152 ) , or in combination with RAC1 inhibitor NSC23766 . Arrowheads point to non - protruding cells and arrow point to protruding cell . Scale bar 20 μm .

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

F) DIC acquisition of fixed Caco-2 cysts treated with ROCK inhibitors alone (Y27632 or H1152), or in combination with RAC1 inhibitor NSC23766. Arrowheads point to non-protruding cells and arrow point to protruding cell. Scale bar 20 μm.


G ) Bar graph representing the percentage of protruding Caco - 2 cysts in control conditions ( non - treated , NT ) or treated with NSC23766 , Y27632 or H1152 ( Means ± SEM of at least 3 independent experiments , unpaired t - test , * * * * p < 0 . 0001 , * * * p < 0 . 001 , * p < 0 .

O O 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 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

G) Bar graph representing the percentage of protruding Caco-2 cysts in control conditions (non-treated, NT) or treated with NSC23766, Y27632 or H1152 (Means ± SEM of at least 3 independent experiments, unpaired t-test, ****p<0.0001, ***p<0.001, *p<0.05


A ) siRNA screen performed in Caco - 2 cysts treated with Y27632 : Caco - 2 cells transfected with control siRNA or pairs of siRNA targeting RhoGEFs were seeded in the modified 3D Boyden Chamber and grown as cysts for 3 days . Then , Caco - 2 cysts were treated with Y27632 and the serum gradient was induced . After 2 days , the number of cells present on the bottom chamber were quantified by automated segmentation after Hoechst staining . The bar graph represents the mean invasion rate . Each condition was duplicated . The red dashed line represent the median invasion rate , the red full line indicates the 3 fold decrease in invasion rate . Three hits were identified : # 2 : ARHGEF18 + RNEF , # 7 : SGEF + ARHGEF16 and # 24 : FARP1 + FARP2 ( Mean ± SD ) .

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 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

A) siRNA screen performed in Caco-2 cysts treated with Y27632: Caco-2 cells transfected with control siRNA or pairs of siRNA targeting RhoGEFs were seeded in the modified 3D Boyden Chamber and grown as cysts for 3 days. Then, Caco-2 cysts were treated with Y27632 and the serum gradient was induced. After 2 days, the number of cells present on the bottom chamber were quantified by automated segmentation after Hoechst staining. The bar graph represents the mean invasion rate. Each condition was duplicated. The red dashed line represent the median invasion rate, the red full line indicates the 3 fold decrease in invasion rate. Three hits were identified: #2:ARHGEF18+RNEF, #7: SGEF+ARHGEF16 and #24: FARP1+FARP2 (Mean ±SD) .


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 ) .

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 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

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 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 )

O O O O O O O O O O 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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 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

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 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 ) .

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 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 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

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 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 ) .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O 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

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 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 .

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 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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O 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 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 .

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

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 , 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 .

O O O O O O 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 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 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 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 - 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 ±

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 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 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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and quantified prior to SDS - PAGE . Western blot analysis confirmed the expression of KLHL3 and CUL3 in normal healthy human aorta .

O O O O O O O O O O O O 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 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 - 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 . )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ( white ; pericyte marker ) and αSMA ( cyan ) using paraffin section from WT ( top panel ) and Glut1iECKO ( bottom panel ) mice .

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 . 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 . )

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

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

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


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

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

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


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

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


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

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

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


E . Western blot analysis for siRNA efficiency of MCT1 .

E. Western blot analysis for siRNA efficiency of MCT1.


G . Western blot analysis for siRNA efficiency of MCT12 .

G. Western blot analysis for siRNA efficiency of MCT12.


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

(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.