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

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 .

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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 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 B-MEASURED_VAR 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 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 .

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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 .

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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 .

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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 .

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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 .

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

O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 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

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 .

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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 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 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

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 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 O O O O O O O O O O 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 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 B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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 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 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 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 B-MEASURED_VAR O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O B-MEASURED_VAR 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 .

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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 B-MEASURED_VAR O B-MEASURED_VAR O B-CONTROLLED_VAR 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 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

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

A. 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 .

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

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


D . 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 B-MEASURED_VAR 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 B-MEASURED_VAR 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

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


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

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

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


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

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 . )

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


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

O O O O 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 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 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

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 . )

O O O O 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 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 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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


A Anti - TK Western blot of ubiquitinated titin samples .

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A Anti-TK Western blot of ubiquitinated titin samples.


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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