A - I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( I ) hepatic phosphorylation of AKT ( p - AKTSer473 ) were analyzed . Compounds were administered by daily subcutaneous injections . To assess insulin - stimulated p - AKTSer473 , insulin ( n = 5 ) or saline ( n = 3 ) was injected 10 min prior to liver sampling . Data represent means ± SEM ; n = 8 in ( A - H ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (I) hepatic phosphorylation of AKT (p-AKTSer473) were analyzed. Compounds were administered by daily subcutaneous injections. To assess insulin-stimulated p-AKTSer473, insulin (n = 5) or saline (n = 3) was injected 10 min prior to liver sampling. Data represent means ± SEM;n = 8 in (A-H); *P < 0.05, **P < 0.01, ***P < 0.001.


A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( A ) Plasma lipoprotein fractions . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (A) Plasma lipoprotein fractions. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( B ) liver cholesterol levels . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (B) liver cholesterol levels. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( C ) expression of genes implicated in hepatic cholesterol and lipoprotein uptake . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (C) expression of genes implicated in hepatic cholesterol and lipoprotein uptake. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . ( D ) expression of genes implicated in cholesterol and bile acid metabolism and excretion were analyzed . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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A-D Cholesterol metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (D) expression of genes implicated in cholesterol and bile acid metabolism and excretion were analyzed. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( A ) body weight . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (A) body weight. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( B ) cumulative food intake . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (B) cumulative food intake. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( C , D ) body composition . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (C, D) body composition. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


A - F 22 days of treatment of DIO male mice with vehicle ( white ) , liraglutide ( 10 nmol / kg ) ( gray ) , RM - 493 ( 3 . 6 μmol / kg ) ( black ) , or liraglutide ( 10 nmol / kg ) and RM - 493 ( 3 . 6 μmol / kg ) ( checkered ) . Effects on ( E , F ) glucose tolerance . Compounds were administered by daily subcutaneous injections . Data represent means ± SEM ; n = 8 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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A-F 22 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). Effects on (E, F) glucose tolerance. Compounds were administered by daily subcutaneous injections. Data represent means ± SEM;n = 8; *P < 0.05, **P < 0.01, ***P < 0.001.


( A ) Size exclusion chromatography profile of NS1 pull - down experiments showing a clear shift after incubation in complete or heat - inactivated human serum ( solid and dotted black lines , respectively ) from the same healthy donor compared to the NS1 protein alone ( blue line ) . NS1 protein interaction partners were identified by SDS - PAGE and N - terminal sequencing as the Apolipoprotein B scaffold of the low - density lipoproteins ( LDL ) in the first SEC elution peak , and the ApoA - I protein scaffold of the high - density lipoproteins ( HDL ) in the second elution peak .

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(A) Size exclusion chromatography profile of NS1 pull-down experiments showing a clear shift after incubation in complete or heat-inactivated human serum (solid and dotted black lines, respectively) from the same healthy donor compared to the NS1 protein alone (blue line). NS1 protein interaction partners were identified by SDS-PAGE and N-terminal sequencing as the Apolipoprotein B scaffold of the low-density lipoproteins (LDL) in the first SEC elution peak, and the ApoA-I protein scaffold of the high-density lipoproteins (HDL) in the second elution peak.


( B ) Biolayer interferometry ( BLI ) profiles corresponding to the binding of NS1 at various concentrations respective to human HDL ( left panel ) and human LDL ( central panel ) particles . The concentration - dependence of the steady - state signal corresponding to the binding of NS1 to HDL ( black dots ) and LDL ( white dots ) is shown on the right - hand side panel . The measurements were replicated at least three times using novel biosensors and samples . Data points and error bars correspond to the mean ± SD .

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(B) Biolayer interferometry (BLI) profiles corresponding to the binding of NS1 at various concentrations respective to human HDL (left panel) and human LDL (central panel) particles. The concentration-dependence of the steady-state signal corresponding to the binding of NS1 to HDL (black dots) and LDL (white dots) is shown on the right-hand side panel. The measurements were replicated at least three times using novel biosensors and samples. Data points and error bars correspond to the mean ± SD.


( C ) Typical sedimentation coefficient distribution of NS1 or human HDL alone or pre - incubated together ( mixture of NS1 and HDL at a 1 : 1 or 5 : 1 mass ratio ) monitored using an interferometric detector . Peaks were integrated for all the detectors ( interference and absorbance at 280nm ) . The calculated stoichiometries are indicated for each peak . Solutions were equilibrated at 20 ° C for 2h before sedimentation velocity analysis .

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(C) Typical sedimentation coefficient distribution of NS1 or human HDL alone or pre-incubated together (mixture of NS1 and HDL at a 1:1 or 5:1 mass ratio) monitored using an interferometric detector. Peaks were integrated for all the detectors (interference and absorbance at 280nm). The calculated stoichiometries are indicated for each peak. Solutions were equilibrated at 20°C for 2h before sedimentation velocity analysis.


( A , B ) Electron microscopy observations from left to right : a representative image , followed by the three most representative classes of ( A ) purified HDL particles and ( B ) NS1 - HDL complexes . White bar : 50 nm , Black bar : 20 nm .

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(A, B) Electron microscopy observations from left to right: a representative image, followed by the three most representative classes of (A) purified HDL particles and (B) NS1-HDL complexes. White bar: 50 nm, Black bar: 20 nm.


( D ) Differential scanning calorimetry ( DSC ) of NS1 alone ( blue line ) or of an NS1 - HDL mixture at a 2 : 1 molar ratio ( orange ) . Of note , the HDL particles alone did not generate any signal in the temperature range tested .

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(D) Differential scanning calorimetry (DSC) of NS1 alone (blue line) or of an NS1-HDL mixture at a 2:1 molar ratio (orange). Of note, the HDL particles alone did not generate any signal in the temperature range tested.


( E ) Binding inhibition of hexameric NS1 to HDL with anti - ApoA - I polyclonal antibodies ( anti - ApoA - I Ab ) measured by BLI .

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(E) Binding inhibition of hexameric NS1 to HDL with anti-ApoA-I polyclonal antibodies (anti-ApoA-I Ab) measured by BLI.


( A - D ) Human primary macrophages were incubated for 24h with the different potential effectors ( NS1 , HDL , mix NS1 - HDL ) or with control suspensions ( PBS buffer , LPS , mix LPS - HDL ) . LPS stimulation was used as a positive control in the presence or absence of HDL and provided values consistent between experiments . Phosphate buffer used in the SEC purification step was used as a negative control . Cell culture supernatants were clarified and tested with a Luminex assay to quantify the amount of ( A ) TNF - α , ( B ) Il - 6 , ( C ) Il - 1ß and ( D ) Il - 10 released in the extracellular medium . Data reported on the graphs correspond to biological replicates of macrophages isolated from four blood donors ( n = 9 for TNF - α , Il - 6 and Il - 1ß , n = 5 for Il - 10 ) . Data represent mean + / - SEM . A Mann - Whitney test was used to assess the statistical significance of differences observed between mean cytokine levels in different cell culture supernatants . Not significant : ns , * p < 0 . 05 , * * * p < 0 . 001 .

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(A-D) Human primary macrophages were incubated for 24h with the different potential effectors (NS1, HDL, mix NS1-HDL) or with control suspensions (PBS buffer, LPS, mix LPS-HDL). LPS stimulation was used as a positive control in the presence or absence of HDL and provided values consistent between experiments. Phosphate buffer used in the SEC purification step was used as a negative control. Cell culture supernatants were clarified and tested with a Luminex assay to quantify the amount of (A) TNF-α, (B) Il-6, (C) Il-1ß and (D) Il-10 released in the extracellular medium. Data reported on the graphs correspond to biological replicates of macrophages isolated from four blood donors (n=9 for TNF-α, Il-6 and Il-1ß, n=5 for Il-10). Data represent mean +/- SEM. A Mann-Whitney test was used to assess the statistical significance of differences observed between mean cytokine levels in different cell culture supernatants. Not significant: ns, * p < 0.05, *** p < 0.001.


levels of ( B ) NS1 , ( C ) NS1 - ApoA - I , ( D ) NS1 - ApoE and ( E ) NS1 - ApoB NS1 - ApoA - I and NS1 - ApoB complexes are representative of NS1 - HDL and NS1 - LDL complex species , respectively , while the NS1 - ApoE - positive complexes remain to be fully characterized . Errors bars indicate SEM .

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levels of (B) NS1, (C) NS1-ApoA-I, (D) NS1-ApoE and (E) NS1-ApoB NS1-ApoA-I and NS1-ApoB complexes are representative of NS1-HDL and NS1-LDL complex species, respectively, while the NS1-ApoE-positive complexes remain to be fully characterized. Errors bars indicate SEM.


levels of ( F ) total cholesterol , ( G ) HDL - cholesterol and ( H ) triglycerides . Errors bars indicate SEM .

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levels of (F) total cholesterol, (G) HDL-cholesterol and (H) triglycerides. Errors bars indicate SEM.


( I ) Purified NS1 from different flaviviruses ( yellow fever , YF ; ZIKA ; West Nile , WN ; Japanese encephalitis , JE ; The Native Antigen Company ) were spiked in normal human plasma and NS1 - ApoA - I complexes further detected by ELISA . Data represent the mean values of two technical replicates .

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(I) Purified NS1 from different flaviviruses (yellow fever, YF; ZIKA; West Nile, WN; Japanese encephalitis, JE; The Native Antigen Company) were spiked in normal human plasma and NS1-ApoA-I complexes further detected by ELISA. Data represent the mean values of two technical replicates.


A ) Shear - dilation relations of Tek - Cre : : Lama5 - / - and Lama4 - / - mesenteric resistance arteries and corresponding wild type controls show a reduced response of Tek - Cre : : Lama5 - / - arteries and an enhanced response of Lama4 - / - arteries . Data shown are mean changes in vessel diameter ( Δ µm ) ± s . e . m . from 8 experiments with 16 wild type and 16 KO arteries ; KO and wild type were analysed as pairs in each experiment . * * P < 0 . 01 , paired t - test .

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A) Shear-dilation relations of Tek-Cre::Lama5-/- and Lama4-/- mesenteric resistance arteries and corresponding wild type controls show a reduced response of Tek-Cre::Lama5-/- arteries and an enhanced response of Lama4-/- arteries. Data shown are mean changes in vessel diameter (Δ µm) ± s.e.m. from 8 experiments with 16 wild type and 16 KO arteries; KO and wild type were analysed as pairs in each experiment. **P<0.01, paired t-test.


The dose - response curves of arteries from wild type , Tek - Cre : : Lama5 - / - ( B ) and Lama4 - / - mice ( C ) and wild type littermates stimulated with methacholine do not show significant differences ( n . s . ) . Data are expressed as percent relaxation of the maximum force developed in presence of 0 . 3 µM U46619 , and are mean values ± s . e . m from 7 experiments with 1 wild type and 1 KO artery in each experiment .

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The dose-response curves of arteries from wild type, Tek-Cre::Lama5-/- (B) and Lama4-/- mice (C) and wild type littermates stimulated with methacholine do not show significant differences (n.s.). Data are expressed as percent relaxation of the maximum force developed in presence of 0.3 µM U46619, and are mean values ± s.e.m from 7 experiments with 1 wild type and 1 KO artery in each experiment.


A ) Three - dimensional digital reconstructions of optical sections through immunofluorescently stained mesenteric resistance arteries from wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice , and optical sections through vessel walls to show the endothelial and underlying smooth muscle layers . DAPI staining permits identification of endothelial cell nuclei , which lie perpendicular to the nuclei of the smooth muscle cells . Laminin α5 is absent from endothelial basement membranes of Tek - Cre : : Lama5 - / - arteries ( arrow ) but is still present in smooth muscle basement membranes ( asterisk ) , while laminin α4 is still detectable in both endothelial and smooth muscle basement membranes . Lama4 - / - arteries lack laminin α4 in both endothelial ( arrow ) and smooth muscle ( asterisk ) basement membranes , but laminin α5 is still detectable . Scale bars are 10 μm .

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A) Three-dimensional digital reconstructions of optical sections through immunofluorescently stained mesenteric resistance arteries from wild type, Tek-Cre::Lama5-/- and Lama4-/- mice, and optical sections through vessel walls to show the endothelial and underlying smooth muscle layers. DAPI staining permits identification of endothelial cell nuclei, which lie perpendicular to the nuclei of the smooth muscle cells. Laminin α5 is absent from endothelial basement membranes of Tek-Cre::Lama5-/- arteries (arrow) but is still present in smooth muscle basement membranes (asterisk), while laminin α4 is still detectable in both endothelial and smooth muscle basement membranes. Lama4-/- arteries lack laminin α4 in both endothelial (arrow) and smooth muscle (asterisk) basement membranes, but laminin α5 is still detectable. Scale bars are 10 μm.


B ) Scanning electron microscopy images of endothelial cell - denuded mesenteric resistance arteries show a comparable topography of the endothelial basement membrane in wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - arteries . The arrows indicate artificial ruptures due to preparation procedure . Scale bars are 100 nm .

B) Scanning electron microscopy images of endothelial cell-denuded mesenteric resistance arteries show a comparable topography of the endothelial basement membrane in wild type, Tek-Cre::Lama5-/- and Lama4-/- arteries. The arrows indicate artificial ruptures due to preparation procedure. Scale bars are 100 nm.


C ) Intravital microscopic quantification of mesenteric arteries sizes in vivo show smaller diameters ( - 24 . 3 % ) in Tek - Cre : : Lama5 - / - arteries and larger diameters ( + 27 . 6 % ) in Lama4 - / - arteries compared to wild type controls . Auto - fluorescence of the internal elastic lamina allowed a good approximation of mesenteric arteries lumen diameter . Scale bar is 100 µm . Data are means ± s . e . m from a minimum of 4 first order mesenteric arteries imaged at least in 4 mice per genotype . * P < 0 . 05 unpaired t - test .

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C) Intravital microscopic quantification of mesenteric arteries sizes in vivo show smaller diameters (-24.3%) in Tek-Cre::Lama5-/- arteries and larger diameters (+27.6%) in Lama4-/- arteries compared to wild type controls. Auto-fluorescence of the internal elastic lamina allowed a good approximation of mesenteric arteries lumen diameter. Scale bar is 100 µm. Data are means ± s.e.m from a minimum of 4 first order mesenteric arteries imaged at least in 4 mice per genotype. * P<0.05 unpaired t-test.


A ) In vitro cell adhesion assays employing HUAECs plated on increasing concentrations of purified laminin 411 and 511 , compared to the non - endothelial cell laminin 111 and fibronectin , showing high levels of HUAECs adhesion to laminin 511 and low binding to laminin 411 .

O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro cell adhesion assays employing HUAECs plated on increasing concentrations of purified laminin 411 and 511, compared to the non-endothelial cell laminin 111 and fibronectin, showing high levels of HUAECs adhesion to laminin 511 and low binding to laminin 411.


B ) Enhanced fold change ( Δ ) in COX2 mRNA expression in HUAECs plated on laminin 511 versus laminin 111 in response to shear . mRNA data are means ± s . e . m of 4 independent experiments with triplicates / experiment , paired t - test .

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

B) Enhanced fold change (Δ) in COX2 mRNA expression in HUAECs plated on laminin 511 versus laminin 111 in response to shear. mRNA data are means ± s.e.m of 4 independent experiments with triplicates/ experiment, paired t-test.


B ) Enhanced fold change ( Δ ) in COX2 protein levels in HUAECs plated on laminin 511 versus laminin 111 in response to shear . Protein quantification data are means ± s . e . m of 7 independent experiments , Mann - Whitney test .

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

B) Enhanced fold change (Δ) in COX2 protein levels in HUAECs plated on laminin 511 versus laminin 111 in response to shear. Protein quantification data are means ± s.e.m of 7 independent experiments, Mann-Whitney test.


C ) Angle histogram of HUAEC orientation after 120 min of 10dyn / cm2 shear stress showing the percent cells at 10 - 90 ° in relation to the direction of flow . Cells were plated on 25 nM laminin 511 and laminin 111 . Data are means ± s . e . m of 6 independent experiments , t - test .

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

C) Angle histogram of HUAEC orientation after 120 min of 10dyn/cm2 shear stress showing the percent cells at 10-90° in relation to the direction of flow. Cells were plated on 25 nM laminin 511 and laminin 111. Data are means ± s.e.m of 6 independent experiments, t-test.


D , E ) Inhibition assays performed at 25nM laminin 511 ( D ) in the presence or absence of function blocking integrin antibodies . Data are means ± s . e . m of 4 experiments with triplicates / experiment , unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E) Inhibition assays performed at 25nM laminin 511 (D) in the presence or absence of function blocking integrin antibodies. Data are means ± s.e.m of 4 experiments with triplicates/ experiment, unpaired t-test . *P<0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.


D , E ) Inhibition assays performed at 25nM laminin 111 , laminin 411 or fibronectin ( E ) in the presence or absence of function blocking integrin antibodies . Data are means ± s . e . m of 4 experiments with triplicates / experiment , unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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D, E) Inhibition assays performed at 25nM laminin 111, laminin 411 or fibronectin (E) in the presence or absence of function blocking integrin antibodies. Data are means ± s.e.m of 4 experiments with triplicates/ experiment, unpaired t-test . *P<0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.


( A ) Double immunofluorescence staining of whole mount wild type mesenteric resistance arteries for β1 integrin and vinculin show colocalization in focal adhesions ( arrow heads ) . Scale bar is 10 µm .

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

(A) Double immunofluorescence staining of whole mount wild type mesenteric resistance arteries for β1 integrin and vinculin show colocalization in focal adhesions (arrow heads). Scale bar is 10 µm.


B ) Vinculin immunofluorescence staining of whole mount wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek - Cre : : Lama5 - / - ( arrow heads ) and larger adhesion complexes in Lama4 - / - vessels ( arrow heads ) . Scale bar is 10 µm . Data are means ± s . e . m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice / genotype . * * * P < 0 . 001 , * * * * P < 0 . 0001 , unpaired t - test .

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B) Vinculin immunofluorescence staining of whole mount wild type, Tek-Cre::Lama5-/- and Lama4-/- mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek-Cre::Lama5-/- (arrow heads) and larger adhesion complexes in Lama4-/- vessels (arrow heads). Scale bar is 10 µm. Data are means ± s.e.m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice/genotype. ***P<0.001, ****P<0.0001, unpaired t-test.


B ) Vinculin immunofluorescence staining of whole mount wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek - Cre : : Lama5 - / - ( arrow heads ) and larger adhesion complexes in Lama4 - / - vessels ( arrow heads ) . Scale bar is 10 µm . Correpsonding quantification of sizes ( C ) and frequency distribution ( D ) of adhesion complexes per endothelial cell . Data are means ± s . e . m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice / genotype . * * * P < 0 . 001 , * * * * P < 0 . 0001 , unpaired t - test .

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B) Vinculin immunofluorescence staining of whole mount wild type, Tek-Cre::Lama5-/- and Lama4-/- mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek-Cre::Lama5-/- (arrow heads) and larger adhesion complexes in Lama4-/- vessels (arrow heads). Scale bar is 10 µm. Correpsonding quantification of sizes (C) and frequency distribution (D) of adhesion complexes per endothelial cell. Data are means ± s.e.m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice/genotype. ***P<0.001, ****P<0.0001, unpaired t-test.


E , F ) The endothelium of excised wild type , Tek - Cre : : Lama5 - / - aortae were analysed by AFM , revealing reduced cortical stiffness in Tek - Cre : : Lama5 - / - vessels ( - 9 . 5 % ) . Data are means ± s . e . m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment . * P < 0 . 05 , * * * * P < 0 . 0001 , unpaired t - test .

E, F) The endothelium of excised wild type, Tek-Cre::Lama5-/- aortae were analysed by AFM, revealing reduced cortical stiffness in Tek-Cre::Lama5-/- vessels (-9.5%). Data are means ± s.e.m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment. *P<0.05, ****P<0.0001, unpaired t-test.


E , F ) The endothelium of excised wild type and Lama4 - / - aortae were analysed by AFM , revealing increased cortical stiffness in Lama4 - / - vessels ( + 2 % ) . Data are means ± s . e . m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment . * P < 0 . 05 , * * * * P < 0 . 0001 , unpaired t - test .

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E, F) The endothelium of excised wild type and Lama4-/- aortae were analysed by AFM, revealing increased cortical stiffness in Lama4-/- vessels (+2%). Data are means ± s.e.m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment. *P<0.05, ****P<0.0001, unpaired t-test.


G ) In vitro AFM measurements of cortical stiffness performed on HUAECs plated on 30 nM purified laminin 511 or 111 , reveal increased cortical stiffness in cells on laminin 511 . Data are mean ± s . e . m from 3 experiments with triplicates / experiments , * P < 0 . 05 , unpaired t - test .

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G) In vitro AFM measurements of cortical stiffness performed on HUAECs plated on 30 nM purified laminin 511 or 111, reveal increased cortical stiffness in cells on laminin 511. Data are mean ± s.e.m from 3 experiments with triplicates/experiments, *P<0.05, unpaired t-test.


A ) A dual pipette - pulling assay was employed to measure cell - cell adhesion strength in HUAECs bound to laminin - coated beads ( asterisk ) or in the absence of beads . Scale bar is 10 µm . B ) Quantification shows higher adhesion strength between cells bound to laminin 511 - coated beads compared to laminin 111 - coated beads or cells not incubated with beads . Measurements could not be made with cells incubated with laminin 411 - coated beads since it was impossible to find cell - bead complexes . Values are means ± s . e . m from 15 cells from 3 independent experiments , unpaired t - test with Welch ' s correction .

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A) A dual pipette-pulling assay was employed to measure cell-cell adhesion strength in HUAECs bound to laminin-coated beads (asterisk) or in the absence of beads. Scale bar is 10 µm. B) Quantification shows higher adhesion strength between cells bound to laminin 511-coated beads compared to laminin 111-coated beads or cells not incubated with beads. Measurements could not be made with cells incubated with laminin 411-coated beads since it was impossible to find cell-bead complexes. Values are means ± s.e.m from 15 cells from 3 independent experiments, unpaired t-test with Welch's correction.


C ) Dual pipette - pulling assay performed in the presence of VE - cadherin blocking antibody , isotope control and soluble laminin 511 , showing almost complete ablation of adhesion strength in the presence of the blocking antibody in all conditions . Similar adhesion strengths were measured between cells in the presence of soluble laminin 511 and cells bound to laminin 511 - coated beads .

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C) Dual pipette-pulling assay performed in the presence of VE-cadherin blocking antibody, isotope control and soluble laminin 511, showing almost complete ablation of adhesion strength in the presence of the blocking antibody in all conditions. Similar adhesion strengths were measured between cells in the presence of soluble laminin 511 and cells bound to laminin 511-coated beads.


D ) Adhesion strength in the presence of soluble laminin 511 was significantly reduced in the presence of integrin β1 blocking antibody ; cell - cell adhesion strength between cells bound to laminin 511 coated beads was significantly reduced in the presence of ROCK , SRC or FAK inhibitors . Values are means ± s . e . m from 11 cells from 3 independent experiments , unpaired t - test with Welch ' s correction . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , n . s . = not significant , n . d . = not determined .

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D) Adhesion strength in the presence of soluble laminin 511 was significantly reduced in the presence of integrin β1 blocking antibody; cell-cell adhesion strength between cells bound to laminin 511 coated beads was significantly reduced in the presence of ROCK, SRC or FAK inhibitors. Values are means ± s.e.m from 11 cells from 3 independent experiments, unpaired t-test with Welch's correction. *P <0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s. = not significant, n.d. = not determined.


A ) Immunofluorescence - based antibody feeding assay performed using HUAECs seeded on laminin 411 , 511 or 111 - coated coverslips , reveals more VE - cadherin at junctions and less in vesicles in cells plated on laminin 511 ( arrow heads mark VE - cadherin positive vesicles ) . Scale bar is 10 µm . B ) Quantification of VE - cadherin positive vesicles / cell in HUAECs plated on laminin 411 , 511 or 111 . Values are means ± s . e . m from 300 cells from 3 independent experiments , unpaired t - test .

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

A) Immunofluorescence-based antibody feeding assay performed using HUAECs seeded on laminin 411, 511 or 111-coated coverslips, reveals more VE-cadherin at junctions and less in vesicles in cells plated on laminin 511 (arrow heads mark VE-cadherin positive vesicles). Scale bar is 10 µm. B) Quantification of VE-cadherin positive vesicles/ cell in HUAECs plated on laminin 411, 511 or 111. Values are means ± s.e.m from 300 cells from 3 independent experiments, unpaired t-test.


C ) Western blot of biotinylated ( surface ) VE - cadherin in HUAECs plated on different laminins versus total VE - cadherin ( input ) confirms more junctionally located VE - cadherin in cells bound to laminin 511 .

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

C) Western blot of biotinylated (surface) VE-cadherin in HUAECs plated on different laminins versus total VE-cadherin (input) confirms more junctionally located VE-cadherin in cells bound to laminin 511.


D ) Quantification of Western blots expressed as relative signal proportions ( surface VE - cadherin / total VE - cadherin ) . Values shown are means ± s . e . m of 6 experiments , unpaired t - test .

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D) Quantification of Western blots expressed as relative signal proportions (surface VE-cadherin/ total VE-cadherin). Values shown are means ± s.e.m of 6 experiments, unpaired t-test.


E ) Western blot of p120 catenincoimmunoprecipitated with VE - cadherin ( input ) from HUAECs plated on different laminins Asterisks are isotope controls . Values shown are means ± s . e . m of 4 experiments , Mann - Whitney test .

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E) Western blot of p120 catenincoimmunoprecipitated with VE-cadherin (input) from HUAECs plated on different laminins Asterisks are isotope controls. Values shown are means ± s.e.m of 4 experiments, Mann-Whitney test.


E ) Western blot of p120 catenin coimmunoprecipitated with VE - cadherin ( input ) from HUAECs plated on different laminins , and ( F ) corresponding quantification of the p120 signal intensity relative to the total VE - cadherin signal . Asterisks are isotope controls . Values shown are means ± s . e . m of 4 experiments , Mann - Whitney test .

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

E) Western blot of p120 catenin coimmunoprecipitated with VE-cadherin (input) from HUAECs plated on different laminins, and (F) corresponding quantification of the p120 signal intensity relative to the total VE-cadherin signal. Asterisks are isotope controls. Values shown are means ± s.e.m of 4 experiments, Mann-Whitney test.


A ) Double immunofluorescence staining for phosphorylated myosin light chain II ( pMLC II ) and VE - cadherin in mesenteric resistance artery endothelium from wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice ( arrow heads ) . Scale bar is 10µm . B ) Quantification of the pMLC II signal along VE - cadherin signal revealed significantly less overlap in Tek - Cre : : Lama5 - / - and more overlap in Lama4 - / - endothelium , compared with wild type controls . The data are normalized to the skeletal length of the VE - cadherin signal . Values shown are means ± s . e . m resulting from quantification of 4 field of view per arteries , 4 mice / genotype . * P < 0 . 05 , * * P < 0 . 01 , paired t - test .

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A) Double immunofluorescence staining for phosphorylated myosin light chain II (pMLC II) and VE-cadherin in mesenteric resistance artery endothelium from wild type, Tek-Cre::Lama5-/- and Lama4-/- mice (arrow heads). Scale bar is 10µm. B) Quantification of the pMLC II signal along VE-cadherin signal revealed significantly less overlap in Tek-Cre::Lama5-/- and more overlap in Lama4-/- endothelium, compared with wild type controls. The data are normalized to the skeletal length of the VE-cadherin signal. Values shown are means ± s.e.m resulting from quantification of 4 field of view per arteries, 4 mice /genotype. *P<0.05, **P<0.01, paired t-test.


A Purified TAP - tagged Rik1 - containing complexes analyzed by SDS - PAGE and silver staining . mock : a mock purification from an untagged strain . Proteins identified by LC - MS / MS are indicated at right , with the number of identified unique peptides in parentheses .

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A Purified TAP-tagged Rik1-containing complexes analyzed by SDS-PAGE and silver staining. mock: a mock purification from an untagged strain. Proteins identified by LC-MS/MS are indicated at right, with the number of identified unique peptides in parentheses.


B In vitro ubiquitylation assays using biotinylated ubiquitin , purified CLRC , and recombinant S . pombe histone H3 as the substrate . Proteins were analyzed by Western blotting ( WB ) using the indicated antibodies . Asterisks indicate ubiquitylated histone H3 species .

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

B In vitro ubiquitylation assays using biotinylated ubiquitin, purified CLRC, and recombinant S. pombe histone H3 as the substrate. Proteins were analyzed by Western blotting (WB) using the indicated antibodies. Asterisks indicate ubiquitylated histone H3 species.


C Ubiquitylation assays using recombinant histone H3 , the N - terminal tail of histone H3 ( residues 1 - 36 ) fused with GST ( H3N - GST ) , or GST alone as the substrate . Both fission yeast ( sp ) and human ( hs ) full - length H3 proteins and H3N - GST fusion proteins were examined . The proteins were analyzed by SDS - PAGE followed by either silver staining or Western blotting with an anti - biotin antibody . Asterisks indicate ubiquitylated histone H3 species .

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C Ubiquitylation assays using recombinant histone H3, the N-terminal tail of histone H3 (residues 1-36) fused with GST (H3N-GST), or GST alone as the substrate. Both fission yeast (sp) and human (hs) full-length H3 proteins and H3N-GST fusion proteins were examined. The proteins were analyzed by SDS-PAGE followed by either silver staining or Western blotting with an anti-biotin antibody. Asterisks indicate ubiquitylated histone H3 species.


A Ubiquitylation assay using H3N - GST . The components included in the assay and the ubiquitylated H3N - GST species are indicated at right .

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A Ubiquitylation assay using H3N-GST. The components included in the assay and the ubiquitylated H3N-GST species are indicated at right.


B MS / MS spectrum of the histone H3 peptide corresponding to residues 10 - 17 . The observed y and b ions and fragment map are shown .

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B MS/MS spectrum of the histone H3 peptide corresponding to residues 10-17. The observed y and b ions and fragment map are shown.


C Ubiquitylation assay using biotinylated ubiquitin and recombinant wild - type H3N - GST ( WT ) and arginine - substituted H3N - GST mutants as substrates . Proteins were analyzed by Western blotting and silver staining . mock : reaction without substrate . Asterisks indicate ubiquitylated H3N - GST proteins .

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C Ubiquitylation assay using biotinylated ubiquitin and recombinant wild-type H3N-GST (WT) and arginine-substituted H3N-GST mutants as substrates. Proteins were analyzed by Western blotting and silver staining. mock: reaction without substrate. Asterisks indicate ubiquitylated H3N-GST proteins.


A Chromatin precipitated with anti - H3K4me2 or anti - H3K9me2 antibodies was analyzed by SDS - PAGE and silver staining . The input sample and mock precipitation using total mouse IgG are also shown . The gel slices indicated by boxes were excised and subjected to LC - MS / MS analysis .

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A Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was analyzed by SDS-PAGE and silver staining. The input sample and mock precipitation using total mouse IgG are also shown. The gel slices indicated by boxes were excised and subjected to LC-MS/MS analysis.


MS / MS spectrum of the histone H3 peptide corresponding to residues 9 - 17 . The observed y and b ions and fragment map are shown . C Summary of the ubiquitylated peptides identified by LC - MS / MS .

MS/MS spectrum of the histone H3 peptide corresponding to residues 9-17. The observed y and b ions and fragment map are shown. C Summary of the ubiquitylated peptides identified by LC-MS/MS.


D Relative abundance of ubiquitylated lysine residues in chromatin - associated histone H3 fractions . Chromatin precipitated with anti - H3K4me2 or anti - H3K9me2 antibodies was subjected to LC - MS / MS analysis , and the abundance of ubiquitylated lysine residues in each fraction is shown Since short peptides cleaved at unmodified lysine residues could not be efficiently detected in the LC - MS / MS analysis , these data do not accurately reflect the abundance including unmodified lysines .

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D Relative abundance of ubiquitylated lysine residues in chromatin-associated histone H3 fractions. Chromatin precipitated with anti-H3K4me2 or anti-H3K9me2 antibodies was subjected to LC-MS/MS analysis, and the abundance of ubiquitylated lysine residues in each fraction is shown Since short peptides cleaved at unmodified lysine residues could not be efficiently detected in the LC-MS/MS analysis, these data do not accurately reflect the abundance including unmodified lysines.


B Heterochromatic silencing assays of wild - type and mutant Hht1 - FH strains . Silencing at the mating - type Kint2 : : ura4 + was evaluated . Ten - fold serial dilutions of the indicated strains were spotted onto non - selective medium ( N / S ) , medium lacking uracil ( - Ura ) , and medium containing 5 - FOA ( 5 - FOA ) .

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B Heterochromatic silencing assays of wild-type and mutant Hht1-FH strains. Silencing at the mating-type Kint2::ura4+ was evaluated. Ten-fold serial dilutions of the indicated strains were spotted onto non-selective medium (N/S), medium lacking uracil (-Ura), and medium containing 5-FOA (5-FOA).


C ChIP analysis of H3K9me2 levels associated with the mating - type loci , relative to the control act1 + locus . Error bars indicate standard errors from three technical replicates .

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C ChIP analysis of H3K9me2 levels associated with the mating-type loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.


D ChIP analysis of Rik1 - myc levels associated with the mating - type cenH and centromeric dg loci , relative to the control act1 + locus . Error bars indicate standard errors from three technical replicates .

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D ChIP analysis of Rik1-myc levels associated with the mating-type cenH and centromeric dg loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.


E Heterochromatic silencing assays comparing the effects of H3 lysine substitution mutants . Silencing at the mating - type kint2 : : ura4 + was

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E Heterochromatic silencing assays comparing the effects of H3 lysine substitution mutants. Silencing at the mating-type kint2::ura4+ was evaluated


In vitro HMTase assays . Recombinant H3N - GST ( WT or K9R ) pre - ubiquitylated by the CLRC was purified and used in the HMTase assay with 6 × His - tagged recombinant Clr4 ( His - Clr4 ) . Proteins were analyzed by SDS - PAGE and Coomassie Brilliant Blue ( CBB ) staining ( left ) and autoradiography ( right ) .

In vitro HMTase assays. Recombinant H3N-GST (WT or K9R) pre-ubiquitylated by the CLRC was purified and used in the HMTase assay with 6×His-tagged recombinant Clr4 (His-Clr4). Proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue (CBB) staining (left) and autoradiography (right).


C , D In vitro HMTase assay using disulfide - linked ubiquitylated histone H3 . H3K14CUb and control histone H3 ( H3C110A ) were used as substrates in the HMTase assay , and analyzed The 3H - methyl group incorporation was measured using a liquid scintillation counter ( D ) . Error bars indicate the standard deviations calculated from three independent experiments .

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C,D In vitro HMTase assay using disulfide-linked ubiquitylated histone H3. H3K14CUb and control histone H3 (H3C110A) were used as substrates in the HMTase assay, and analyzed The 3H-methyl group incorporation was measured using a liquid scintillation counter (D). Error bars indicate the standard deviations calculated from three independent experiments.


B Recombinant proteins were resolved by 10 % SDS - PAGE and visualized by CBB staining .

B Recombinant proteins were resolved by 10% SDS-PAGE and visualized by CBB staining.


C In vitro HMTase assay using disulfide - linked ubiquitylated histone H3 . H3K14CUb and control histone H3 ( H3C110A ) were used as substrates in the HMTase assay , and

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C In vitro HMTase assay using disulfide-linked ubiquitylated histone H3. H3K14CUb and control histone H3 (H3C110A) were used as substrates in the HMTase assay, and analyzed


D In vitro pulldown assays of GST - Clr4 proteins with biotinylated ubiquitin . Bound ubiquitin was analyzed by Western blotting with HRP - conjugated streptavidin ( left ) . Precipitated GST proteins were analyzed by SDS - PAGE and CBB staining ( right ) .

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D In vitro pulldown assays of GST-Clr4 proteins with biotinylated ubiquitin. Bound ubiquitin was analyzed by Western blotting with HRP-conjugated streptavidin (left). Precipitated GST proteins were analyzed by SDS-PAGE and CBB staining (right).


F Whole cell lysates prepared from control cells , cells expressing FLAG - tagged wild - type Clr4 ( F - clr4 ) or mutant Clr4 ( F - clr4 ∆ 63 - 127 ) , or clr4 ∆ cells were subjected to immunoblotting using the indicated antibodies .

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F Whole cell lysates prepared from control cells, cells expressing FLAG-tagged wild-type Clr4 (F-clr4) or mutant Clr4 (F-clr4∆63-127), or clr4∆ cells were subjected to immunoblotting using the indicated antibodies.


G Heterochromatic silencing assays of wild - type and clr4 mutant strains . Silencing at the mating - type Kint2 : : ura4 + was evaluated .

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G Heterochromatic silencing assays of wild-type and clr4 mutant strains. Silencing at the mating-type Kint2::ura4+ was evaluated.


H ChIP analysis of H3K9me2 levels associated with the silencing marker gene inserted in the mating - type loci ( Kint2 : : ura4 + ) and the mating - type cenH and centromeric dg loci , relative to the control act1 + locus . Error bars indicate standard errors from three technical replicates .

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H ChIP analysis of H3K9me2 levels associated with the silencing marker gene inserted in the mating-type loci (Kint2::ura4+) and the mating-type cenH and centromeric dg loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.


I The levels of Kint2 : : ura4 + , mating - type cenH , and centromeric dg transcripts were quantified by RT - qPCR and calculated as fold change to the wild - type expression . Error bars indicate standard errors from three technical replicates .

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I The levels of Kint2::ura4+, mating-type cenH, and centromeric dg transcripts were quantified by RT-qPCR and calculated as fold change to the wild-type expression. Error bars indicate standard errors from three technical replicates.


A Representative images of Bem1 - GFP and the PS marker LactC2 - GFP in wild type and cho1 ∆ cells . Images show average intensity projections of deconvolved z - stacks . Black and white pixels were inverted to improve contrast for display . B Frequency of cells displaying polarized Bem1 - GFP signal in wild type and cho1 ∆ cells . Values are means ± SD , n > 100 cells in each of 3 independent experiments . Paired Student ' s t - tests were performed .

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A Representative images of Bem1-GFP and the PS marker LactC2-GFP in wild type and cho1∆ cells. Images show average intensity projections of deconvolved z-stacks. Black and white pixels were inverted to improve contrast for display. B Frequency of cells displaying polarized Bem1-GFP signal in wild type and cho1∆ cells. Values are means ± SD, n > 100 cells in each of 3 independent experiments. Paired Student's t-tests were performed.


C Imaging of the PI4P probe GFP - 2xPHOsh2 in wild type and cho1 ∆ cells . Images show average intensity projections of deconvolved z - stacks in which black and white pixels were inverted .

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C Imaging of the PI4P probe GFP-2xPHOsh2 in wild type and cho1∆ cells. Images show average intensity projections of deconvolved z-stacks in which black and white pixels were inverted.


D Scatter dot plot showing PI4P levels at the plasma membrane ( see experimental procedure for details of the quantification ) in wild type and cho1 ∆ cells . Values are means ± SD , n > 100 cells , observed over 3 experiments . Mann - Whitney tests were performed .

D Scatter dot plot showing PI4P levels at the plasma membrane (see experimental procedure for details of the quantification) in wild type and cho1∆ cells. Values are means ± SD, n > 100 cells, observed over 3 experiments. Mann-Whitney tests were performed.


E Ratio of PI4P average fluorescence intensity in the bud versus mother cell in the cho1 ∆ mutant . Values are means ± SD , n > 100 cells , observed over 3 experiments . A value > 1 indicates enrichment of the fluorescence in the bud .

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E Ratio of PI4P average fluorescence intensity in the bud versus mother cell in the cho1∆ mutant. Values are means ± SD, n > 100 cells, observed over 3 experiments. A value > 1 indicates enrichment of the fluorescence in the bud.


F Images of Bem1 - GFP in cho1 ∆ 9xMyc - AID - stt4 cells after 30 min treatment with or without 0 . 5 mM auxin . Images are average intensity projections of deconvolved z - stacks .

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F Images of Bem1-GFP in cho1∆ 9xMyc-AID-stt4 cells after 30 min treatment with or without 0.5 mM auxin. Images are average intensity projections of deconvolved z-stacks.


G Frequency of cells with polarized Bem1 - GFP signal or Bem1 - GFP in puncta in cho1 ∆ 9xMyc - AID - stt4 cells treated with or without auxin as shown in F . Values are means ± SD , n > 100 cells in each of 6 independent experiments . Paired Student ' s t - tests were performed .

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G Frequency of cells with polarized Bem1-GFP signal or Bem1-GFP in puncta in cho1∆ 9xMyc-AID-stt4 cells treated with or without auxin as shown in F. Values are means ± SD, n > 100 cells in each of 6 independent experiments. Paired Student's t-tests were performed.


H Images of Bem1 - GFP ( cyan ) and Cdc24 - mCherry ( red ) signals in cho1 ∆ 9xMyc - AID - stt4 cells with or without auxin . Images display maximum intensity projections of z - stacks .

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H Images of Bem1-GFP (cyan) and Cdc24-mCherry (red) signals in cho1∆ 9xMyc-AID-stt4 cells with or without auxin. Images display maximum intensity projections of z-stacks.


I Maximum intensity projections of z - stacks displaying Bem1 - GFP in the mutants indicated . Bem1 - GFP was imaged in mss4 - 102 cho1 ∆ cells after shifting to the restrictive temperature for 30 minutes .

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I Maximum intensity projections of z-stacks displaying Bem1-GFP in the mutants indicated. Bem1-GFP was imaged in mss4-102 cho1∆ cells after shifting to the restrictive temperature for 30 minutes.


J Frequency of cells with polarized Bem1 - GFP signal after incubation at the time and temperature shown . Values are means ± SD , n > 200 cells in each of 3 independent experiments . Paired Student ' s t - tests were performed .

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J Frequency of cells with polarized Bem1-GFP signal after incubation at the time and temperature shown. Values are means ± SD, n > 200 cells in each of 3 independent experiments. Paired Student's t-tests were performed.


B Upper panel . SDS - PAGE stained with Coomassie blue in which Bem1 is indicated . Lower panel . The liposomes were composed of 100 % phosphatidylcholine ( PC ) , 80 % PC and 20 % phosphatidylethanolamine ( PE ) , 95 % PC and 5 % phosphatidic acid ( PA ) , 95 % PC and 5 % phosphoatidylinositol ( PI ) , 95 % PC and 5 % PI ( 4 , 5 ) P2 ( PI ( 4 , 5 ) P2 ) , 95 % PC and 5 % PI4P ( PI4P ) , 80 % PC and 20 % phosphatidylserine ( PS ) or 75 % PC 20 % PS 5 % PI4P ( PS + PI4P ) . ( S ) supernatant , ( P ) pellet .

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B Upper panel. SDS-PAGE stained with Coomassie blue in which Bem1 is indicated. Lower panel. The liposomes were composed of 100% phosphatidylcholine (PC), 80% PC and 20% phosphatidylethanolamine (PE), 95% PC and 5% phosphatidic acid (PA), 95% PC and 5% phosphoatidylinositol (PI), 95% PC and 5% PI(4,5)P2 (PI(4,5)P2), 95% PC and 5% PI4P (PI4P), 80% PC and 20% phosphatidylserine (PS) or 75% PC 20% PS 5% PI4P (PS+PI4P). (S) supernatant, (P) pellet.


C Liposomes composed of either 100 % PC ± 40 % ergosterol or 75 % PC , 20 % PS , 5 % Bem1 ± 40 % ergosterol replacing PC were incubated with Bem1 and treated as in panel ( B ) .

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C Liposomes composed of either 100% PC ± 40% ergosterol or 75% PC, 20% PS, 5% Bem1 ± 40% ergosterol replacing PC were incubated with Bem1 and treated as in panel (B).


E Percentage of the indicated bem1 constructs associated with liposomes containing 75 % PC , 20 % PS and 5 % PI4P .

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E Percentage of the indicated bem1 constructs associated with liposomes containing 75% PC, 20% PS and 5% PI4P.


F Lipid ordering determined by 2H solid - state NMR analysis of liposomes containing POPC - d31 / POPS ( 4 : 1 molar ratio ) in the presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) and simulation of 2H solid - state NMR spectra were applied to measure individual quadrupolar splittings for POPC - d31 and determine order parameter accurately .

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

F Lipid ordering determined by 2H solid-state NMR analysis of liposomes containing POPC-d31/POPS (4:1 molar ratio) in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately.


Lipid ordering determined by 2H solid - state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) and simulation of 2H solid - state NMR spectra were applied to measure individual quadrupolar splittings for POPC - d31 and determine order parameter accurately . containing POPC - d31 / PI4P ( 19 : 1 molar ratio ) .

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

Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/PI4P (19:1 molar ratio).


Lipid ordering determined by 2H solid - state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) and simulation of 2H solid - state NMR spectra were applied to measure individual quadrupolar splittings for POPC - d31 and determine order parameter accurately . containing POPC - d31 / POPC / PI4P ( 15 : 4 : 1 molar ratio ) .

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

Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/POPC/PI4P (15:4:1 molar ratio).


Lipid ordering determined by 2H solid - state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) and simulation of 2H solid - state NMR spectra were applied to measure individual quadrupolar splittings for POPC - d31 and determine order parameter accurately . containing POPC - d31 / POPS / Yeast lipid extract / ergosterol ( 1 : 1 : 1 : 2 molar ratio ) .

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 O O O O O

Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/POPS/Yeast lipid extract/ergosterol (1:1:1:2 molar ratio).


B Percentage of the different full - length bem1 bc mutants associated with liposomes containing 75 % PC 20 % PS and 5 % PI4P .

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B Percentage of the different full-length bem1 bc mutants associated with liposomes containing 75% PC 20% PS and 5% PI4P.


C Fluorescence intensity change associated with the nucleotide exchange of GDP - Cdc42 for mant - GTP Cdc42 . Fluorescence was measured after the addition of GDP - Cdc42 to reactions containing Mant - GTP ( 100 nM ) , GMP - PNP ( 100 µM ) and the proteins indicated .

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C Fluorescence intensity change associated with the nucleotide exchange of GDP-Cdc42 for mant-GTP Cdc42. Fluorescence was measured after the addition of GDP-Cdc42 to reactions containing Mant-GTP (100 nM), GMP-PNP (100 µM) and the proteins indicated.


D Ten - fold serial dilutions of cells and subsequent colony formation on the indicated plates , where expression of wild type GALp - CDC42 is either induced in the presence of Gal or repressed in Dex . Note how mutation of the wild type Cdc42 ( KKSKK ) to MMSMM is lethal ( see blue box ) , whereas appending the Bem1 BC - 1 motif to this cdc42 mutant restores viability ( red box ) .

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D Ten-fold serial dilutions of cells and subsequent colony formation on the indicated plates, where expression of wild type GALp-CDC42 is either induced in the presence of Gal or repressed in Dex. Note how mutation of the wild type Cdc42 (KKSKK) to MMSMM is lethal (see blue box), whereas appending the Bem1 BC-1 motif to this cdc42 mutant restores viability (red box).


E Representative images of the mEOS - cdc42 mutants signal ( cyan ) after inducing the expression of GAL1p - CDC42 in the presence of Gal or repressing it in the presence of Dex . The cells in the blue and red boxes correspond to the cells in the blue and red box in panel ( D ) . Images are average fluorescence intensity projections in which black and white pixels have been inverted .

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E Representative images of the mEOS-cdc42 mutants signal (cyan) after inducing the expression of GAL1p-CDC42 in the presence of Gal or repressing it in the presence of Dex. The cells in the blue and red boxes correspond to the cells in the blue and red box in panel (D). Images are average fluorescence intensity projections in which black and white pixels have been inverted.


A Ten - fold serial dilutions of the indicated mutant cells and subsequent colony formation at the temperatures indicated . Note how mutations on both the BC motifs and PX in bem1 compromise growth at the restrictive temperature .

A Ten-fold serial dilutions of the indicated mutant cells and subsequent colony formation at the temperatures indicated. Note how mutations on both the BC motifs and PX in bem1 compromise growth at the restrictive temperature.


B Western blots probed with anti - HA antibody to detect the indicated bem1 constructs tagged with 3XHA ( top panel ) . Detection of Ade13 was used as loading control ( bottom panel ) .

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B Western blots probed with anti-HA antibody to detect the indicated bem1 constructs tagged with 3XHA (top panel). Detection of Ade13 was used as loading control (bottom panel).


C Representative images of the indicated bem1 mutants tagged with GFP at the BEM1 locus . The inverted fluorescence images are average intensity projections .

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C Representative images of the indicated bem1 mutants tagged with GFP at the BEM1 locus. The inverted fluorescence images are average intensity projections.


D Frequency of cells with polarized Bem1 - GFP . E Scatter dot plot showing the fluorescence levels of the indicated bem1 strains at the cell pole . The measurements were made on average intensity projections .

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D Frequency of cells with polarized Bem1-GFP. E Scatter dot plot showing the fluorescence levels of the indicated bem1 strains at the cell pole. The measurements were made on average intensity projections.


B Percentage of full - length Cdc24 and cdc24 ph domain mutant ( cdc24 ph ) associated with liposomes containing 75 % PC 20 % PS and 5 % PI4P .

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B Percentage of full-length Cdc24 and cdc24 ph domain mutant (cdc24 ph) associated with liposomes containing 75% PC 20% PS and 5% PI4P.


C Percentage of Cdc24 and cdc24 ph mutant associated with liposomes of the composition shown in ( B ) in the presence of the indicated bem1 protein . Note how additive mutations in the Bem1 lipid binding sequences reduce the percentage of Cdc24 associated with the liposomes .

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C Percentage of Cdc24 and cdc24 ph mutant associated with liposomes of the composition shown in (B) in the presence of the indicated bem1 protein. Note how additive mutations in the Bem1 lipid binding sequences reduce the percentage of Cdc24 associated with the liposomes.


D Percentage of Cdc24 and cdc24 ph associated with liposomes of the composition shown in ( B ) as a function of the indicated bem1 protein concentration . The curves denote the regression fit of the data to equation 2 in the materials and methods .

O O O O O O O O O O O O O O O O O O O O O O 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 Percentage of Cdc24 and cdc24 ph associated with liposomes of the composition shown in (B) as a function of the indicated bem1 protein concentration. The curves denote the regression fit of the data to equation 2 in the materials and methods.


E Ten - fold serial dilutions and subsequent colony formation of the indicated mutant cells at the indicated temperatures .

E Ten-fold serial dilutions and subsequent colony formation of the indicated mutant cells at the indicated temperatures.


F DIC images of the indicated bem1 and cdc24 mutants showing the increased morphological defects ensuing from loss of lipid tethering in the bem1 and cdc24 mutants .

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F DIC images of the indicated bem1 and cdc24 mutants showing the increased morphological defects ensuing from loss of lipid tethering in the bem1 and cdc24 mutants.


G Western blots probed with anti - HA antibody to detect Cdc24 or cdc24 ph tagged with 3XHA in the indicated mutant strains ( top panel ) . Detection of Ade13 was used as a loading control ( bottom panel ) .

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

G Western blots probed with anti-HA antibody to detect Cdc24 or cdc24 ph tagged with 3XHA in the indicated mutant strains (top panel). Detection of Ade13 was used as a loading control (bottom panel).


B Global average MSD curves of mEOS - Cdc42 in the strains indicated at the Pole ( P ) and Non - Pole ( NP ) of cells . Trajectories longer than 6 frames were analyzed . Number of trajectories analyzed : BEM1 ( n = 11 cells : Non Pole : 1814 tracks ; Pole : 706 ) ; bem1 bc - 14E px ( n = 10 cells : Non Pole : 1793 tracks ; Pole : 908 ) ; bem1 bc - 14E ( n = 13 cells : Non Pole : 1714 tracks ; Pole : 975 ) . Error bars display s . e . m . C D coefficients of mEOS - Cdc42 in the strains indicated ( in box - plots displaying the median ( line ) , the 25 - 75 percentiles ( box ) and the mean ( cross ) ) , which were compared using a non - parametric , two - tailed Mann - Whitney rank sum test .

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B Global average MSD curves of mEOS-Cdc42 in the strains indicated at the Pole (P) and Non-Pole (NP) of cells. Trajectories longer than 6 frames were analyzed. Number of trajectories analyzed: BEM1 (n = 11 cells: Non Pole: 1814 tracks; Pole: 706); bem1 bc-14E px (n = 10 cells: Non Pole: 1793 tracks; Pole: 908); bem1 bc-14E (n = 13 cells: Non Pole: 1714 tracks; Pole: 975). Error bars display s.e.m. C D coefficients of mEOS-Cdc42 in the strains indicated (in box-plots displaying the median (line), the 25-75 percentiles (box) and the mean (cross)), which were compared using a non-parametric, two-tailed Mann-Whitney rank sum test.


D SR - Tesseler images of mEOS - Cdc42 nanocluster organization in BEM1 ( 5312 localizations shown in image ) and bem1 bc - 14E px cells ( 3739 localizations shown ) . Insets show mEOS - Cdc42 after 491 nm widelfield laser excitation to identify the cell pole . Scale bars : 2 μm . A zoom of the pole region shows the organization of the detected nanoclusters , circled in light blue , in the strains indicated . Scale bar in the zoom : 60 nm .

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D SR-Tesseler images of mEOS-Cdc42 nanocluster organization in BEM1 (5312 localizations shown in image) and bem1 bc-14E px cells (3739 localizations shown). Insets show mEOS-Cdc42 after 491 nm widelfield laser excitation to identify the cell pole. Scale bars: 2 μm. A zoom of the pole region shows the organization of the detected nanoclusters, circled in light blue, in the strains indicated. Scale bar in the zoom: 60 nm.


E Distribution of nanocluster area at the pole ( P ) and non - pole ( NP ) regions of BEM1 ( diameter NP : 59 nm ± 1 nm ( s . e . m ) ; P : 74 nm ± 3 . 2 nm ) ( n = 16 cells . NP : 570 clusters ; P : 162 clusters ) and bem1 bc - 14E px cells ( diameter NP : 59 nm ± 1 . 5 nm ( s . e . m ) ; P : 57 nm ± 2 . 4 nm ) ( n = 15 cells . NP : 421 clusters ; P : 141 clusters ) . Data are presented as scatter dot - plots displaying the median as a line and the 25 - 75 percentiles . Data were compared using non - parametric , two - tailed Mann - Whitney rank sum test .

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E Distribution of nanocluster area at the pole (P) and non-pole (NP) regions of BEM1 (diameter NP: 59 nm ± 1 nm (s.e.m); P: 74 nm ± 3.2 nm) (n = 16 cells. NP: 570 clusters; P: 162 clusters) and bem1 bc-14E px cells (diameter NP: 59 nm ± 1.5 nm (s.e.m); P: 57 nm ± 2.4 nm) (n = 15 cells. NP: 421 clusters; P: 141 clusters). Data are presented as scatter dot-plots displaying the median as a line and the 25-75 percentiles. Data were compared using non-parametric, two-tailed Mann-Whitney rank sum test.


F Active Cdc42 - GTP levels were quantified in the strains indicated using a gic2 ( 1 - 208 ) - yeGFP probe . Values are mean ± SD for n = 30 cells observed over 2 experiments . Data were compared using a Mann - Whitney test .

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F Active Cdc42-GTP levels were quantified in the strains indicated using a gic2(1-208)-yeGFP probe. Values are mean ± SD for n = 30 cells observed over 2 experiments. Data were compared using a Mann-Whitney test.


Mitochondria - enriched fractions were isolated from HdhQ7 or HdhQ111 cells . mtHSF1 was examined by WB analysis . VDAC was analyzed as a loading control . ( n = 6 biological replicates ) .

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Mitochondria-enriched fractions were isolated from HdhQ7 or HdhQ111 cells. mtHSF1 was examined by WB analysis. VDAC was analyzed as a loading control. (n=6 biological replicates).


HdhQ7 WT striatal cells were exposed to 5 mM 3 - NP for 2 h . mtHSF1 levels were determined by immunoblotting . VDAC was used as a loading control . ( n = 5 biological replicates ) .

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HdhQ7 WT striatal cells were exposed to 5 mM 3-NP for 2 h. mtHSF1 levels were determined by immunoblotting. VDAC was used as a loading control. (n=5 biological replicates).


HSF1 was measured in the mitochondria - enriched fractions of the HD transgenic YAC128 mice ( 9 months old ) or age - matched littermates . ( n = 5 biological replicates ) .

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HSF1 was measured in the mitochondria-enriched fractions of the HD transgenic YAC128 mice (9 months old) or age-matched littermates. (n=5 biological replicates).


mtHSF1 was analyzed in HD patient fibroblasts ( GM04208 , GM04222 , and GM21756 ) and fibroblasts from healthy individuals . ( n = 5 for Con1 / GM04208 and Con2 / GM04222 groups ; n = 3 for Con3 / GM21756 group ) .

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mtHSF1 was analyzed in HD patient fibroblasts (GM04208, GM04222, and GM21756) and fibroblasts from healthy individuals. (n = 5 for Con1/ GM04208 and Con2/ GM04222 groups; n = 3 for Con3/ GM21756 group).


Representative immunoblot of HSF1 levels in mitochondria isolated from patient - derived iPSCs and control iPSCs . ( n = 3 biological replicates ) .

Representative immunoblot of HSF1 levels in mitochondria isolated from patient-derived iPSCs and control iPSCs. (n=3 biological replicates).


Representative images of striatal organoids . Left , immunostaining for the MSN marker DARPP32 ( green ) and the radial glial marker SOX2 ( red ) in striatal organoids at D60 . Middle , immunostaining for the LGE progenitor markers Ctip2 ( green ) and GSH2 ( red ) at D30 . Right panel , immunostaining for the neuronal marker NeuN ( green ) and the proliferation marker Ki67 ( red ) at D30 . The images are representative of 12 independent differentiation experiments of H9 and IMR90 - 4 cells . HO = Hoechst ( blue ) . The scale bar represents 100 µm .

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Representative images of striatal organoids. Left, immunostaining for the MSN marker DARPP32 (green) and the radial glial marker SOX2 (red) in striatal organoids at D60. Middle, immunostaining for the LGE progenitor markers Ctip2 (green) and GSH2 (red) at D30. Right panel, immunostaining for the neuronal marker NeuN (green) and the proliferation marker Ki67 (red) at D30. The images are representative of 12 independent differentiation experiments of H9 and IMR90-4 cells. HO= Hoechst (blue). The scale bar represents 100 µm.


Representative images and scatterplot confirming the increased HSF1 ( green ) translocation to mitochondria ( Tom20 , red ) . The data were obtained from 3 independent biological experiments . Con : n = 15 organoids ; HD : n = 14 organoids . Two - tailed unpaired t - test ; Means ± SEMs . Images were taken using Structured Illumination Microscopy ( SIM ) .

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Representative images and scatterplot confirming the increased HSF1 (green) translocation to mitochondria (Tom20, red). The data were obtained from 3 independent biological experiments. Con: n=15 organoids; HD: n=14 organoids. Two-tailed unpaired t-test; Means ± SEMs. Images were taken using Structured Illumination Microscopy (SIM).


The subcellular localization of HSF1 in HD striatal organoids was observed by immunoelectron microscopy . The arrows ( red ) mark HSF1 in mitochondria . The negative control lacked the HSF1 antibody . The scale bar represents 200 nm .

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The subcellular localization of HSF1 in HD striatal organoids was observed by immunoelectron microscopy. The arrows (red) mark HSF1 in mitochondria. The negative control lacked the HSF1 antibody. The scale bar represents 200 nm.


HTT was knocked down in HdhQ7 or HdhQ111 cells by lentiviral infection . The protein levels of HSF1 were detected in mitochondrial fractions . ( n = 3 biological replicates ) .

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HTT was knocked down in HdhQ7 or HdhQ111 cells by lentiviral infection. The protein levels of HSF1 were detected in mitochondrial fractions. (n=3 biological replicates).


HdhQ7 cells were transfected with Flag - mtHSF1 and stained with anti - Tom20 ( green ) and anti - Flag ( red ) antibodies . Mitochondrial morphology was examined by confocal microscopy . Scatterplot with bar showed the percentage of cells with mitochondrial fragmentation . The scale bar represents 40 µm . ( n = 3 biological replicates , at least 100 cells per group were counted ) .

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HdhQ7 cells were transfected with Flag-mtHSF1 and stained with anti-Tom20 (green) and anti-Flag (red) antibodies. Mitochondrial morphology was examined by confocal microscopy. Scatterplot with bar showed the percentage of cells with mitochondrial fragmentation. The scale bar represents 40 µm. (n=3 biological replicates, at least 100 cells per group were counted).


The total protein levels of p - Drp1 S616 , p - Drp1S637 , Drp1 , OPA1 , MFN2 , Flag - mtHSF1 and actin ( loading control ) were determined by WB analysis . ( n = 3 biological replicates ) .

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The total protein levels of p-Drp1 S616, p-Drp1S637, Drp1, OPA1, MFN2, Flag-mtHSF1 and actin (loading control) were determined by WB analysis. (n=3 biological replicates).


Brain sections were stained with an anti - Tom20 antibody ( red ) , an anti - Flag antibody ( green ) and DAPI ( blue ) . Mitochondrial morphology was analyzed in Flag + or Flag - regions by confocal microscopy . The scale bar presents 2 mm , and the enlarged image scale bar is 40 µm . ( n = 6 mice / group ) .

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Brain sections were stained with an anti-Tom20 antibody (red), an anti-Flag antibody (green) and DAPI (blue). Mitochondrial morphology was analyzed in Flag+ or Flag- regions by confocal microscopy. The scale bar presents 2 mm, and the enlarged image scale bar is 40 µm. (n=6 mice/group).


Striata injected with AAVs were lysed . p - Drp1 S616 was tested by immunoblotting . ( n = 5 mice / group ) .

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Striata injected with AAVs were lysed. p-Drp1 S616 was tested by immunoblotting. (n=5 mice/group).


Mitochondrial morphology was analyzed by electron microscopy ( EM ) in mice injected with AAV - Con or AAV - mtHSF1 . Scatterplot showed the length of mitochondria . The scale bar presents 4 µm , and the enlarged image scale bar is 400 nm . ( n = 3 mice / group ) . AAV - Con group , n = 268 mitochondria were measured ; AAV - Flag - mtHSF1 group , n = 179 mitochondria were measured .

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Mitochondrial morphology was analyzed by electron microscopy (EM) in mice injected with AAV-Con or AAV-mtHSF1. Scatterplot showed the length of mitochondria. The scale bar presents 4 µm, and the enlarged image scale bar is 400 nm. (n=3 mice/group). AAV-Con group, n=268 mitochondria were measured; AAV-Flag-mtHSF1 group, n=179 mitochondria were measured.


Representative images and scatterplot of mitochondrial length ( Tom20 , red ) in Flag + or Flag - cells . The data were obtained from 3 independent biological experiments . Flag - : n = 154 mitochondria ; Flag + : n = 132 mitochondria .

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Representative images and scatterplot of mitochondrial length (Tom20, red) in Flag+ or Flag- cells. The data were obtained from 3 independent biological experiments. Flag-: n=154 mitochondria; Flag+: n=132 mitochondria.


Representative images and scatterplot showing that the number of p - Drp1 S616 puncta in Flag + cells was greater than that in Flag - cells . The data were obtained from 3 independent biological experiments . Flag - : n = 70 cells from 10 organoids ; Flag + : n = 55 cells from 10 organoids . The images were obtained using structured illumination microscopy ( SIM ) .

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Representative images and scatterplot showing that the number of p-Drp1 S616 puncta in Flag+ cells was greater than that in Flag- cells. The data were obtained from 3 independent biological experiments. Flag-: n=70 cells from 10 organoids; Flag+: n=55 cells from 10 organoids. The images were obtained using structured illumination microscopy (SIM).


The mtDNA content was examined with primers of D - loop , mtCO1 , mtCO2 and mtND2 by qPCR in control or Flag - mtHSF1 - expressing striatal cells . Scatterplot with bar showed the relative percentage of mtDNA content . ( n = 3 biological replicates ) .

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The mtDNA content was examined with primers of D-loop, mtCO1, mtCO2 and mtND2 by qPCR in control or Flag-mtHSF1-expressing striatal cells. Scatterplot with bar showed the relative percentage of mtDNA content. (n=3 biological replicates).


Measurement of mitochondrial respiratory activity by Seahorse analysis . ( n = 9 - 10 biological replicates )

Measurement of mitochondrial respiratory activity by Seahorse analysis. (n=9-10 biological replicates)


Cells were treated with EtBr for six days to delete mtDNA . mtDNA repopulation efficiency was measured by qPCR after withdrawal of EtBr . ( n = 3 biological replicates ) .

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Cells were treated with EtBr for six days to delete mtDNA. mtDNA repopulation efficiency was measured by qPCR after withdrawal of EtBr. (n=3 biological replicates).


D - E . Cell death was determined by detection of LDH release into the medium and cleavage of PARP . Scatterplot with bar showed the relative percentage of LDH release . ( n = 3 biological replicates ) .

D-E. Cell death was determined by detection of LDH release into the medium and cleavage of PARP. Scatterplot with bar showed the relative percentage of LDH release. (n=3 biological replicates).


qPCR analysis of the mtDNA content of AAV - Con - injected mice or AAV - mtHSF1 - injected mice . Scatterplot with bar showed the relative percentage of mtDNA content ( n = 3 mice / group ) .

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qPCR analysis of the mtDNA content of AAV-Con-injected mice or AAV-mtHSF1-injected mice. Scatterplot with bar showed the relative percentage of mtDNA content (n=3 mice/group).


Cleaved - Caspase3 in AAV - Con - or AAV - mtHSF1 - injected mice was detected by WB analysis . ( n = 5 mice / group ) .

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Cleaved-Caspase3 in AAV-Con- or AAV-mtHSF1-injected mice was detected by WB analysis. (n=5 mice/group).


Representative images and scatterplot showing the number of mtDNA ( purple ) colocalized with mitochondria ( Tom20 , green ) in striatal organoids injected with AAV - Con or AAV - mtHSF1 . The data were obtained from 3 independent biological experiments . Con : n = 65 cells from 20 organoids ; Flag - mtHSF1 : n = 70 cells from 20 organoids . Two - tailed unpaired t - test was used . Images were captured by Structured Illumination Microscopy ( SIM ) .

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Representative images and scatterplot showing the number of mtDNA (purple) colocalized with mitochondria (Tom20, green) in striatal organoids injected with AAV-Con or AAV-mtHSF1. The data were obtained from 3 independent biological experiments. Con: n=65 cells from 20 organoids; Flag-mtHSF1: n=70 cells from 20 organoids. Two-tailed unpaired t-test was used. Images were captured by Structured Illumination Microscopy (SIM).


The typical nuclear fragmentation morphology of AAV - Con - ( GFP ) or AAV - mtHSF1 - injected striatal organoids was measured . Scatterplot showed the quantitative result of nuclear fragmentation . The arrows ( yellow ) mark fragmented nuclei . Two - tailed unpaired t - test was used . The data were obtained from 3 independent biological experiments . Con : n = 14 organoids ; Flag - mtHSF1 : n = 15 organoids .

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The typical nuclear fragmentation morphology of AAV-Con- (GFP) or AAV-mtHSF1-injected striatal organoids was measured. Scatterplot showed the quantitative result of nuclear fragmentation. The arrows (yellow) mark fragmented nuclei. Two-tailed unpaired t-test was used. The data were obtained from 3 independent biological experiments. Con: n=14 organoids; Flag-mtHSF1: n=15 organoids.


Total cell lysates were subjected to immunoprecipitation with an anti - HSF1 antibody followed by WB analysis . ( n = 3 biological replicates ) .

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Total cell lysates were subjected to immunoprecipitation with an anti-HSF1 antibody followed by WB analysis. (n=3 biological replicates).


HdhQ7 or HdhQ111 cell extracts were incubated with DSS ( 1 mM ) for 30 min at RT . SSBP1 oligomers were detected by immunoblotting with an anti - SSBP1 antibody . ( n = 3 biological replicates ) .

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HdhQ7 or HdhQ111 cell extracts were incubated with DSS (1 mM) for 30 min at RT. SSBP1 oligomers were detected by immunoblotting with an anti-SSBP1 antibody. (n=3 biological replicates).


SSBP1 oligomers in control and patient fibroblasts ( GM04208 and GM21756 ) were analyzed . ( n = 3 biological replicates ) .

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SSBP1 oligomers in control and patient fibroblasts (GM04208 and GM21756) were analyzed. (n=3 biological replicates).


SSBP1 oligomers were measured in WT mice or YAC128 mice . ( n = 3 mice / group ) .

SSBP1 oligomers were measured in WT mice or YAC128 mice. (n=3 mice/group).


Cells were transfected with an empty vector or Flag - mtHSF1 for 48 h . The total cell lysates were harvested and crosslinked with DSS . SSBP1 oligomers were tested by immunoblotting . ( n = 4 biological replicates ) .

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Cells were transfected with an empty vector or Flag-mtHSF1 for 48 h. The total cell lysates were harvested and crosslinked with DSS. SSBP1 oligomers were tested by immunoblotting. (n=4 biological replicates).


Flag - mtHSF1 was expressed in the striata of WT mice for three weeks . SSBP1 oligomers were determined by immunoblotting . ( n = 5 mice / group ) .

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Flag-mtHSF1 was expressed in the striata of WT mice for three weeks. SSBP1 oligomers were determined by immunoblotting. (n=5 mice/group).


Flag - mtHSF1 or sh - SSBP1 was delivered into cells via lentiviral infection . The expression of SSBP1 and mtHSF1 was detected by WB analysis . The mtDNA content was examined by qPCR analysis . ( n = 3 biological replicates ) .

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Flag-mtHSF1 or sh-SSBP1 was delivered into cells via lentiviral infection. The expression of SSBP1 and mtHSF1 was detected by WB analysis. The mtDNA content was examined by qPCR analysis. (n=3 biological replicates).


Primary neurons expressing Flag - mtHSF1 were stained with anti - DARPP32 ( green ) and anti - Flag ( red ) antibodies . The arrows ( white ) mark Flag + or Flag - primary neurons . The neurite length of Flag + or Flag - neurons was measured by Image J and showed by scatterplot . The scale bar represents 50 µm . ( n = 155 for Flag - group and n = 154 for Flag + group ) .

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Primary neurons expressing Flag-mtHSF1 were stained with anti-DARPP32 (green) and anti-Flag (red) antibodies. The arrows (white) mark Flag+ or Flag- primary neurons. The neurite length of Flag+ or Flag- neurons was measured by Image J and showed by scatterplot. The scale bar represents 50 µm. (n = 155 for Flag- group and n = 154 for Flag+ group).