Figure 3A - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-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.


Figure 4Western 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Western 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.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.


Figure 5Representative 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Representative 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.


Figure 6Continuous 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 ± SEMIn 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Continuous 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 ± SEMIn 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.


Figure 7Western 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Western 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.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.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.


Figure 1A Normal lung cells ( IMR90 and MRC5 ) and lung cancer cells ( A549 and H1975 ) were treated with various concentrations of PM2 . 5 for 24 h , and cell viability was assessed by a trypan blue assay . The values are the mean ± SD of three independent experiments . B H1975 and A549 cells were treated with various concentrations of PM2 . 5 for 24 h , and their cell lysates were analyzed for phosphor - ERK ( pERK ) , phosphor - AKT ( pAKT ) , phosphor - STAT3 ( pSTAT3 ) , phosphor - EGFR ( pEGFR ) , ERK , AKT , STAT3 , and EGFR by Western blotting . β - actin served as the loading control . The results shown are from one of three similar experiments ( left panel ) . The relative expression levels of pEGFR , pAKT , pSTAT3 , and pERK were quantified by normalized with β - actin and are shown at the right panel . The values are the mean ± SD of three independent experiments . * p < 0 . 05 , as analyzed with one - sample t - test and compared with untreated cells .

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Figure 1A Normal lung cells (IMR90 and MRC5) and lung cancer cells (A549 and H1975) were treated with various concentrations of PM2.5 for 24 h, and cell viability was assessed by a trypan blue assay. The values are the mean ± SD of three independent experiments.B H1975 and A549 cells were treated with various concentrations of PM2.5 for 24 h, and their cell lysates were analyzed for phosphor-ERK (pERK), phosphor-AKT (pAKT), phosphor-STAT3 (pSTAT3), phosphor-EGFR (pEGFR), ERK, AKT, STAT3, and EGFR by Western blotting. β-actin served as the loading control. The results shown are from one of three similar experiments (left panel). The relative expression levels of pEGFR, pAKT, pSTAT3, and pERK were quantified by normalized with β-actin and are shown at the right panel. The values are the mean ± SD of three independent experiments. *p < 0.05, as analyzed with one-sample t-test and compared with untreated cells.


Figure 2A , B H1975 and A549 cells were treated with PM2 . 5 at 50 μg / ml for 90 days , and the proliferation of the treated cells was assessed by trypan blue assays . C , D H1975 and A549 cells were exposed to PM2 . 5 at 50 μg / ml for different lengths of time , and the cell lysates of treated cells were assessed for phosphorylated EGFR ( pEGFR ) and EGFR by Western blotting . β - actin served as the loading control . E , F H1975 and A549 cells were treated with PM2 . 5 at 50 μg / ml for 90 days . The anchorage - independent growth was assessed by a soft agar colony formation assay . The number of colonies was scored , and the data are presented as the relative colony formation ability .

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Figure 2A, B H1975 and A549 cells were treated with PM2.5 at 50 μg/ml for 90 days, and the proliferation of the treated cells was assessed by trypan blue assays.C, D H1975 and A549 cells were exposed to PM2.5 at 50 μg/ml for different lengths of time, and the cell lysates of treated cells were assessed for phosphorylated EGFR (pEGFR) and EGFR by Western blotting. β-actin served as the loading control.E, F H1975 and A549 cells were treated with PM2.5 at 50 μg/ml for 90 days. The anchorage-independent growth was assessed by a soft agar colony formation assay. The number of colonies was scored, and the data are presented as the relative colony formation ability.


Figure 3H1975 cells were exposed to 50 μg / ml PM2 . 5 for 90 days . Both unexposed and exposed cells were injected subcutaneously into the flank of each mouse ( n = six per group ) . The tumor volume and excised tumor weight were measured . The results shown in ( A ) are presented as the means ± SDs of six mice . * p < 0 . 05 and * * p < 0 . 01 , compared with untreated group . ( A ) p values were determined by two - way repeated measures ANOVA with pairwise comparison of post - hoc analysis with Benjamini - Hochberg ( BH ) correction . H1975 cells were exposed to 50 μg / ml PM2 . 5 for 90 days . Both unexposed and exposed cells were injected subcutaneously into the flank of each mouse ( n = six per group ) . C . IHC staining of excised tumors for Ki - 67 , IL18 , and TMPRSS2 is shown in ( C ) . Scale bars , 50 μm .

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Figure 3H1975 cells were exposed to 50 μg/ml PM2.5 for 90 days. Both unexposed and exposed cells were injected subcutaneously into the flank of each mouse (n = six per group). The tumor volume and excised tumor weight were measured. The results shown in (A) are presented as the means ± SDs of six mice. *p < 0.05 and **p < 0.01, compared with untreated group. (A) p values were determined by two-way repeated measures ANOVA with pairwise comparison of post-hoc analysis with Benjamini-Hochberg (BH) correction.H1975 cells were exposed to 50 μg/ml PM2.5 for 90 days. Both unexposed and exposed cells were injected subcutaneously into the flank of each mouse (n = six per group). C. IHC staining of excised tumors for Ki-67, IL18, and TMPRSS2 is shown in (C). Scale bars, 50 μm.


Figure 4H1975 cells were treated with 50 μg / ml PM2 . 5 for 90 days , and the expression levels of TMPRSS2 mRNA were determined by real - time RT ‒ PCR ( A ) respectively . ( A ) p values were determined by one - sample t - test . C The proteins from whole cell lysates ( WCL ) and nuclear fractions ( CN ) were analyzed for AhR , Lamin B ( nuclei marker ) , and Vimentin ( cytoplasm marker ) by Western blotting . The data shown represent the mean ± SD of three independent experiments . * p < 0 . 05 and * * p < 0 . 01 , and * * * p < 0 . 001 , compared with untreated cells . # p < 0 . 05 , compared with PM2 . 5 treated cells . E Western blot analysis of TMPRSS2 expression in cells treated with CH223191 at 10 μM for 48 h . The relative expression level of TMPRSS2 was quantified by normalized with β - actin and are shown at the right panel . : The data shown represent the mean ± SD of three independent experiments . * p < 0 . 05 and * * p < 0 . 01 , and * * * p < 0 . 001 , compared with untreated cells . # p < 0 . 05 , compared with PM2 . 5 treated cells .

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Figure 4H1975 cells were treated with 50 μg/ml PM2.5 for 90 days, and the expression levels of TMPRSS2 mRNA were determined by real-time RT‒PCR (A) respectively. (A) p values were determined by one-sample t-test.C The proteins from whole cell lysates (WCL) and nuclear fractions (CN) were analyzed for AhR, Lamin B (nuclei marker), and Vimentin (cytoplasm marker) by Western blotting. The data shown represent the mean ± SD of three independent experiments. *p < 0.05 and **p< 0.01, and ***p < 0.001, compared with untreated cells. #p < 0.05, compared with PM2.5 treated cells.E Western blot analysis of TMPRSS2 expression in cells treated with CH223191 at 10 μM for 48 h. The relative expression level of TMPRSS2 was quantified by normalized with β-actin and are shown at the right panel. : The data shown represent the mean ± SD of three independent experiments. *p < 0.05 and **p< 0.01, and ***p < 0.001, compared with untreated cells. #p < 0.05, compared with PM2.5 treated cells.


Figure 5A Expression of TMPRSS2 was examined by Western blots of four lung cancer cell lines ( H460 , A546 , H1299 , and H1975 ) and two normal fibroblasts ( MRC5 and IMR90 ) . The results shown in ( A are from one of three similar experiments . β - actin was used as the loading control . H1975 cells were infected with sh - TMPRSS2 ( sh - TMPRSS2 - 1 and sh - TMPRSS2 - 2 ) or empty vector ( Vector ) . The stable clones of TMPRSS2 knockdown cells were analyzed and their ability to perform anchorage - independent growth in soft agar ( C ) . The data shown in ( C ) represent the means ± SDs from three independent experiments . ( C ) p values were determined by one - sample t - test .

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Figure 5A Expression of TMPRSS2 was examined by Western blots of four lung cancer cell lines (H460, A546, H1299, and H1975) and two normal fibroblasts (MRC5 and IMR90). The results shown in (A are from one of three similar experiments. β-actin was used as the loading control.H1975 cells were infected with sh-TMPRSS2 (sh-TMPRSS2-1 and sh-TMPRSS2-2) or empty vector (Vector). The stable clones of TMPRSS2 knockdown cells were analyzed and their ability to perform anchorage-independent growth in soft agar (C). The data shown in (C) represent the means ± SDs from three independent experiments. (C) p values were determined by one-sample t-test.


Figure 6A - C H1299 cells were transfected with TMPRSS2 - HA or the empty vector ( Vector ) . After 48 h , the transfected cells were assayed for the expression of TMPRSS2 ( A ) and for the induction of IL18 by qRT ‒ PCR ( B ) and Western blotting ( C ) . The results shown in ( A , C are from one of three similar experiments . β - actin was used as the loading control . The expression level of IL18 was examined in A549 cells exposed to PM2 . 5 at 50 μg / ml for 90 days Western blotting The results shown in H ) are from one of three similar experiments . β - actin was used as the loading control . The data shown in H ) was normalized to β - actin from three independent experiments .

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Figure 6A-C H1299 cells were transfected with TMPRSS2-HA or the empty vector (Vector). After 48 h, the transfected cells were assayed for the expression of TMPRSS2 (A) and for the induction of IL18 by qRT‒PCR (B) and Western blotting (C). The results shown in (A, C are from one of three similar experiments. β-actin was used as the loading control.The expression level of IL18 was examined in A549 cells exposed to PM2.5 at 50 μg/ml for 90 days Western blotting The results shown in H) are from one of three similar experiments. β-actin was used as the loading control. The data shown in H) was normalized to β-actin from three independent experiments.


Figure 7A IHC staining of TMPRSS2 , IL18 , and nuclear AhR in a representative normal lung tissue section . Scale bars , 2 . 5 mm ( low magnification ) and 100 μm ( high magnification ) . B IHC staining of TMPRSS2 , IL18 , and nuclear AhR in lung cancer tissues that displayed high or low expression of TMPRSS2 . Scale bars , 2 . 5 mm ( low magnification ) and 100 μm ( high magnification ) .

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Figure 7A IHC staining of TMPRSS2, IL18, and nuclear AhR in a representative normal lung tissue section. Scale bars, 2.5 mm (low magnification) and 100 μm (high magnification).B IHC staining of TMPRSS2, IL18, and nuclear AhR in lung cancer tissues that displayed high or low expression of TMPRSS2. Scale bars, 2.5 mm (low magnification) and 100 μm (high magnification).


Figure 2B Average seizure score of KA - treated WT and Fxr2 KO mice over time from t = 0 ( injection time ) to 2 h . * p < 0 . 05 compared to WT mice ( two - way Repeated Measures ANOVA ; interaction effect of time and genotype ; F1 , 666 = 10 . 05 , p = 0 . 0016 ) . KA - treated WT mice ( n = 16 ) , KA - treated Fxr2 KO mice ( n = 12 ) . Data are presented as mean ± Standard Error of the Mean ( SEM ) . D Average seizure score of pilocarpine - treated WT and Fxr2 KO mice over time from t = 0 ( injection time ) to 2 h ( two - way Repeated Measures ANOVA ) . Pilocarpine - treated WT mice ( n = 6 ) , pilocarpine - treated Fxr2 KO mice ( n = 6 ) . Data are presented as mean ± SEM .

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Figure 2B Average seizure score of KA-treated WT and Fxr2 KO mice over time from t=0 (injection time) to 2 h. * p < 0.05 compared to WT mice (two-way Repeated Measures ANOVA; interaction effect of time and genotype; F1,666 = 10.05, p = 0.0016). KA-treated WT mice (n=16), KA-treated Fxr2 KO mice (n=12). Data are presented as mean ± Standard Error of the Mean (SEM). D Average seizure score of pilocarpine-treated WT and Fxr2 KO mice over time from t=0 (injection time) to 2 h (two-way Repeated Measures ANOVA). Pilocarpine-treated WT mice (n=6), pilocarpine-treated Fxr2 KO mice (n=6). Data are presented as mean ± SEM.


Figure 3B PSD - 95 mRNA had lower read counts in Fxr2 KO samples ( n = 3 ) compared to WT samples ( n = 3 ) ( + adjusted p < 0 . 1 , two - tailed Student ' s t - test ) . Data are presented as mean ± SEM .

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Figure 3B PSD-95 mRNA had lower read counts in Fxr2 KO samples (n=3) compared to WT samples (n=3) (+ adjusted p < 0.1, two-tailed Student's t-test). Data are presented as mean ± SEM.


Figure 5A Representative Western blot and quantification of GluK5 , PSD - 95 , GluN2B and mGluR5 proteins . Endogenous GluK5 , PSD - 95 , GluN2B and mGluR5 in Fxr2 KO mice ( n = 9 ) compared to WT mice ( n = 11 ) . * p < 0 . 05 , * * p < 0 . 01 , significant difference between WT and Fxr2 KO ( two - tailed Student ' s t - test : GluK5 : t18 = 0 . 87 , p = 0 . 394 ; PSD - 95 : t18 = 2 . 29 , p < 0 . 05 ; GluN2B : t18 = 3 . 09 , p < 0 . 01 ; mGluR5 : t18 = 3 . 74 , p < 0 . 01 ) . Quantified bands are highlighted with an asterisk or squared bracket . Data are presented as mean ± SEM .

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Figure 5A Representative Western blot and quantification of GluK5, PSD-95, GluN2B and mGluR5 proteins. Endogenous GluK5, PSD-95, GluN2B and mGluR5 in Fxr2 KO mice (n=9) compared to WT mice (n=11). * p < 0.05, ** p < 0.01, significant difference between WT and Fxr2 KO (two-tailed Student's t-test: GluK5: t18 = 0.87, p = 0.394; PSD-95: t18 = 2.29, p < 0.05; GluN2B: t18 = 3.09, p < 0.01; mGluR5: t18 = 3.74, p < 0.01). Quantified bands are highlighted with an asterisk or squared bracket. Data are presented as mean ± SEM.


Figure 6A Bar plot showing phospho - ERK1 / 2 over total ERK1 / 2 levels in vehicle - and KA - treated WT ( closed and open black circles , vehicle - treated WT n = 6 ; KA - treated WT n = 6 ) and Fxr2 KO mice ( closed and open magenta circles , vehicle - treated Fxr2 KO n = 5 ; KA - treated Fxr2 KO n = 6 ) . * * * p < 0 . 001 comparison between vehicle - and KA - treated WT ( two - way ANOVA : interaction effect between genotype and treatment : F1 , 19 = 24 . 66 , p < 0 . 001 ) . Data are presented as mean ± SEM . B Phospho - ERK1 / 2 levels in vehicle - and pilocarpine - treated WT ( n = 4 - 6 ) and Fxr2 KO mice ( n = 4 - 6 ) . Both genotypes responded with long - lasting seizures upon pilocarpine administration * p < 0 . 05 ( two - way ANOVA : main effect for treatment : F1 , 16 = 10 . 98 , p < 0 . 01 ) . Data are presented as mean ± SEM .

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Figure 6A Bar plot showing phospho-ERK1/2 over total ERK1/2 levels in vehicle- and KA-treated WT (closed and open black circles, vehicle-treated WT n=6; KA-treated WT n=6) and Fxr2 KO mice (closed and open magenta circles, vehicle-treated Fxr2 KO n=5; KA-treated Fxr2 KO n=6). *** p < 0.001 comparison between vehicle- and KA-treated WT (two-way ANOVA: interaction effect between genotype and treatment: F1,19 = 24.66, p < 0.001). Data are presented as mean ± SEM. B Phospho-ERK1/2 levels in vehicle- and pilocarpine-treated WT (n=4-6) and Fxr2 KO mice (n=4-6). Both genotypes responded with long-lasting seizures upon pilocarpine administration * p < 0.05 (two-way ANOVA: main effect for treatment: F1,16 = 10.98, p < 0.01). Data are presented as mean ± SEM.


Figure 1B Recombinant MAVS - Region III - dimer ( lanes 1 - 4 ) , trimer ( lanes 5 - 8 ) or tetramer ( lanes 9 - 12 ) were subjected to IRF3 dimerization assay respectively in vitro . Increasing doses of recombinant MAVS - Region III were used and shown in Fig EV1C . Immunoblotting analysis was performed following Native - PAGE for the reaction mixture . C pcDNA3 - based constructs expressing MAVS - Region III - dimer , trimer and tetramer were transfected into HEK293T cells respectively . ISG54 induction were measured thirty - six hours after transfection by quantitative PCR ( qPCR ) . D Recombinant MAVS - Region III - dimer or tetramer were immunoprecipitated with anti - FLAG M2 beads with or without a preincubation with S100 from HEK293T cells following an experimental procedure outlined in Fig EV1G . The IP products were resolved in SDS - PAGE and a specific protein band pulled - down with MAVS - Region III - tetramer was indicated by an arrow . TRAF3IP3 was identified in the specific band by mass spectrometry . E Protein samples as described in ( D ) were subjected to immunoblotting analysis . The TRAF3IP3 band was indicated by an arrow and the asterisk indicated nonspecific bandsF pcDNA3 - FLAG - TRAF3IP3 ( full - length and truncated forms ) and pcDNA3 - HA - MAVS were transfected into HEK293T cells , which were harvested thirty - six hours post transfection . Co - IP experiments were performed with anti - HA antibody , followed by immunoblotting . G Human cells , including THP1 , HeLa , HEK293 , HEK293T as well as Traf3ip3 - / - HEK293T , were lysed and subjected to immunoblotting to examine TRAF3IP3 expression .

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Figure 1B Recombinant MAVS-Region III-dimer (lanes 1-4), trimer (lanes 5-8) or tetramer (lanes 9-12) were subjected to IRF3 dimerization assay respectively in vitro. Increasing doses of recombinant MAVS-Region III were used and shown in Fig EV1C. Immunoblotting analysis was performed following Native-PAGE for the reaction mixture.C pcDNA3-based constructs expressing MAVS-Region III-dimer, trimer and tetramer were transfected into HEK293T cells respectively. ISG54 induction were measured thirty-six hours after transfection by quantitative PCR (qPCR).D Recombinant MAVS-Region III-dimer or tetramer were immunoprecipitated with anti-FLAG M2 beads with or without a preincubation with S100 from HEK293T cells following an experimental procedure outlined in Fig EV1G. The IP products were resolved in SDS-PAGE and a specific protein band pulled-down with MAVS-Region III-tetramer was indicated by an arrow. TRAF3IP3 was identified in the specific band by mass spectrometry.E Protein samples as described in (D) were subjected to immunoblotting analysis. The TRAF3IP3 band was indicated by an arrow and the asterisk indicated nonspecific bandsF pcDNA3-FLAG-TRAF3IP3 (full-length and truncated forms) and pcDNA3-HA-MAVS were transfected into HEK293T cells, which were harvested thirty-six hours post transfection. Co-IP experiments were performed with anti-HA antibody, followed by immunoblotting.G Human cells, including THP1, HeLa, HEK293, HEK293T as well as Traf3ip3-/- HEK293T, were lysed and subjected to immunoblotting to examine TRAF3IP3 expression.


Figure 2A Three pairs of small interfering RNA against TRAF3IP3 were transfected into HEK293T cells respectively and immunoblotting were then employed to examine the knockdown efficiency . A pair of scrambled RNA oligoes was transfected and signified as con . B Three pairs of si - TRAF3IP3 together with IFNβ - luciferase reporter were transfected into HEK 293T cells . Twenty - four hours after transfection , the cells were infected with ( SeV ) or without ( mock ) Sendai virus , which were harvested six hours post infection to measure the luciferase activity . C Si - TRAF3IP3 ( oligo # 1 ) were transfected into HEK 293 cells , which were then treated with mock , VSV , SeV , poly ( I : C ) or HSV - 1 . After various treatments , IFNB inductions were measured by qPCR . D Immunoblotting to detect TRAF3IP3 in wild type and Traf3ip3 - / - HEK293T cells . E Wild type and Traf3ip3 - / - HEK293T cells were treated with or without VSV , SeV or poly ( I : C ) as indicated for twelve hours . IFNB induction was measured by qPCR . F Wild type and Traf3ip3 - / - HEK293T cells were infected with GFP - VSV . Fluorescent images were taken to examine GFP - VSV proliferation at eight hours post infection ( left ) . Scale bar represents 10 micrometers . GFP - VSV titers were quantitated by plaque assay ( right ) . G Various expression constructs were transfected into WT and Traf3ip3 - / - HEK293T cells . Thirty - six hours after transfection , IFNB induction was measured by qPCR . H Mavs - / - HEK293T cells were transfected with empty vector , pcDNA - FLAG - MAVS , pcDNA - FLAG - MAVS - ( Region III only ) , or pcDNA - FLAG - MAVS - ( ΔRegion III ) , together with pcDNA3 - FLAG - TRAF3IP3 or not as indicated . Twenty - four hours after transfection , cells were infected with SeV for twelve hours . IFNB induction was measured by qPCR .

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Figure 2A Three pairs of small interfering RNA against TRAF3IP3 were transfected into HEK293T cells respectively and immunoblotting were then employed to examine the knockdown efficiency. A pair of scrambled RNA oligoes was transfected and signified as con.B Three pairs of si-TRAF3IP3 together with IFNβ-luciferase reporter were transfected into HEK 293T cells. Twenty-four hours after transfection, the cells were infected with (SeV) or without (mock) Sendai virus, which were harvested six hours post infection to measure the luciferase activity.C Si-TRAF3IP3 (oligo #1) were transfected into HEK 293 cells, which were then treated with mock, VSV, SeV, poly(I:C) or HSV-1. After various treatments, IFNB inductions were measured by qPCR.D Immunoblotting to detect TRAF3IP3 in wild type and Traf3ip3-/- HEK293T cells.E Wild type and Traf3ip3-/- HEK293T cells were treated with or without VSV, SeV or poly (I:C) as indicated for twelve hours. IFNB induction was measured by qPCR.F Wild type and Traf3ip3-/- HEK293T cells were infected with GFP-VSV. Fluorescent images were taken to examine GFP-VSV proliferation at eight hours post infection (left). Scale bar represents 10 micrometers. GFP-VSV titers were quantitated by plaque assay (right).G Various expression constructs were transfected into WT and Traf3ip3-/- HEK293T cells. Thirty-six hours after transfection, IFNB induction was measured by qPCR.H Mavs-/- HEK293T cells were transfected with empty vector, pcDNA-FLAG-MAVS, pcDNA-FLAG-MAVS-(Region III only), or pcDNA-FLAG-MAVS-(ΔRegion III), together with pcDNA3-FLAG-TRAF3IP3 or not as indicated. Twenty-four hours after transfection, cells were infected with SeV for twelve hours. IFNB induction was measured by qPCR.


Figure 3A The experiment was performed as described in Fig 2C . Instead of IFNB , ISG54 and IL6 inductions were measured by qPCR . B HEK293 cells were transfected with si - TRAF3IP3 oligos and then treated with various stimuli . Following subcellular fractionation , S5 fractions were obtained and subjected to native PAGE for detecting IRF3 dimer by immunoblotting . The whole cell lysates were also subjected to SDS - PAGE for anti - IRF3 and tubulin immunoblotting . C The experiment was performed as described in Fig 2E . ISG54 and IL6 induction was measured by qPCR . D Wild type and Traf3ip3 - / - HEK293T cells were infected with or without VSV . At indicated time points , Cells were collected and analyzed by immunoblotting . Following subcellular fractionation , S5 fractions were subjected to native PAGE to examine IRF3 dimer , and P5 fractions contained mitochondria were subjected to SDD - AGE to examine MAVS aggregation . The mitochondrial outer membrane protein Prohibitin was immunoblotted as an internal control .

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Figure 3A The experiment was performed as described in Fig 2C. Instead of IFNB, ISG54 and IL6 inductions were measured by qPCR.B HEK293 cells were transfected with si-TRAF3IP3 oligos and then treated with various stimuli. Following subcellular fractionation, S5 fractions were obtained and subjected to native PAGE for detecting IRF3 dimer by immunoblotting. The whole cell lysates were also subjected to SDS-PAGE for anti- IRF3 and tubulin immunoblotting.C The experiment was performed as described in Fig 2E. ISG54 and IL6 induction was measured by qPCR.D Wild type and Traf3ip3-/- HEK293T cells were infected with or without VSV. At indicated time points, Cells were collected and analyzed by immunoblotting. Following subcellular fractionation, S5 fractions were subjected to native PAGE to examine IRF3 dimer, and P5 fractions contained mitochondria were subjected to SDD-AGE to examine MAVS aggregation. The mitochondrial outer membrane protein Prohibitin was immunoblotted as an internal control.


Figure 4A pcDNA - FLAG - TRADD and pcDNA - FLAG - TRAF3IP3 were transfected into HEK293T cells at increasing amount respectively . ISG54 and IL6 induction were measured thirty - six hours post - transfection by qPCR . Expression levels were shown in Fig EV4B . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 NS indicates no statistically significant difference . B HEK293T cells were transfected with pcDNA3 - HA - TRAF3IP3 , in combination with pcDNA3 - FLAG - RIG - I , MAVS , TBK1 , IRF3 , TRAF2 , TRAF3 , TRAF5 , or TRAF6 as indicated . Thirty - six hours after transfection , cells were collected and anti - HA immunoprecipitations were performed . IP products were subjected to immunoblotting . C shRNA targeting TRAF3IP3 ( sh - TRAF3IP3 ) was transduced into Mavs - / - HEK293T cells . Twenty - four hours after transduction , cells were treated with puromycin ( 2 μg / mL ) for forty - eight hours and then transfected with pcDNA3 - FLAG - MAVS - ( Region III only ) . Twenty - four hours after transfection , cells were infected with or without VSV for twelve hours . Cells were then collected and subjected to immunoprecipitation assay and immunoblotting . D Wild type and Traf3ip3 - / - HEK293T cells were transfected with pcDNA3 - FLAG - MAVS - ( Region III only ) . Twenty - four hours post - transfection , cells were infected with or without VSV . Twelve hours post - infection , cells were collected and subjected to immunoprecipitation assay and immunoblotting .

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Figure 4A pcDNA-FLAG-TRADD and pcDNA-FLAG-TRAF3IP3 were transfected into HEK293T cells at increasing amount respectively. ISG54 and IL6 induction were measured thirty-six hours post-transfection by qPCR. Expression levels were shown in Fig EV4B. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05, **P<0.01 and ***P<0.001 NS indicates no statistically significant difference.B HEK293T cells were transfected with pcDNA3-HA-TRAF3IP3, in combination with pcDNA3-FLAG- RIG-I, MAVS, TBK1, IRF3, TRAF2, TRAF3, TRAF5, or TRAF6 as indicated. Thirty-six hours after transfection, cells were collected and anti-HA immunoprecipitations were performed. IP products were subjected to immunoblotting.C shRNA targeting TRAF3IP3 (sh-TRAF3IP3) was transduced into Mavs-/- HEK293T cells. Twenty-four hours after transduction, cells were treated with puromycin (2 μg/mL) for forty-eight hours and then transfected with pcDNA3-FLAG-MAVS-(Region III only). Twenty-four hours after transfection, cells were infected with or without VSV for twelve hours. Cells were then collected and subjected to immunoprecipitation assay and immunoblotting.D Wild type and Traf3ip3-/- HEK293T cells were transfected with pcDNA3-FLAG-MAVS-(Region III only). Twenty-four hours post-transfection, cells were infected with or without VSV. Twelve hours post-infection, cells were collected and subjected to immunoprecipitation assay and immunoblotting.


Figure 5A Subcellular fractionation was performed for HEK293T cells infected with or without SeV to get three fractions , i . e . , cytoplasm , membrane and nuclei extracts , which were then subjected to Immunoblotting analysis as indicated . B Membranous fraction obtained in Fig 5A were further separated into mitochondria , Golgi and ER extracts as described in methods , followed by immunoblotting analysis . C - E Immunofluorescent microscopic imaging for TRAF3IP3 . HeLa cells were transfected with pcDNA3 - FLAG - TRAF3IP3 . Twenty - four hours after transfection , cells were infected with or without SeV for twelve hours . Anti - FLAG M2 ( FITC ) was used for immunofluorescence staining of FLAG - TRAF3IP3 . Nuclei were stained with DAPI . Mitochondria was stained with Mitotracker Red ( C ) . Calnexin ( an ER protein ) was stained for ER ( D ) . GM130 ( a Golgi protein ) was stained for Golgi ( E ) . Scale bar represents 5 micrometers . F Quantitative analysis of yellow color as shown in Fig 5C , D and E , indicating TRAF3IP3 localization on various organelles with or without virus infection . The quantification was performed with ImageJ . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * * * P < 0 . 001 . NS indicates no statistically significant difference .

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Figure 5A Subcellular fractionation was performed for HEK293T cells infected with or without SeV to get three fractions, i.e., cytoplasm, membrane and nuclei extracts, which were then subjected to Immunoblotting analysis as indicated.B Membranous fraction obtained in Fig 5A were further separated into mitochondria, Golgi and ER extracts as described in methods, followed by immunoblotting analysis.C-E Immunofluorescent microscopic imaging for TRAF3IP3. HeLa cells were transfected with pcDNA3-FLAG-TRAF3IP3. Twenty-four hours after transfection, cells were infected with or without SeV for twelve hours. Anti-FLAG M2 (FITC) was used for immunofluorescence staining of FLAG-TRAF3IP3. Nuclei were stained with DAPI. Mitochondria was stained with Mitotracker Red (C). Calnexin (an ER protein) was stained for ER (D). GM130 (a Golgi protein) was stained for Golgi (E). Scale bar represents 5 micrometers. F Quantitative analysis of yellow color as shown in Fig 5C, D and E, indicating TRAF3IP3 localization on various organelles with or without virus infection. The quantification was performed with ImageJ. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. ***P<0.001. NS indicates no statistically significant difference.


Figure 6BMDMs from wild type , Traf3ip3 - / - and Mavs - / - mice were treated with or without VSV , SeV , poly ( I : C ) or HSV - 1 respectively as indicated for six hours . Ifnb ( A ) , Ifna4 ( B ) , Il6 induction was measured respectively by qPCR . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference . BMDMs from wild type , Traf3ip3 - / - and Mavs - / - mice were treated with or without VSV , SeV , poly ( I : C ) or HSV - 1 respectively as indicated for six hours . Il6 ( C ) , Isg54 ( D ) , and Cxcl10 ( E ) induction was measured respectively by qPCR . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference . F BMDMs from wild type , Traf3ip3 - / - and Mavs - / - mice were infected with or without VSV for six hours . Cells were then collected and following subcellular fractionation , S5 fractions were subjected to native PAGE to examine IRF3 dimer , and P5 fractions contained mitochondria were subjected to SDD - AGE to examine MAVS aggregation .

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Figure 6BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were treated with or without VSV, SeV, poly (I:C) or HSV-1 respectively as indicated for six hours. Ifnb (A), Ifna4 (B), Il6 induction was measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were treated with or without VSV, SeV, poly (I:C) or HSV-1 respectively as indicated for six hours. Il6 (C), Isg54 (D), and Cxcl10 (E) induction was measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.F BMDMs from wild type, Traf3ip3-/- and Mavs-/- mice were infected with or without VSV for six hours. Cells were then collected and following subcellular fractionation, S5 fractions were subjected to native PAGE to examine IRF3 dimer, and P5 fractions contained mitochondria were subjected to SDD-AGE to examine MAVS aggregation.


Figure 7Wild type , Traf3ip3 - / - and Mavs - / - mice ( three littermates per group ) were infected intravenously with VSV at a MOI of 2 × 108 . The spleens were collected 4 hours or 8 hours after infection and Ifna4 ( A ) , Ifnb ( B ) , Isg54 ( C ) and Il6 ( D ) inductions were measured respectively by qPCR . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference . Wild type , Traf3ip3 - / - and Mavs - / - mice ( three littermates per group ) were infected intravenously with VSV at a MOI of 2 × 108 . The spleens were collected 4 hours or 8 hours after infection and VSV RNA levels were measured by qPCR ( E ) . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference . Wild type , Traf3ip3 - / - and Mavs - / - mice ( three littermates per group ) were infected intravenously with VSV at a MOI of 2 × 108 . The sera were collected from the treated mice and used for the measurement of IFNβ production by ELISA ( F ) . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference . G Wild type , Traf3ip3 - / - and Mavs - / - mice ( n = 6 each ) were injected with VSV via tail vein injection at 5 × 107 pfu per mouse , and the survival rates were monitored for 15 days . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 .

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Figure 7Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The spleens were collected 4 hours or 8 hours after infection and Ifna4 (A), Ifnb (B), Isg54 (C) and Il6 (D) inductions were measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The spleens were collected 4 hours or 8 hours after infection and VSV RNA levels were measured by qPCR (E). All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The sera were collected from the treated mice and used for the measurement of IFNβ production by ELISA (F). All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.G Wild type, Traf3ip3-/- and Mavs-/- mice (n=6 each) were injected with VSV via tail vein injection at 5×107 pfu per mouse, and the survival rates were monitored for 15 days. P values were determined by unpaired two-tailed Student's t-test. *P<0.05


Figure 1 ( F ) Malate dehydrogenase ( MDH ; 0 . 2 µM ) was incubated at 40 ° C in the absence ( black ) or presence of 0 . 5 µM WT HSP27 ( red ) or 0 . 5 µM P182L HSP27 ( blue ) . ( G ) Insulin ( 40 µM ) was incubated at 40 ° C in the absence ( black ) or presence of 20 µM WT HSP27 ( red ) or 20 µM P182L HSP27 ( blue ) . The y - axes depict the absorbance at 340 nm due to the formation of large aggregates . The solid lines represent the average of three replicates with the filled area reflecting ± one standard deviation .

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Figure 1(F) Malate dehydrogenase (MDH; 0.2 µM) was incubated at 40 °C in the absence (black) or presence of 0.5 µM WT HSP27 (red) or 0.5 µM P182L HSP27 (blue). (G) Insulin (40 µM) was incubated at 40 °C in the absence (black) or presence of 20 µM WT HSP27 (red) or 20 µM P182L HSP27 (blue). The y-axes depict the absorbance at 340 nm due to the formation of large aggregates. The solid lines represent the average of three replicates with the filled area reflecting ± one standard deviation.


Figure 2 ( A ) SEC - MALS data from WT HSP27 and the P182L variant , with cHSP27 included for comparison . All samples were injected at 40 µM concentration ( monomer ) . The respective average molecular masses are 14 MDa , 470 kDa , and 18 kDa for P182L , WT HSP27 , and cHSP27 . The normalized UV signals are plotted on the left y - axis as solid lines and the molecular masses are plotted on the right y - axis as circles . ( B ) Fractionation of HeLa cells overexpressing either HSP27 or the P182L mutant by a 10 % to 80 % sucrose gradient . Western blot against anti - V5 is shown . ( C ) Expression of V5 - epitope - tagged HSP27 ( WT or P182L mutant ) in HSP27 knock - out HeLa cells and immunostaining of HSP27 . The scale bars correspond to the non - expanded dimensions , derived by dividing by the sample expansion factor of 4 . 3 . ( D ) The P182L mutation decreases the density of HSP27 spots upon detection of local fluorescence intensity maxima and quantification of the number of detected spots in expanded samples ( n = 30 ) . The error bar is the standard deviation for the different cytoplasmic regions . ( E ) Distribution of the intensity of individual spots ( n = 3342 ) after Gaussian fitting of fluorescence intensity in the neighborhood of detected spots . ( F ) Immunostaining of HSP27 ( white ) in motor neurons differentiated from CMT patient - derived iPSCs that harbor the heterozygous P182L mutation reveals large , cytoplasmic aggregates ( center and right panels ) . The control motor neurons that contain WT HSP27 do not show evidence of any aggregates . The nucleus is stained with Hoechst ( blue ) . Scale bar , 10 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) SEC-MALS data from WT HSP27 and the P182L variant, with cHSP27 included for comparison. All samples were injected at 40 µM concentration (monomer). The respective average molecular masses are 14 MDa, 470 kDa, and 18 kDa for P182L, WT HSP27, and cHSP27. The normalized UV signals are plotted on the left y-axis as solid lines and the molecular masses are plotted on the right y-axis as circles.(B) Fractionation of HeLa cells overexpressing either HSP27 or the P182L mutant by a 10% to 80% sucrose gradient. Western blot against anti-V5 is shown.(C) Expression of V5-epitope-tagged HSP27 (WT or P182L mutant) in HSP27 knock-out HeLa cells and immunostaining of HSP27. The scale bars correspond to the non-expanded dimensions, derived by dividing by the sample expansion factor of 4.3. (D) The P182L mutation decreases the density of HSP27 spots upon detection of local fluorescence intensity maxima and quantification of the number of detected spots in expanded samples (n = 30). The error bar is the standard deviation for the different cytoplasmic regions. (E) Distribution of the intensity of individual spots (n = 3342) after Gaussian fitting of fluorescence intensity in the neighborhood of detected spots.(F) Immunostaining of HSP27 (white) in motor neurons differentiated from CMT patient-derived iPSCs that harbor the heterozygous P182L mutation reveals large, cytoplasmic aggregates (center and right panels). The control motor neurons that contain WT HSP27 do not show evidence of any aggregates. The nucleus is stained with Hoechst (blue). Scale bar, 10 µm.


Figure 3 ( A ) Primary sequences of the WT peptide ( red ) and P182L peptide ( blue ) . The IxI / V motif is indicated with white text . Below : regions of 2D 1H - 15N HSQC spectra of 15N - labeled cHSP27 ( black ) in the presence of increasing amounts of WT ( red ) or P182L peptide ( blue ) . Resonances in the β4 and β8 strands broaden and disappear upon peptide binding , indicative of intermediate exchange . ( B ) Zoomed - in regions of resonances during the peptide titrations . The color bars indicate the amount of added WT or P182L peptide . ( C ) The combined 1HN and 15N chemical shift changes , denoted as chemical shift perturbation ( CSP ) , are plotted as a function of added peptide concentration . ( D ) CSPs shown as a function of residue number for the WT ( red ) and P182L peptide ( blue ) . ( E ) X - ray structure of cHSP27 bound to the WT peptide ( PDB : 4mjh ) , shown in white sticks , with the results from ( D ) plotted onto the structure .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Primary sequences of the WT peptide (red) and P182L peptide (blue). The IxI/V motif is indicated with white text. Below: regions of 2D 1H-15N HSQC spectra of 15N-labeled cHSP27 (black) in the presence of increasing amounts of WT (red) or P182L peptide (blue). Resonances in the β4 and β8 strands broaden and disappear upon peptide binding, indicative of intermediate exchange. (B) Zoomed-in regions of resonances during the peptide titrations. The color bars indicate the amount of added WT or P182L peptide. (C) The combined 1HN and 15N chemical shift changes, denoted as chemical shift perturbation (CSP), are plotted as a function of added peptide concentration. (D) CSPs shown as a function of residue number for the WT (red) and P182L peptide (blue). (E) X-ray structure of cHSP27 bound to the WT peptide (PDB: 4mjh), shown in white sticks, with the results from (D) plotted onto the structure.


Figure 4 ( A , B ) i . 15N CPMG RD data for residues in the β4 strand ( T113 ) and the β8 strand ( S154 ) in the absence of added peptide . CPMG data are shown for the same residues in the presence of ii . ca . 2 % cHSP27 - WT peptide complex or iii . ca . 2 % of the cHSP27 - P182L peptide complex . Uncertainties in R2 , eff values are derived from the standard deviations of fitted peak intensities obtained from duplicated νCPMG values . ( C ) i . The association ( kon ) and dissociation ( koff ) rates for WT ( red ) and P182L ( blue ) IxI / V peptide binding to the ACD , calculated directly from the CPMG RD data . Residues that are impacted by the presence of a small amount of peptide are indicated by red ( ii . , WT ) and blue ( iii . , P182L ) spheres . In both cases , residues in similar ACD regions show enhanced Rex , indicative of chemical exchange at similar sites .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A, B) i. 15N CPMG RD data for residues in the β4 strand (T113) and the β8 strand (S154) in the absence of added peptide. CPMG data are shown for the same residues in the presence of ii. ca. 2% cHSP27-WT peptide complex or iii. ca. 2% of the cHSP27-P182L peptide complex. Uncertainties in R2,eff values are derived from the standard deviations of fitted peak intensities obtained from duplicated νCPMG values. (C) i. The association (kon) and dissociation (koff) rates for WT (red) and P182L (blue) IxI/V peptide binding to the ACD, calculated directly from the CPMG RD data. Residues that are impacted by the presence of a small amount of peptide are indicated by red (ii., WT) and blue (iii., P182L) spheres. In both cases, residues in similar ACD regions show enhanced Rex, indicative of chemical exchange at similar sites.


Figure 5 ( B ) Volcano plot representing the interactors that are enriched for the P182L mutant versus GFP as a negative control , obtained with affinity - enrichment mass spectrometry . Proteins that co - immunoprecipitated significantly more ( p < 0 . 05 ) with the P182L variant are shown in the upper right quadrant . Interactors with one or more [ I / V ] - x - [ I / V ] motifs are displayed in red while other significantly enriched interactors are displayed in black . ( C - D ) Co - immunoprecipitation from HeLa cells stably overexpressing V5 - epitope - tagged HSP27 ( WT or P182L mutant ) using anti - V5 beads . Co - immunoprecipitation was quantified and presented as a percentage relative to the P182L variant ( n = 3 ) . The mean ± one standard deviation ( n = 3 ) is shown . Right : the location of Pro182 ( red ) in the crystal structure of peptide - bound cHSP27 ( PDB : 4mjh ) and in silico creation of the P182L mutation ( blue ) . The peptide is shown in white sticks . P182L specifically weakens binding of the HSP27 IxI / V motif . ( E ) Co - immunoprecipitation from HeLa cells stably overexpressing V5 - epitope - tagged HSP27 ( WT or P182L mutant ) and transiently transfected with BAG3 - eGFP ( WT or IPV - mutant ) using anti - GFP beads . Western blots for anti - GFP ( BAG3 ) , V5 ( HSPB1 ) , and α - tubulin . NS indicates a non - specific band . Note that detection of BAG3 ( anti - GFP ) and HSP27 ( anti - V5 ) occurs with different antibodies . The locations of the IPV - to - GPG mutations in BAG3 are depicted below . ( F ) Co - immunoprecipitation from HeLa cells transiently transfected with V5 - epitope - tagged HSP27 ( P182L or S155Q / P182L mutant ) using anti - V5 beads ( n = 2 ) . Right : the location of Ser155 ( red ) in the crystal structure of peptide - bound cHSP27 ( PDB : 4mjh ) and in silico creation of the S155Q mutation ( purple ) showing a clash with the bound peptide . The peptide is shown in white sticks . S155Q disrupts binding of all IxI / V motifs .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Volcano plot representing the interactors that are enriched for the P182L mutant versus GFP as a negative control, obtained with affinity-enrichment mass spectrometry. Proteins that co-immunoprecipitated significantly more (p<0.05) with the P182L variant are shown in the upper right quadrant. Interactors with one or more [I/V]-x-[I/V] motifs are displayed in red while other significantly enriched interactors are displayed in black.(C-D) Co-immunoprecipitation from HeLa cells stably overexpressing V5-epitope-tagged HSP27 (WT or P182L mutant) using anti-V5 beads. Co-immunoprecipitation was quantified and presented as a percentage relative to the P182L variant (n=3). The mean ± one standard deviation (n=3) is shown. Right: the location of Pro182 (red) in the crystal structure of peptide-bound cHSP27 (PDB: 4mjh) and in silico creation of the P182L mutation (blue). The peptide is shown in white sticks. P182L specifically weakens binding of the HSP27 IxI/V motif.(E) Co-immunoprecipitation from HeLa cells stably overexpressing V5-epitope-tagged HSP27 (WT or P182L mutant) and transiently transfected with BAG3-eGFP (WT or IPV-mutant) using anti-GFP beads. Western blots for anti-GFP (BAG3), V5 (HSPB1), and α-tubulin. NS indicates a non-specific band. Note that detection of BAG3 (anti-GFP) and HSP27 (anti-V5) occurs with different antibodies. The locations of the IPV-to-GPG mutations in BAG3 are depicted below.(F) Co-immunoprecipitation from HeLa cells transiently transfected with V5-epitope-tagged HSP27 (P182L or S155Q/P182L mutant) using anti-V5 beads (n=2). Right: the location of Ser155 (red) in the crystal structure of peptide-bound cHSP27 (PDB: 4mjh) and in silico creation of the S155Q mutation (purple) showing a clash with the bound peptide. The peptide is shown in white sticks.S155Q disrupts binding of all IxI/V motifs.


Figure 1 ( D ) Immunostaining of PGCLCs d5 cryosections for SOX2 ( magenta ) and TFAP2C ( cyan ) . Barplot indicates the quantification of SOX2 + cells , TFAP2C + cells , and SOX2 + / TFAP2C + . n = 1150 cells , from n = 3 separate inductions , using two biological clones . The white squares represent the position of the magnified region at the bottom . Scale bar , 50 µm and 10 µm for the magnified region .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(D) Immunostaining of PGCLCs d5 cryosections for SOX2 (magenta) and TFAP2C (cyan). Barplot indicates the quantification of SOX2+ cells, TFAP2C+ cells, and SOX2+/TFAP2C+. n=1150 cells, from n=3 separate inductions, using two biological clones. The white squares represent the position of the magnified region at the bottom. Scale bar, 50 µm and 10 µm for the magnified region.


Figure 2 ( C ) MA plot of differential gene expression changes between XGFP - and XGFP + PGCLCs as determined by RNA - seq . Log2 - mean expression ( log2 - normalised counts from DESeq2 ) on the x - axis and the log2 - fold change on the y - axis are shown . Significantly upregulated and downregulated genes are highlighted in red and green respectively . False Discovery Rate ( FDR ) < 0 . 001 . Non - significant genes with log2 - mean expression between 0 and 0 . 2 were removed for easier plot visualisation . ( D ) Expression levels ( normalised DEseq2 counts ) of selected differentially expressed genes between XGFP - and XGFP + PGCLCs during the differentiation time course . Genes with FDR < 0 . 001 were considered significantly differentially expressed . Points indicate expression of individual biological replicates . ( E ) Heatmap of RNA - seq normalised counts showing the Zscore across PGCLC induction timepoints of 31 manually selected and manually ordered marker genes belonging to the categories reported on the side . ( G ) FACS analysis of cell cycle using DAPI . Numbers indicate the percentage of cells in G1 , S and G2 / M respectively . ( H ) Alkaline phosphatase staining for ESCs , XGFP + PGCLCs and XGFP - PGCLCs grown for 7 days in 2i / LIF medium on immortalised mouse embryonic fibroblasts . ( I ) Barplot indicating the absolute numbers of Alkaline Phosphatase ( AP ) positive colonies in each cell type after 7 days of culture in 2i / LIF medium on immortalised mouse embryonic fibroblasts . Y - axis is in square root scale ( sqrt ) for better plot visualisation . Each white dot represents one technical replicate .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(C) MA plot of differential gene expression changes between XGFP- and XGFP+ PGCLCs as determined by RNA-seq. Log2-mean expression (log2-normalised counts from DESeq2) on the x-axis and the log2-fold change on the y-axis are shown. Significantly upregulated and downregulated genes are highlighted in red and green respectively. False Discovery Rate (FDR) < 0.001. Non-significant genes with log2-mean expression between 0 and 0.2 were removed for easier plot visualisation.(D) Expression levels (normalised DEseq2 counts) of selected differentially expressed genes between XGFP- and XGFP+ PGCLCs during the differentiation time course. Genes with FDR < 0.001 were considered significantly differentially expressed. Points indicate expression of individual biological replicates.(E) Heatmap of RNA-seq normalised counts showing the Zscore across PGCLC induction timepoints of 31 manually selected and manually ordered marker genes belonging to the categories reported on the side.(G) FACS analysis of cell cycle using DAPI. Numbers indicate the percentage of cells in G1, S and G2/M respectively.(H) Alkaline phosphatase staining for ESCs, XGFP+ PGCLCs and XGFP- PGCLCs grown for 7 days in 2i/LIF medium on immortalised mouse embryonic fibroblasts. (I) Barplot indicating the absolute numbers of Alkaline Phosphatase (AP) positive colonies in each cell type after 7 days of culture in 2i/LIF medium on immortalised mouse embryonic fibroblasts. Y-axis is in square root scale (sqrt) for better plot visualisation. Each white dot represents one technical replicate.


Figure 4 ( H ) Immunofluorescence images of SYCP3 ( red ) , DAZL ( yellow ) and DAPI at agg11 of IVDi tissue maturation from XGFP - PGCLCs . IVDi = in vitro differentiation . White squares indicate the positions of the magnified section shown below . Top panel scale bar = 100 μm . Middle panel scale bar = 10 μm . Bottom panel scale bar = 50 μm . ( I ) Quantification of SYCP3 + cells ( oocytes in cyst and primary follicles ) in IVDi tissues at agg11 . Each dot represents one IVDi tissue performed in 3 biological replicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(H) Immunofluorescence images of SYCP3 (red), DAZL (yellow) and DAPI at agg11 of IVDi tissue maturation from XGFP- PGCLCs. IVDi = in vitro differentiation. White squares indicate the positions of the magnified section shown below. Top panel scale bar = 100 μm. Middle panel scale bar = 10 μm. Bottom panel scale bar = 50 μm. (I) Quantification of SYCP3+ cells (oocytes in cyst and primary follicles) in IVDi tissues at agg11. Each dot represents one IVDi tissue performed in 3 biological replicates.


Figure 6 ( A ) Schematic representation of the m220 stromal feeders expansion culture to compare the meiotic capacity of XGFP - and XGFP + PGCLCs . Meiosis is induced via addition of Retinoic Acid ( RA ) and Bone Morphogenetic Protein 2 ( BMP2 ) . c0 = starting day of culture , c9 = culture day 9 and last day of culture . ( B ) Representative images for the expression of XGFP ( green ) and SYCP3 ( red ) in germ cells at c5 , c7 and c9 from XGFP + and XGFP - PGCLCs . All cells with SYCP3 signal irrespective of localization pattern or intensity were scored as SYCP3 + cells . Cells were counterstained with DAPI ( grey ) . Scale bars = 10 μm . ( C ) Number of SYCP3 + cells per m220 culture day originating from XGFP + and XGFP - PGCLCs . Each white dot represents a biological replicate ( n = 3 ) . P - values shown are from a two - sided Mann - Whitney U test . n . s . = not significant . ( D ) Percentage of XGFP + cells among SYCP3 + cells at the indicated m220 culture day , originating from XGFP + or XGFP - PGCLCs . Green bars show constitutive GFP expression in XGFP + PGCLC - derived cells , while striped bars signify XGFP reactivation in XGFP - cells . Each white dot represents a biological replicate ( n = 3 ) . ( E ) Representative images showing stages of meiotic prophase I from culture day 9 ( c9 ) germ cells from XGFP + and XGFP - PGCLCs . c9 germ cells were spread and immunostained for SYCP3 ( red ) , and γH2AX ( grey ) . Scale bars = 10 µm . ( F ) Quantification of meiotic progression in culture day ( c9 ) expanded germ cells derived from XGFP + and XGFP - PGCLCs . The graphs show the percentages of the meiotic stage for SYCP3 + cells . Lepto , leptotene ; Zygo , zygotene ; Pachy , pachytene ; Diplo , diplotene . Numbers indicate detected cells per meiotic stage and original XGFP status . ( G ) Representative tile scan merged images of XGFP ( green ) and Xtomato ( red ) in germ cells at c7 from XGFP + and XGFP - PGCLCs . Cells were counterstained with DAPI ( grey ) . Scale Bar = 1 mm . The white squares contain a magnified image of the region depicted by the white dotted squares ( Scale Bar = 1 µm ) . ( H ) Density ridge plot showing size of cellular aggregates in ( G ) measured using the XTomato signal . X - Axis shows area in µm2 scaled as log2 . Dashed line represents the mean . XFGP - mean = 1320 µm2 , XGFP + mean = 5514 µm2 . XGFP - n = 297 aggregates , XGFP + n = 373 aggregates . P - value from two - sample unpaired Wilcoxon - Mann - Whitney test with R defaults .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Schematic representation of the m220 stromal feeders expansion culture to compare the meiotic capacity of XGFP- and XGFP+ PGCLCs. Meiosis is induced via addition of Retinoic Acid (RA) and Bone Morphogenetic Protein 2 (BMP2). c0 = starting day of culture, c9 = culture day 9 and last day of culture. (B) Representative images for the expression of XGFP (green) and SYCP3 (red) in germ cells at c5, c7 and c9 from XGFP+ and XGFP- PGCLCs. All cells with SYCP3 signal irrespective of localization pattern or intensity were scored as SYCP3+ cells. Cells were counterstained with DAPI (grey). Scale bars = 10 μm. (C) Number of SYCP3+ cells per m220 culture day originating from XGFP+ and XGFP- PGCLCs. Each white dot represents a biological replicate (n = 3). P-values shown are from a two-sided Mann-Whitney U test. n.s. = not significant. (D) Percentage of XGFP+ cells among SYCP3+ cells at the indicated m220 culture day, originating from XGFP+ or XGFP- PGCLCs. Green bars show constitutive GFP expression in XGFP+ PGCLC-derived cells, while striped bars signify XGFP reactivation in XGFP- cells. Each white dot represents a biological replicate (n = 3).(E) Representative images showing stages of meiotic prophase I from culture day 9 (c9) germ cells from XGFP+ and XGFP- PGCLCs. c9 germ cells were spread and immunostained for SYCP3 (red), and γH2AX (grey). Scale bars = 10 µm. (F) Quantification of meiotic progression in culture day (c9) expanded germ cells derived from XGFP+ and XGFP- PGCLCs. The graphs show the percentages of the meiotic stage for SYCP3+ cells. Lepto, leptotene; Zygo, zygotene; Pachy, pachytene; Diplo, diplotene. Numbers indicate detected cells per meiotic stage and original XGFP status.(G) Representative tile scan merged images of XGFP (green) and Xtomato (red) in germ cells at c7 from XGFP+ and XGFP- PGCLCs. Cells were counterstained with DAPI (grey). Scale Bar = 1 mm. The white squares contain a magnified image of the region depicted by the white dotted squares (Scale Bar = 1 µm). (H) Density ridge plot showing size of cellular aggregates in (G) measured using the XTomato signal. X-Axis shows area in µm2 scaled as log2. Dashed line represents the mean. XFGP- mean = 1320 µm2, XGFP+ mean = 5514 µm2. XGFP- n = 297 aggregates, XGFP+ n = 373 aggregates. P-value from two-sample unpaired Wilcoxon-Mann-Whitney test with R defaults.