Permeabilized HEK293T expressing wild type ( WT ) or edited ( H112Q or H112Y ) OSCP subunit were suspended in KSCN - media at pH 7 . 4 ( left traces ) or pH 6 . 5 ( right traces ) PTP opening was measured as decrease in absorbance at 540 nm . Where indicated ( arrows ) 0 . 1 mM Ca2 + in absence of further additions ( black traces ) or in presence of 0 . 5 mM DPC ( red traces ) or 2 µM CsA ( blue traces ) was added . bottom panel reports difference between absorbance readings before addition of Ca2 + at end of swelling phase . Data are mean ± s . e . of 5 experiments ( triplicate samples ) for Ca2 + alone ( black bars ) Ca2 + + DPC ( red bars ) of 3 experiments ( duplicate samples ) for Ca2 + + CsA ( blue bars ) . Solid bars , pH 7 . 4 , hatched bars , pH 6 .

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Permeabilized HEK293T cells expressing wild type (WT) or edited (H112Q or H112Y) OSCP subunit were suspended in KSCN-based media at pH 7.4 (left traces) or pH 6.5 (right traces) and PTP opening was measured as the decrease in absorbance at 540 nm. Where indicated (arrows) 0.1 mM Ca2+ in the absence of further additions (black traces) or in the presence of 0.5 mM DPC (red traces) or 2 µM CsA (blue traces) was added. The bottom panel reports the difference between the absorbance readings before the addition of Ca2+ and at the end of the swelling phase. Data are mean ± s.e. of 5 experiments (triplicate samples) for Ca2+ alone (black bars) and Ca2+ + DPC (red bars) and of 3 experiments (duplicate samples) for Ca2+ + CsA (blue bars). Solid bars, pH 7.4, hatched bars, pH 6.5


Mitochondria were prepared from , suspended in sucrose buffer at pH 7 . 4 ( solid bars ) or 6 . 5 ( hatched bars ) in absence ( black bars ) or presence ( blue bars ) of 1 . 6 µM CsA , extracted with 1 % ( w / v ) digitonin immunoprecipitated with an F - ATP synthase antibody . immunoprecipitates were subjected to 12 % SDS - PAGE followed by Western blotting for CyPD α subunit of F - ATP synthase . Each immunodetected band was analyzed by densitometry , between CyPD α subunit was measured expressed relative to obtained in absence of CsA at pH 7 . 4 , which was taken as 100 % . Data are average ± s . e . of 6 independent experiments .

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Mitochondria were prepared from cells, suspended in sucrose buffer at pH 7.4 (solid bars) or 6.5 (hatched bars) in the absence (black bars) or presence (blue bars) of 1.6 µM CsA, extracted with 1% (w/v) digitonin and immunoprecipitated with an F-ATP synthase antibody. The immunoprecipitates were subjected to 12% SDS-PAGE followed by Western blotting for CyPD and α subunit of F-ATP synthase. Each immunodetected band was analyzed by densitometry, the ratio between CyPD and α subunit was measured and expressed relative to the ratio obtained in absence of CsA at pH 7.4, which was taken as 100%. Data are average ± s.e. of 6 independent experiments.


HEK293T were incubated in a balanced salt solution containing 2 - deoxyglucose at pH 7 . 4 ( solid bars ) or 6 . 5 ( hatched bars ) anoxia induced by incubation in a BD GasPak EZ Anaerobe - generating Pouch System ( BD Biosciences ) . was measured on LDH release ( average ± s . e . from 8 independent experiments ) . In panels p values calculated with Student t are shown ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) .

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HEK293T cells were incubated in a balanced salt solution containing 2-deoxyglucose at pH 7.4 (solid bars) or 6.5 (hatched bars) and anoxia induced by incubation in a BD GasPak EZ Anaerobe Gas-generating Pouch System (BD Biosciences). Cell death was measured based on LDH release (average ± s.e. from 8 independent experiments). In all panels the p values calculated with the Student t test are shown (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).


Representative current traces recorded at V ( pipette ) + 10 mV from mitoplasts derived from wild type ( WT ) or H112Q OSCP mutation - harboring .

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Representative current traces recorded at V(pipette) +10 mV from mitoplasts derived from wild type (WT) or H112Q OSCP mutation-harboring cells.


Channel activity ( top ) corresponding amplitude histograms ( bottom ) recorded in wild type mitochondria before ( WT pH 7 . 3 ) after acidification to pH 6 . 5 ( WT pH 6 . 5 ) . Amplitude histograms were obtained from 120 second - long current traces for corresponding pH values panels are from same experiment .

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Channel activity (top) and corresponding amplitude histograms (bottom) recorded in wild type mitochondria before (WT pH 7.3) and after acidification to pH 6.5 (WT pH 6.5). Amplitude histograms were obtained from 120 second-long current traces for the corresponding pH values and the panels are from the same experiment.


Recordings as in panel B from mitoplasts of harboring OSCP H112Q replacement . Left panel , pH 7 . 3 ; middle panel , 440 seconds after acidification to pH 6 , 5 ; right panel , 30 seconds after addition of 5 mM Ba2 + at pH 6 . 5 . Amplitude histograms were obtained from 100 second - long segments panels are from same experiment . Note that both in panels B C well - visible subconductance states can be detected in amplitude histograms ( peaks between open o2 o3 on left panels ) . Gaussian fitting has been obtained using Origin Program set . In panels c , closed ; o1 , o2 , o3 open states with discrete subconductance .

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Recordings as in panel B from mitoplasts of cells harboring the OSCP H112Q replacement. Left panel, pH 7.3; middle panel, 440 seconds after acidification to pH 6,5; right panel, 30 seconds after the addition of 5 mM Ba2+ at pH 6.5. Amplitude histograms were obtained from 100 second-long segments and the panels are from the same experiment. Note that both in panels B and C well-visible subconductance states can be detected in the amplitude histograms (peaks between open levels o2 and o3 on left panels). Gaussian fitting has been obtained using the Origin Program set. In all panels c, closed; o1, o2, o3 open states with discrete subconductance.


left panel depicts a cryo - EM atomic model of monomeric bovine F - ATP synthase ( pdb 5ara ) ( excluding subunits e , f , g , DAPIT , 6 . 8PL A6L ) with OSCP ( cyan ) subunit b ( yellow ) highlighted on structure of F - ATP synthase in lower part of panel . region H112 - E115 is shown as van der Waals spheres ( inflated for better visibility ) . backbone of stretch T130 - K135 is shown in red van der Waals spheres . top part of panel shows results of restrained molecular dynamics simulations of isolated lateral stalk ( OSCP subunits b , d , F6 ) displaying a large number of conformations in this region . Snapshots taken at 1 ns intervals along last 15 ns of trajectory are shown for fragment 112 - HRGE - 115 for deprotonated ( HIS ) protonated form ( HIS + ) . Only for protonated H112 a salt bridge with a well defined turn or helix arrangement at H112 - E115 is found in a large number of MD conformations . larger conformational variability of deprotonated form can also be appreciated . right panel reports a comparison of OSCP across 16 out of 197 species ( HRGE is boxed in red ; Sauria sequences are in thin black box ) . Conservation , quality of aligment consensus at each position in alignment of vertebrate sequences in RefSeq , excluding Sauria most divergent sequences , are described in documentation of program JalView [ 67 ] .

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

The left panel depicts a cryo-EM based atomic model of monomeric bovine F-ATP synthase (pdb 5ara) (excluding subunits e, f, g, DAPIT, 6.8PL and A6L) with OSCP (cyan) and subunit b (yellow) highlighted on the structure of F-ATP synthase in the lower part of the panel. The region H112-E115 is shown as van der Waals spheres (inflated for better visibility). The backbone of the stretch T130-K135 is shown in red van der Waals spheres. The top part of the panel shows the results of restrained molecular dynamics simulations of the isolated lateral stalk (OSCP and subunits b, d, F6) displaying a large number of conformations in this region. Snapshots taken at 1 ns intervals along the last 15 ns of the trajectory are shown for the fragment 112-HRGE-115 for the deprotonated (HIS) and the protonated form (HIS+). Only for protonated H112 a salt bridge with a well defined turn or helix arrangement at H112-E115 is found in a large number of MD conformations. The larger conformational variability of the deprotonated form can also be appreciated. The right panel reports a sequence comparison of OSCP across 16 out of 197 species (HRGE is boxed in red; Sauria sequences are in the thin black box). Conservation, quality of the aligment and consensus at each position in the alignment of all vertebrate sequences in RefSeq, excluding Sauria and most divergent sequences, are described in the documentation of the program JalView [67].


( A ) Relative expression of BBSome subunits in indicated organs T measured by qPCR . CT values of BBS genes were normalized to geometric mean of CT values of reference genes Gapdh , Tubb2a , Eef1a1 . expression are normalized to those of brain ( = 1 ) . Mean + SD . Three biological replicates ( result of each biological replicate is a median of three technical replicates ) .

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(A) Relative expression of the BBSome subunits in the indicated organs and T cells measured by qPCR. CT values of the BBS genes were normalized to the geometric mean of the CT values of reference genes Gapdh, Tubb2a, and Eef1a1. The expression levels are normalized to those of brain (=1). Mean+SD. Three biological replicates (the result of each biological replicate is a median of three technical replicates).


( B ) Immunoblot of BBS4 expression in T B lymphocytes isolated from lymph nodes spleen of C57Bl / 6 mouse . β - actin served as a loading control .

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(B) Immunoblot analysis of BBS4 expression in T and B lymphocytes isolated from the lymph nodes and spleen of C57Bl/6 mouse. β-actin staining served as a loading control.


( D ) Immunoblot of BBS4 expression in testes thymi lysates of Bbs4 + / + ( WT ) , Bbs4GT / + , Bbs4GT / GT mice . β - actin serves as a loading control . A representative experiment out of three biological replicates in total is shown .

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(D) Immunoblot analysis of BBS4 expression in testes and thymi lysates of Bbs4+/+ (WT), Bbs4GT/+, and Bbs4GT/GT mice. β-actin staining serves as a loading control. A representative experiment out of three biological replicates in total is shown.


( E ) Immunoblot of BBS4 expression in brain lysates of Bbs4 + / + , Bbs4GT / GT , Bbs4KO / KO mice . β - actin served as a loading control . A representative experiment out of two biological replicates is shown .

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(E) Immunoblot analysis of BBS4 expression in the brain lysates of Bbs4+/+, Bbs4GT/GT, and Bbs4KO/KO mice. β-actin staining served as a loading control. A representative experiment out of two biological replicates is shown.


( F ) Hematoxylin eosin of sections of seminiferous tubules from ~ 4 old Bbs4 + / + ( n = 2 mice ) Bbs4GT / GT ( n = 4 ) . Scale bar , 100 µm . Representative are shown .

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(F) Hematoxylin and eosin staining of sections of seminiferous tubules from ~4 weeks old Bbs4+/+ (n=2 mice) and Bbs4GT/GT (n=4) males. Scale bar, 100 µm. Representative images are shown.


( G ) Genotypic of Bbs4 + / + , Bbs4GT / + or Bbs4KO / + ( Het ) , Bbs4GT / GT ( GT ) or Bbs4KO / KO ( KO ) offspring at weaning from mating of Bbs4GT / + ( n = 145 pups ) or Bbs4 + / KO ( n = 168 pups ) parents . Binomial was used for statistical comparison of observed distribution to expected Mendelian .

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(G) Genotypic ratio of Bbs4+/+, Bbs4GT/+ or Bbs4KO/+ (Het), and Bbs4GT/GT (GT) or Bbs4KO/KO (KO) offspring at weaning from mating of Bbs4GT/+ (n=145 pups) or Bbs4+/KO (n=168 pups) parents. Binomial test was used for statistical comparison of the observed distribution to the expected Mendelian ratio.


( H ) Bbs4 + / + Bbs4GT / GT at 20 of . Representative litter out of seven in total . ( I ) Bbs4 + / + Bbs4KO / KO at 20 of . Representative litter out of five in total .

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(H) Bbs4+/+ and Bbs4GT/GT female littermates at 20 weeks of age. Representative litter out of seven in total. (I) Bbs4+/+ and Bbs4KO/KO female littermates at 20 weeks of age. Representative litter out of five in total.


( J ) Growth curves of Bbs4 - deficient mice , mean ± SD is shown . : Bbs4 + / + ( n = 12 mice ) , Bbs4GT / GT ( n = 12 ) , Bbs4KO / KO ( n = 6 ) . : Bbs4 + / + ( n = 6 mice ) , Bbs4GT / GT ( n = 7 ) , Bbs4KO / KO ( n = 5 ) .

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(J) Growth curves of Bbs4-deficient mice, mean ± SD is shown. Females: Bbs4+/+ (n=12 mice), Bbs4GT/GT (n=12), Bbs4KO/KO (n=6). Males: Bbs4+/+ (n=6 mice), Bbs4GT/GT (n=7), Bbs4KO/KO (n=5).


( K ) Leptin in blood plasma taken from young adult ( 7 - 8 ) or mid - ( 14 - 20 ) mice . Young adult mice : Bbs4 + / + ( n = 4 mice ) , Bbs4KO / KO ( n = 3 ) , analyzed in two independent experiments . Mean + . Mid - adult mice : Bbs4 + / + ( n = 7 mice ) , Bbs4KO / KO ( n = 5 ) , Bbs4GT / GT ( n = 3 ) , analyzed in 4 independent experiments . Kruskal - Wallis was used for statistical . Mean + .

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(K) Leptin concentration in blood plasma taken from young adult (7-8 weeks) or mid-age (14-20 weeks) mice. Young adult mice: Bbs4+/+ (n=4 mice), Bbs4KO/KO (n=3), analyzed in two independent experiments. Mean+SEM. Mid-age adult mice: Bbs4+/+ (n=7 mice), Bbs4KO/KO (n=5), Bbs4GT/GT (n=3), analyzed in 4 independent experiments. Kruskal-Wallis tests was used for the statistical analysis. Mean+SEM.


isolated from bone marrow ( BM ) of 18 - 25 old Bbs4 + / + ( WT ) , Bbs4GT / GT , Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT Bbs4WT / WT controls ) or eight ( Bbs4KO / KO Bbs4WT / WT controls ) independent experiments were performed . ( A ) of B220low positive in bone marrow . Up : Bbs4 + / + n = 9 , Bbs4GT / GT n = 10 . Down : Bbs4 + / + ( n = 11 ) , Bbs4KO / KO ( n = 10 ) . Statistical significance was calculated using two - Mann - Whitney . Medians are shown .

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Cells isolated from the bone marrow (BM) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (A) Percentage of B220low positive cells in the bone marrow . Up: Bbs4+/+ n=9, Bbs4GT/GT n=10. Down: Bbs4+/+ (n =11), Bbs4KO/KO (n=10). Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.


isolated from bone marrow ( BM ) of 18 - 25 old Bbs4 + / + ( WT ) , Bbs4GT / GT , Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT Bbs4WT / WT controls ) or eight ( Bbs4KO / KO Bbs4WT / WT controls ) independent experiments were performed . ( B ) of B - precursors ( IgM - IgD - ) in bone marrow . Gated on viable B220 + . Up : Bbs4 + / + ( n = 9 ) , Bbs4GT / GT ( n = 10 ) . Down : Bbs4 + / + ( n = 11 ) , Bbs4KO / KO ( n = 10 ) . Statistical significance was calculated using two - Mann - Whitney . Medians are shown .

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Cells isolated from the bone marrow (BM) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (B) Percentage of B-cell precursors (IgM- IgD-) in the bone marrow. Gated on viable B220+ cells. Up: Bbs4+/+ (n=9), Bbs4GT/GT (n=10). Down: Bbs4+/+ (n =11), Bbs4KO/KO (n=10). Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.


isolated from bone marrow ( BM ) of 18 - 25 old Bbs4 + / + ( WT ) , Bbs4GT / GT , Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT Bbs4WT / WT controls ) or eight ( Bbs4KO / KO Bbs4WT / WT controls ) independent experiments were performed . ( C ) of pre - B ( CD43 - CD24high ) in bone marrow of Bbs4 + / + ( n = 11 ) Bbs4KO / KO mice ( n = 10 ) . Gated on viable B220 + IgM - IgD - . Statistical significance was calculated using two - Mann - Whitney . Medians are shown .

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Cells isolated from the bone marrow (BM) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (C) Percentage of pre-B cells (CD43- CD24high) in the bone marrow of Bbs4+/+ (n=11) and Bbs4KO/KO mice (n=10). Gated on viable B220+ IgM- IgD- cells. Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.


isolated from spleens ( SPL ) of 18 - 25 old Bbs4 + / + ( WT ) , Bbs4GT / GT , Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT Bbs4WT / WT controls ) or eight ( Bbs4KO / KO Bbs4WT / WT controls ) independent experiments were performed . ( D ) of late mature ( IgM - IgD + ) B in spleen of Bbs4 + / + ( n = 11 ) Bbs4KO / KO mice ( n = 10 ) Gated on viable CD19 + . Statistical significance was calculated using two - Mann - Whitney . Medians are shown .

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Cells isolated from the spleens (SPL) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (D) Percentage of late mature (IgM- IgD+) B cells in the spleen of Bbs4+/+ (n=11) and Bbs4KO/KO mice (n=10) Gated on viable CD19+ cells. Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.


isolated from spleens ( SPL ) of 18 - 25 old Bbs4 + / + ( WT ) , Bbs4GT / GT , Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT Bbs4WT / WT controls ) or eight ( Bbs4KO / KO Bbs4WT / WT controls ) independent experiments were performed . ( E ) An alternative of experiment shown in ( D ) . Geometric mean ( MFI ) of BV421 ( IgM ) on IgD + B was determined . Mean of MFI values for each genotype per experiment was quantified , obtained values were normalized to Bbs4 + / + ( = 1 ) for each experiment . Two - One Sample Wilcoxon Signed Rank was used for statistical .

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

Cells isolated from the spleens (SPL) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (E) An alternative analysis of the experiment shown in (D). Geometric mean fluorescence intensity (MFI) of BV421 (IgM) on IgD+ B cells was determined. Mean of MFI values for each genotype per experiment was quantified, and obtained values were normalized to Bbs4+/+ (=1) for each experiment. Two-tailed One Sample Wilcoxon Signed Rank Test was used for the statistical analysis.


isolated from spleens ( SPL ) of 18 - 25 old Bbs4 + / + ( WT ) , Bbs4GT / GT , Bbs4KO / KO mice were analyzed by flow cytometry . Number of analyzed mice ( n ) is indicated . Six ( Bbs4GT / GT Bbs4WT / WT controls ) or eight ( Bbs4KO / KO Bbs4WT / WT controls ) independent experiments were performed . ( F ) of splenic marginal zone ( MZ ) B ( CD23 - CD1d + ) in Bbs4 + / + controls ( n = 10 ) Bbs4KO / KO mice ( n = 9 ) was determined . Gated on viable CD19 + , IgD - IgM + , CD138 - . Statistical significance was calculated using two - Mann - Whitney . Medians are shown .

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

Cells isolated from the spleens (SPL) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed. (F) Percentage of splenic marginal zone (MZ) B cells (CD23- CD1d+) in Bbs4+/+ controls (n=10) and Bbs4KO/KO mice (n=9) was determined. Gated on viable CD19+, IgD- IgM+, CD138- cells. Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.


( A , B ) Splenocytes isolated from Bbs4 + / + ( n = 4 mice ) Bbs4GT / GT ( n = 3 ) B - transgenic B1 - 8 were activated with 4 - hydroxy - 3 - nitrophenylacetic acid succinimide ester - loaded T2 - Kb in three independent experiments . of activated B ( gated as CD69 + B220 + IgLλ + viable ) in samples without T2 - Kb ( negative control ) , in samples with T2 - Kb added in ratios 1 : 10 or 1 : 3 was determined by flow cytometry . ( A ) Representative mice are shown . ( B ) Mean ± . Statistical significance was calculated using two - Mann - Whitney .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,B) Splenocytes isolated from Bbs4+/+ (n=4 mice) and Bbs4GT/GT (n=3) B-cell transgenic B1-8 littermates were activated with 4-hydroxy-3-nitrophenylacetic acid succinimide ester-loaded T2-Kb cells in three independent experiments. Percentage of activated B cells (gated as CD69+ B220+ IgLλ+ viable cells) in the samples without T2-Kb (negative control), and in the samples with T2-Kb added in ratios 1:10 or 1:3 was determined by flow cytometry. (A) Representative mice are shown. (B) Mean±SEM. Statistical significance was calculated using two-tailed Mann-Whitney test.


( C ) B from 18 - 20 old Bbs4KO / KO ( red circle , n = 3 mice ) , control mice ( Bbs4 + / + , grey square , n = 4 , or Bbs4 + / KO , grey square , n = 2 ) were activated with F ( ab ' ) 2 - goat anti - mouse IgM ( Mu chain ) . of activated B ( gated as CD69 + CD19 + viable ) was determined by flow cytometry . Representative experiment out of three biological replicates is shown . Mean ± . Statistical significance was calculated using two - Mann - Whitney .

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

(C) B cells from 18-20 weeks old Bbs4KO/KO (red circle, n=3 mice), and control mice (Bbs4+/+, dark grey square, n=4, or Bbs4+/KO, light grey square, n=2) were activated with F(ab')2-goat anti-mouse IgM (Mu chain). Percentage of activated B cells (gated as CD69+ CD19+ viable cells) was determined by flow cytometry. Representative experiment out of three biological replicates is shown. Mean±SEM. Statistical significance was calculated using two-tailed Mann-Whitney test.


( D ) CFSE - loaded T isolated from lymph nodes of Bbs4FL / FL OT - I Rag2KO / KO ( OT - I ) CD4 - Cre Bbs4FL / FL OT - I Rag2KO / KO ( Bbs4 cKO , OT - I ) were incubated with DDAO - labeled WT splenocytes loaded with OVA peptide or with indicated altered peptide ligands for 20 min . of T conjugated with APCs was determined by flow cytometry . Four biological replicates were performed . Mean + . Statistical significance was calculated using two - Mann - Whitney , p > 0 . 05 for peptides .

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

(D) CFSE-loaded T cells isolated from lymph nodes of Bbs4FL/FL OT-I Rag2KO/KO (OT-I) and CD4-Cre Bbs4FL/FL OT-I Rag2KO/KO (Bbs4 cKO, OT-I) littermates were incubated with DDAO-labeled WT splenocytes loaded with OVA peptide or with the indicated altered peptide ligands for 20 min. Percentage of T cells conjugated with the APCs was determined by flow cytometry. Four biological replicates were performed. Mean+SEM. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all peptides.


500 or 1000 T isolated from lymph nodes of Bbs4FL / FL OT - I Rag2KO / KO ( OT - I ) CD4 - Cre Bbs4FL / FL OT - I Rag2KO / KO ( Bbs4 cKO , OT - I ) were adoptively transferred into RIP . OVA mice followed by infection with Listeria monocytogenes expressing ovalbumin ( LM - OVA ) on next day . ( E ) Glucose level in urine of mice was monitored on a daily basis . mouse was considered diabetic when it had urine glucose level ≥ 1000 mg / dL for two consecutive days . Statistical significance was calculated by Log - rank ( Mantel - Cox ) .

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

500 or 1000 T cells isolated from lymph nodes of Bbs4FL/FL OT-I Rag2KO/KO (OT-I) and CD4-Cre Bbs4FL/FL OT-I Rag2KO/KO (Bbs4 cKO, OT-I) littermates were adoptively transferred into RIP.OVA mice followed by infection with Listeria monocytogenes expressing ovalbumin (LM-OVA) on the next day. (E) Glucose level in the urine of mice was monitored on a daily basis. The mouse was considered diabetic when it had urine glucose level ≥ 1000 mg/dL for two consecutive days. Statistical significance was calculated by Log-rank (Mantel-Cox) test.


500 or 1000 T isolated from lymph nodes of Bbs4FL / FL OT - I Rag2KO / KO ( OT - I ) CD4 - Cre Bbs4FL / FL OT - I Rag2KO / KO ( Bbs4 cKO , OT - I ) were adoptively transferred into RIP . OVA mice followed by infection with Listeria monocytogenes expressing ovalbumin ( LM - OVA ) on next day . ( F ) Glucose in blood on day 7 post - infection . 500 OT - I ctrl ( n = 13 mice ) , 500 OT - I Bbs4 cKO ( n = 13 ) , 1000 OT - I ctrl ( n = 9 ) , 1000 OT - I Bbs4 cKO ( n = 11 ) , analyzed in four independent experiments , mean is shown . Statistical significance was calculated using two - Mann - Whitney .

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

500 or 1000 T cells isolated from lymph nodes of Bbs4FL/FL OT-I Rag2KO/KO (OT-I) and CD4-Cre Bbs4FL/FL OT-I Rag2KO/KO (Bbs4 cKO, OT-I) littermates were adoptively transferred into RIP.OVA mice followed by infection with Listeria monocytogenes expressing ovalbumin (LM-OVA) on the next day. (F) Glucose concentration in blood on day 7 post-infection. 500 OT-I ctrl (n=13 mice), 500 OT-I Bbs4 cKO (n=13), 1000 OT-I ctrl (n=9), 1000 OT-I Bbs4 cKO (n=11), analyzed in four independent experiments, mean is shown. Statistical significance was calculated using two-tailed Mann-Whitney test.


isolated from bone marrow of Bbs4FL / FL ( control ) Vav - iCre Bbs4FL / FL ( cKO ) mice were analyzed by flow cytometry . ( A ) of B220low in bone marrow . Representative experiment out of four in total is shown . Bbs4FL / FL ( n = 6 mice ) , Vav - iCre Bbs4FL / FL ( n = 9 ) . Representative experiment out of six in total is shown . Statistical significance was calculated using two - Mann - Whitney , p > 0 . 05 for . Median is shown .

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

Cells isolated from bone marrow of Bbs4FL/FL (control) and Vav-iCre Bbs4FL/FL (cKO) mice were analyzed by flow cytometry. (A) Percentage of B220low in bone marrow. Representative experiment out of four in total is shown. Bbs4FL/FL (n=6 mice), Vav-iCre Bbs4FL/FL (n=9). Representative experiment out of six in total is shown. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all tests. Median is shown.


isolated from bone marrow of Bbs4FL / FL ( control ) Vav - iCre Bbs4FL / FL ( cKO ) mice were analyzed by flow cytometry . ( B ) of B - precursors ( B220 + , IgM - IgD - ) in bone marrow . Representative experiment out of four in total is shown . Bbs4FL / FL ( n = 6 mice ) , Vav - iCre Bbs4FL / FL ( n = 9 ) . Representative experiment out of six in total is shown . Statistical significance was calculated using two - Mann - Whitney , p > 0 . 05 for . Median is shown .

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

Cells isolated from bone marrow of Bbs4FL/FL (control) and Vav-iCre Bbs4FL/FL (cKO) mice were analyzed by flow cytometry. (B) Percentage of B-cell precursors (B220+, IgM- IgD-) in the bone marrow. Representative experiment out of four in total is shown. Bbs4FL/FL (n=6 mice), Vav-iCre Bbs4FL/FL (n=9). Representative experiment out of six in total is shown. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all tests. Median is shown.


isolated from , lymph nodes spleen of Bbs4FL / FL ( control ) Vav - iCre Bbs4FL / FL ( cKO ) mice were analyzed by flow cytometry . ( C ) of CD19 + B in spleens lymph nodes of Vav - iCre Bbs4FL / FL ( n = 12 mice ) Bbs4FL / FL ( n = 9 ) mice , six independent experiments . Representative experiment out of six in total is shown . Statistical significance was calculated using two - Mann - Whitney , p > 0 . 05 for . Median is shown .

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

Cells isolated from , lymph nodes and spleen of Bbs4FL/FL (control) and Vav-iCre Bbs4FL/FL (cKO) mice were analyzed by flow cytometry. (C) Percentage of CD19+ B cells in spleens and lymph nodes of Vav-iCre Bbs4FL/FL (n=12 mice) and Bbs4FL/FL (n=9) mice, six independent experiments. Representative experiment out of six in total is shown. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all tests. Median is shown.


isolated from spleen of Bbs4FL / FL ( control ) Vav - iCre Bbs4FL / FL ( cKO ) mice were analyzed by flow cytometry . D ) of late mature B ( CD19 + , IgM - , IgD + ) in spleens of Vav - iCre Bbs4FL / FL ( n = 12 mice ) , Bbs4FL / FL mice ( n = 9 ) . Representative experiment out of six in total is shown . Statistical significance was calculated using two - Mann - Whitney , p > 0 . 05 for . Median is shown .

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

Cells isolated from spleen of Bbs4FL/FL (control) and Vav-iCre Bbs4FL/FL (cKO) mice were analyzed by flow cytometry. D) Percentage of late mature B cells (CD19+, IgM-, IgD+) in spleens of Vav-iCre Bbs4FL/FL (n=12 mice), and Bbs4FL/FL mice (n=9). Representative experiment out of six in total is shown. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all tests. Median is shown.


( E ) Expression of Cxcl12 Il - 7 in bone marrow from Bbs4 + / + Bbs18 + / + ( WT ) mice ( black circles , n = 7 mice ) , Bbs4 + / KO ( Het , green circle , n = 3 ) , Bbs18 + / KO ( Het , blue circle , n = 3 ) , Bbs4KO / KO ( green square , n = 5 ) , Bbs18KO / KO ( blue square , n = 2 ) was analyzed by RT - qPCR in five independent experiments . expression was normalized to Gapdh . statistical significance of difference between BBSome - deficient mice ( Bbs4KO / KO Bbs18KO / KO ) controls were calculated using two - Mann - Whitney . Median is shown .

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

(E) Expression of Cxcl12 and Il-7 in the bone marrow from Bbs4+/+ Bbs18+/+ (WT) mice (black circles, n=7 mice), Bbs4+/KO (Het, green circle, n=3), Bbs18+/KO (Het, blue circle, n=3), Bbs4KO/KO (green square, n=5), and Bbs18KO/KO (blue square, n=2) was analyzed by RT-qPCR in five independent experiments. The expression was normalized to Gapdh. The statistical significance of the difference between the BBSome-deficient mice (Bbs4KO/KO and Bbs18KO/KO) and controls were calculated using two-tailed Mann-Whitney test. Median is shown.


( F ) MEF lines were derived from a single Bbs4 + / + ( WT ) or a single Bbs4KO / KO . expression of Cxcl12 in these lines was analyzed by RT - qPCR . expression was normalized to Gapdh to Bbs4 + / + MEFs ( = 1 ) for each experiment . Three biological replicates . One sample t was used for statistical .

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

(F) MEF cell lines were derived from a single Bbs4+/+ (WT) or a single Bbs4KO/KO embryos. The expression of Cxcl12 in these cell lines was analyzed by RT-qPCR. The expression was normalized to Gapdh and to Bbs4+/+ MEFs (=1) for each experiment. Three biological replicates. One sample t test was used for the statistical analysis.


( G ) Expression of Cxcl12 canonical - WNT responsive gene Alpl in untreated ( NT ) ST2 , or ST2 treated with WNT3A or WNT5A was analyzed by RT - qPCR . expression was normalized to Gapdh untreated ST2 sample ( = 1 ) for each experiment . Four biological replicates were performed . One sample t was used for statistical .

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

(G) Expression of Cxcl12 and canonical-WNT responsive gene Alpl in untreated (NT) ST2 cells, or ST2 cells treated with WNT3A or WNT5A was analyzed by RT-qPCR. The expression was normalized to Gapdh and untreated ST2 sample (=1) for each experiment. Four biological replicates were performed. One sample t test was used for the statistical analysis.


( A Results of blood of BBS from Guy ' s Hospital Great Ormond Street Hospital were extracted from medical records compared to two sets of healthy controls obtained from UK Biobank . BMI - random controls were - gender - matched to set of BBS , with random BMIs . BMI - matched controls were matched for , gender , BMI . BBS n = 43 for parameters blood n = 41 for parameters Neutrophils , Lymphocytes , Monocytes , Eosinophils Both data sets of healthy controls were selected as 10 - fold larger than set of BBS . Median is shown . Kruskal - Wallis was used for statistical in (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Results of the blood tests of BBS patients from the Guy's Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank. The BMI-random controls were age- and gender-matched to the set of BBS patients, with random BMIs. The BMI-matched controls were matched for age, gender, and BMI. BBS patients n=43 for parameters White blood cell count n=41 for parameters Neutrophils, Lymphocytes, Monocytes, Eosinophils Both data sets of healthy controls were selected as 10-fold larger than the set of BBS patients. Median is shown. Kruskal-Wallis test was used for the statistical analysis in (A


( B Indicated parameters were measured using blood from 20 - 21 old Bbs4 + / + ( WT ) ( n = 15 ) , Bbs4KO / KO ( n = 7 ) , Bbs4GT / GT ( n = 12 ) mice . Kruskal - Wallis with Dunn ' s Multiple Comparison Post - was used for statistical . Median is shown .

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

(B Indicated parameters were measured using the blood from 20-21 weeks old Bbs4+/+ (WT) (n=15), Bbs4KO/KO (n=7), and Bbs4GT/GT (n=12) mice. Kruskal-Wallis test with Dunn's Multiple Comparison Post-tests was used for the statistical analysis. Median is shown.


Results of blood of BBS from Guy ' s Hospital Great Ormond Street Hospital were extracted from medical records compared to two sets of healthy controls obtained from UK Biobank . BMI - random controls were - gender - matched to set of BBS , with random BMIs . BMI - matched controls were matched for , gender , BMI . In ( C ) , of or controls having CRP ≥ 5 were compared using Fisher ' s exact with post - hoc Sidak correction for multiple comparisons , BBS n = 42 .

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

Results of the blood tests of BBS patients from the Guy's Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank. The BMI-random controls were age- and gender-matched to the set of BBS patients, with random BMIs. The BMI-matched controls were matched for age, gender, and BMI. In (C), the percentage of patients or controls having CRP≥5 were compared using Fisher's exact test with post-hoc Sidak correction for multiple comparisons, BBS patients n=42.


Results of blood of BBS from Guy ' s Hospital Great Ormond Street Hospital were extracted from medical records compared to two sets of healthy controls obtained from UK Biobank . BMI - random controls were - gender - matched to set of BBS , with random BMIs . BMI - matched controls were matched for , gender , BMI . BBS n = 43 for parameters Mean volume , Hemoglobin n = 39 for parameters Red Blood , Mean Hemoglobin red distribution width ( RDW ) . Both data sets of healthy controls were selected as 10 - fold larger than set of BBS . Median is shown . Kruskal - Wallis was used for statistical

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

Results of the blood tests of BBS patients from the Guy's Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank. The BMI-random controls were age- and gender-matched to the set of BBS patients, with random BMIs. The BMI-matched controls were matched for age, gender, and BMI. BBS patients n=43 for parameters Mean cell volume, Hemoglobin n=39 for parameters Red Blood cells, Mean cell Hemoglobin and red cell distribution width (RDW). Both data sets of healthy controls were selected as 10-fold larger than the set of BBS patients. Median is shown. Kruskal-Wallis test was used for the statistical analysis in


Indicated parameters were measured using blood from 20 - 21 old Bbs4 + / + ( WT ) ( n = 15 ) , Bbs4KO / KO ( n = 7 ) , Bbs4GT / GT ( n = 12 ) mice . Kruskal - Wallis with Dunn ' s Multiple Comparison Post - was used for statistical . Median is shown .

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

Indicated parameters were measured using the blood from 20-21 weeks old Bbs4+/+ (WT) (n=15), Bbs4KO/KO (n=7), and Bbs4GT/GT (n=12) mice. Kruskal-Wallis test with Dunn's Multiple Comparison Post-tests was used for the statistical analysis. Median is shown.


Results of blood of BBS from Guy ' s Hospital Great Ormond Street Hospital were extracted from medical records compared to two sets of healthy controls obtained from UK Biobank . BMI - random controls were - gender - matched to set of BBS , with random BMIs . BMI - matched controls were matched for , gender , BMI . BBS n = 43 for parameters Platelets ; Both data sets of healthy controls were selected as 10 - fold larger than set of BBS . Median is shown . Kruskal - Wallis was used for statistical in F ) .

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Results of the blood tests of BBS patients from the Guy's Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank. The BMI-random controls were age- and gender-matched to the set of BBS patients, with random BMIs. The BMI-matched controls were matched for age, gender, and BMI. BBS patients n=43 for parameters Platelets; Both data sets of healthy controls were selected as 10-fold larger than the set of BBS patients. Median is shown. Kruskal-Wallis test was used for the statistical analysis in F).


G ) Indicated parameters were measured using blood from 20 - 21 old Bbs4 + / + ( WT ) ( n = 15 ) , Bbs4KO / KO ( n = 7 ) , Bbs4GT / GT ( n = 12 ) mice . Kruskal - Wallis with Dunn ' s Multiple Comparison Post - was used for statistical . Median is shown .

G) Indicated parameters were measured using the blood from 20-21 weeks old Bbs4+/+ (WT) (n=15), Bbs4KO/KO (n=7), and Bbs4GT/GT (n=12) mice. Kruskal-Wallis test with Dunn's Multiple Comparison Post-tests was used for the statistical analysis. Median is shown.


( a ) MCF7 stably expressing Myc - TEV - Flag - Beclin 1 ( MEF - Beclin 1 ) parental controls were lysed subjected to MEF - tag - purification . bound to MEF - Beclin 1 were isolated detected by SDS - PAGE silver . Seven bands specific for MEF - Beclin 1 are numbered . ( b ) Six bands were identified by nanoflow LC - MS / MS MASCOT software .

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(a) MCF7 cells stably expressing Myc-TEV-Flag-Beclin 1 (MEF-Beclin 1) and parental controls were lysed and subjected to MEF-tag-based purification. Proteins bound to MEF-Beclin 1 were isolated and detected by SDS-PAGE and silver staining. Seven bands specific for MEF-Beclin 1 are numbered. (b) Six bands were identified by nanoflow LC-MS/MS and MASCOT software.


( c ) MCF7 were lysed subjected to immunoprecipitation using indicated antibodies .

(c) MCF7 cells were lysed and subjected to immunoprecipitation using the indicated antibodies.


( d ) Differential subcellular fractionation . Post - supernatant of A549 lysates were spun at 20 , 000g for 10 min supernatant was further spun at 100 , 000g for 60 min . Equivalent quantities of each supernatant pellets from same number of were subjected to western blotting with indicated antibodies , including anti - Sec61β ( ER ) , cathepsin D ( lysosome ) , EEA1 ( early endosome ) , transferrin receptor ( plasma recycling endosome ) GAPDH ( cytosol ) .

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(d) Differential subcellular fractionation. Post-nuclear supernatant of A549 cell lysates were spun at 20,000g for 10 min and the supernatant was further spun at 100,000g for 60 min. Equivalent quantities of each supernatant and pellets from the same number of cells were subjected to western blotting with the indicated antibodies, including anti-Sec61β (ER), cathepsin D (lysosome), EEA1 (early endosome), transferrin receptor (plasma membrane and recycling endosome) and GAPDH (cytosol).


( e ) Gel filtration . supernatant fraction of A549 ( or Atg14L knockdown ) lysates were subjected to a Superose 6 column each fraction was immunoblotted with indicated antibody ( see Supplementary Information , Fig . S2e ) . Relative amounts of each fraction determined by densitometry were plotted . Vo , void fraction . elution pattern of each was reproduced in several experiments . Full scans of gel blots in a , c d are available in Supplementary Information , Fig . S6 .

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

(e) Gel filtration analysis. The supernatant fraction of A549 cell (or Atg14L knockdown) lysates were subjected to a Superose 6 column and each fraction was immunoblotted with the indicated antibody (see Supplementary Information, Fig. S2e). Relative amounts of each fraction determined by densitometry were plotted. Vo, void fraction. The elution pattern of each protein was reproduced in several experiments. Full scans of the gel and blots in a, c and d are available in Supplementary Information, Fig. S6.


( a ) A549 were infected with adenovirus harbouring shRNA targeting Atg14L , Rubicon , UVRAG or a control ( luciferase ) . lysates were subjected to immunoblotting with antibodies indicated . asterisk in Rubicon blot represents a non - specific cross - reacting band .

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(a) A549 cells were infected with adenovirus harbouring shRNA targeting Atg14L, Rubicon, UVRAG or a control (luciferase). Cell lysates were subjected to immunoblotting with the antibodies indicated. The asterisk in the Rubicon blot represents a non-specific cross-reacting band.


( b ) of GFP - positive dots per . were taken from shown in c number of GFP - positive dots was determined . At least 140 were counted per experiment values are mean ± s . d . of three independent experiments . ( c ) A monoclonal A549 stably expressing GFP - LC3 was infected with adenovirus harbouring shRNA targeting Atg14L , Rubicon or control ( luciferase ) . were cultured under nutrient - rich or starvation conditions . GFP was observed by confocal microscopy . Scale bar , 10 μm . full scan of blot in a is available in Supplementary Information , Fig . S6 .

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(b) Quantification of GFP-positive dots per cell. Images were taken from cells shown in c and the number of GFP-positive dots was determined. At least 140 cells were counted per experiment and the values are mean ± s.d. of three independent experiments. (c) A monoclonal A549 cell line stably expressing GFP-LC3 was infected with adenovirus harbouring shRNA targeting Atg14L, Rubicon or control (luciferase). The cells were cultured under nutrient-rich or starvation conditions. GFP was observed by confocal laser microscopy. Scale bar, 10 μm. The full scan of the blot in a is available in Supplementary Information, Fig. S6.


( a ) Southern blot of genomic isolated from wild - type ( WT , mAtg14L + / + ) , mAtg14L + / - knockout ( KO , mAtg14L - / - ) ES after digestion with PstI . Details of probe are shown in Supplementary Information , Fig . S3 .

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(a) Southern blot analysis of genomic DNA isolated from wild-type (WT, mAtg14L+/+) , mAtg14L+/- and knockout (KO, mAtg14L-/-) ES cells after digestion with PstI. Details of the probe are shown in Supplementary Information, Fig. S3.


( c ) effect of Atg14L knockout on p62 turnover . WT , mAtg14L − / − , mAtg14L − / − + Atg14L , mAtg5 − / − ES were cultured in nutrient - rich ( N ) or starvation medium ( S ) for 3 h . lysates were subjected to western blotting with antibodies indicated .

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(c) The effect of Atg14L knockout on p62 turnover. WT, mAtg14L−/−, mAtg14L−/− + Atg14L, and mAtg5−/− ES cells were cultured in nutrient-rich (N) or starvation medium (S) for 3 h. Cell lysates were subjected to western blotting with the antibodies indicated.


( d ) Degradation of long - lived is reduced in Atg14L knockout . WT , mAtg14L − / − , mAtg5 − / − ES were cultured in N or S medium or S medium with wortmannin ( W , 100 nM ) for 3 h degradation of long - lived was measured . data are mean ± s . d . of three independent experiments .

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(d) Degradation of long-lived proteins is reduced in Atg14L knockout cells. WT, mAtg14L−/−, and mAtg5−/− ES cells were cultured in N or S medium or S medium with wortmannin (W, 100 nM) for 3 h and degradation of long-lived proteins was measured. The data are the mean ± s.d. of three independent experiments.


( e ) effect of Atg14L knockout on LC3 turnover . WT , mAtg14L − / − mAtg5 − / − ES were cultured in N or S medium for 3 h in presence or absence of E64d ( 50 μg ml − 1 ) pepstatin A ( 50 μg ml − 1 ) . lysates were subjected to western blotting with antibodies indicated . Full scans of blots in b , c e are available in Supplementary Information , Fig . S6 .

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

(e) The effect of Atg14L knockout on LC3 turnover. WT, mAtg14L−/− and mAtg5−/− ES cells were cultured in N or S medium for 3 h in the presence or absence of E64d (50 μg ml−1) and pepstatin A (50 μg ml−1). Cell lysates were subjected to western blotting with the antibodies indicated. Full scans of the blots in b, c and e are available in Supplementary Information, Fig. S6.


( a ) effect of Atg14L knockout ( mAtg14 − / − ) on Atg16L dot formation . Wild - type ( WT ) mAtg14 − / − ES were cultured in nutrient - rich or starvation medium for 3 h . were fixed subjected to immunofluorescence with an anti - Atg16L antibody . of Atg16L ( grey - scale ) Atg16L merged with Hoechst ( colour ) are shown . ( c ) of number of Atg16L dots in a . data are mean ± s . d . of three independent experiments .

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

(a) The effect of Atg14L knockout (mAtg14−/−) on Atg16L dot formation. Wild-type (WT) and mAtg14−/− ES cells were cultured in nutrient-rich or starvation medium for 3 h. Cells were fixed and subjected to immunofluorescence with an anti-Atg16L antibody. Images of Atg16L staining (grey-scale) and Atg16L merged with Hoechst staining (colour) are shown.(c) Quantification of the number of Atg16L dots in a. The data are the mean ± s.d. of three independent experiments.


( b ) effect of Atg14L knockout on LC3 dot formation . WT mAtg14L − / − ES were cultured in nutrient - rich or starvation medium for 3 h . were fixed subjected to immunofluorescence with anti - LC3 antibody . of LC3 ( grey - scale ) LC3 merged with Hoechst ( colour ) are shown . Scale bars , 20 μm . ( d ) of number of LC3 dots in b . data are mean ± s . d . of three independent experiments . For each of experiments in c d at least 13 were counted .

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

(b) The effect of Atg14L knockout on LC3 dot formation. WT and mAtg14L−/− ES cells were cultured in nutrient-rich or starvation medium for 3 h. Cells were fixed and subjected to immunofluorescence with anti-LC3 antibody. Images of LC3 staining (grey-scale) and LC3 merged with Hoechst staining (colour) are shown. Scale bars, 20 μm. (d) Quantification of the number of LC3 dots in b. The data are the mean ± s.d. of three independent experiments. For each of the experiments in c and d at least 13 cells were counted.


( a ) Immunofluorescence using an anti - Atg16L antibody . A549 were transfected with adenovirus expressing GFP - Atg14L cultured in nutrient - rich or starvation medium for 4 h . were fixed stained with an anti - Atg16L antibody .

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(a) Immunofluorescence using an anti-Atg16L antibody. A549 cells were transfected with adenovirus expressing GFP-Atg14L and cultured in nutrient-rich or starvation medium for 4 h. Cells were fixed and stained with an anti-Atg16L antibody.


( b ) Immunofluorescence with an anti - LC3 antibody . A549 were transfected with adenovirus expressing GFP - Atg14L cultured in starvation medium for 4 h . were fixed stained with an anti - LC3 antibody . Scale bar , 10 μm .

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(b) Immunofluorescence with an anti-LC3 antibody. A549 cells were transfected with adenovirus expressing GFP-Atg14L and cultured in starvation medium for 4 h. Cells were fixed and stained with an anti-LC3 antibody. Scale bar, 10 μm.


( c ) Colocalization efficiency in a b . of colocalization between Atg14L Atg16L or LC3 - positive dot is shown . For example , ( Atg14L × Atg16L ) / Atg16L means of colocalization of Atg14L Atg16L to of Atg16L - positive . For colocalization between Atg14L Atg16L data are mean ± s . d . of 25 . For colocalization between Atg14L LC3 data are mean ± s . d . of 20 .

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(c) Colocalization efficiency in a and b. The percentage of colocalization between Atg14L and Atg16L or LC3-positive dot is shown. For example, (Atg14L×Atg16L)/Atg16L means the ratio of colocalization of Atg14L and Atg16L puncta to all of the Atg16L-positive puncta. For colocalization between Atg14L and Atg16L the data are mean ± s.d. of 25 cells. For colocalization between Atg14L and LC3 the data are mean ± s.d. of 20 cells.


d ) Immunofluorescence with an anti - calnexin ( ER ) antibody . A549 transfected with adenovirus - GFP - Atg14L . After 48 h , were fixed immunostained with calnexin ( ER ) . Scale bar , 10 μm .

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d) Immunofluorescence with an anti-calnexin (ER) antibody. A549 cells transfected with adenovirus-based GFP-Atg14L. After 48 h, the cells were fixed and immunostained with calnexin (ER).Scale bar, 10 μm.


( e ) Immuno - EM of GFP - Atg14L . A549 transfected with adenovirus - vector GFP or GFP - Atg14L were subjected to immuno - EM using an anti - GFP antibody . Scale bar , 500 nm . arrows indicate ER arrowheads indicate autophagosome .

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(e) Immuno-EM analysis of GFP-Atg14L. A549 cells transfected with adenovirus-vector based GFP or GFP-Atg14L were subjected to immuno-EM analysis using an anti-GFP antibody. Scale bar, 500 nm. The arrows indicate the ER membrane and the arrowheads indicate the autophagosome membrane.


( a ) Double knockdown of Rubicon Atg14L . A549 stably expressing GFP - LC3 were infected with adenovirus expressing either shRNA against Rubicon Atg14L or control shRNA ( luciferase ) . image is available in Supplementary Information , Fig . S5a . GFP - LC3 were counted in nutrient - rich ( N ) or starvation ( S ) conditions . data are mean ± s . d . of three independent experiments ; at least 23 were counted for each experiment .

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(a) Double knockdown of Rubicon and Atg14L. A549 cells stably expressing GFP-LC3 were infected with adenovirus expressing either shRNA against Rubicon and Atg14L or control shRNA (luciferase). The image is available in Supplementary Information, Fig. S5a. The GFP-LC3 puncta were counted in nutrient-rich (N) or starvation (S) conditions. The data are mean ± s.d. of three independent experiments; at least 23 cells were counted for each experiment.


( b ) A549 were infected with adenovirus harbouring shRNA for Rubicon or control ( luciferase ) . were cultured in N or S medium in presence or absence of E64d ( 50 μg ml − 1 ) pepstatin A ( 50 μg ml − 1 ) for 1 - 4 h . lysates were subjected to western blotting with antibodies indicated .

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(b) A549 cells were infected with adenovirus harbouring shRNA for Rubicon or control (luciferase). The cells were cultured in N or S medium in the presence or absence of E64d (50 μg ml−1) and pepstatin A (50 μg ml−1) for 1-4 h. Cell lysates were subjected to western blotting with the antibodies indicated.


( c ) of Atg16L in Rubicon knockdown . are available in Supplementary Information , Fig . S5c . data are mean ± s . d . , n = 60 ( 60 , sample of Medium : N , shRNA : Control ) , n = 68 ( 68 , sample of Medium : S , shRNA : Control ) , n = 78 ( 78 , sample of Medium : N , shRNA : Rubicon ) , n = 83 ( 83 sample of Medium : S , shRNA : Rubicon ) .

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(c) Quantification of Atg16L puncta in Rubicon knockdown cells. The images are available in Supplementary Information, Fig. S5c. The data are mean ± s.d., n = 60 (60 cells, sample of Medium: N, shRNA: Control), n = 68 (68 cells, sample of Medium: S, shRNA: Control), n = 78 (78 cells, sample of Medium: N, shRNA: Rubicon), n = 83 (83 cells sample of Medium: S, shRNA: Rubicon).


d ) Bulk degradation activity of Rubicon knockdown . bulk degradation activity in control Rubicon knockdown in nutrient - rich medium was measured . Wortmannin ( W , 100 nM ) was added as a negative control . data are mean ± s . d . of three independent experiments .

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d) Bulk degradation activity of Rubicon knockdown cells. The bulk degradation activity in control and Rubicon knockdown cells in nutrient-rich medium was measured. Wortmannin (W, 100 nM) was added as a negative control. The data are mean ± s.d. of three independent experiments.


( e ) A549 infected with adenovirus harbouring shRNA for Rubicon control ( luciferase ) were cultured in N or S medium for 4 h lysates were subjected to immunoblotting with antibodies indicated . Full scans of blots in b e are available in Supplementary Information , Fig . S6 .

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(e) A549 cells infected with adenovirus harbouring shRNA for Rubicon and control (luciferase) were cultured in N or S medium for 4 h and cell lysates were subjected to immunoblotting with the antibodies indicated. Full scans of the blots in b and e are available in Supplementary Information, Fig. S6.


( a ) A549 transfected with a GFP - Rubicon expression plasmid were subjected to immunofluorescence with various organelle marker antibodies . Scale bar , 10 μm .

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(a) A549 cells transfected with a GFP-Rubicon expression plasmid and were subjected to immunofluorescence with various organelle marker antibodies. Scale bar, 10 μm.


( b ) A549 stably expressing GFP - LC3 were subjected to Rubicon knockdown ( KD ) then transfected with mStrawberry , mStrawberry - Rubicon or mStrawberry - Rubicon mutant lacking Beclin 1 binding region ( Δ393849 ) . with strong expression of mStrawberry - Rubicon showed an overexpression - dependent dominant - negative phenotype ; therefore , we selected with modest expression . number of GFP - LC3 was counted mean ± s . d . are shown . mStrawberry , n = 12 ( 12 ) ; mStrawberry - Mm - Rubicon , n = 14 ( 14 ) ; mStrawberry - Mm - RubiconΔ393 - 849 , n = 13 ( 13 ) .

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(b) A549 cells stably expressing GFP-LC3 were subjected to Rubicon knockdown (KD) and then transfected with mStrawberry, mStrawberry-Rubicon or mStrawberry-Rubicon mutant lacking the Beclin 1 binding region (Δ393849). Cells with strong expression of mStrawberry-Rubicon showed an overexpression-dependent dominant-negative phenotype; therefore, we selected cells with modest expression. The number of GFP-LC3 puncta was counted and mean ± s.d. are shown. mStrawberry, n = 12 (12 cells); mStrawberry-Mm-Rubicon, n = 14 (14 cells); mStrawberry-Mm-RubiconΔ393-849, n = 13 (13 cells).


( a ) maturation of autophagosomes is enhanced in Rubicon knockdown . A549 stably expressing GFP - LC3 , with Rubicon or control shRNA , were incubated in nutrient - rich medium with or without protease inhibitors ( PI ) E64d ( 50 μg ml − 1 ) pepstatin A ( 50 μg ml − 1 ) treatment for 4 h . were stained with an anti - Lamp1 antibody ( upper panels ) colocalization efficiency between GFP - LC3 Lamp1 was counted ( lower panel ) . data are mean ± s . d . of 30 .

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

(a) The maturation of autophagosomes is enhanced in Rubicon knockdown cells. A549 cells stably expressing GFP-LC3, with Rubicon or control shRNA, were incubated in nutrient-rich medium with or without the protease inhibitors (PI) E64d (50 μg ml−1) and pepstatin A (50 μg ml−1) treatment for 4 h. The cells were stained with an anti-Lamp1 antibody (upper panels) and colocalization efficiency between GFP-LC3 and Lamp1 was counted (lower panel). The data are mean ± s.d. of 30 cells.


( b ) . Electron microscopy of Rubicon knockdown . Control ( luciferase ) or Rubicon shRNA A549 grown in nutrient - rich medium was subjected to electron microscopy ( left panels ) . arrows indicate autophagosomes arrowheads indicate autolysosomes . Scale bar , 1 μm . Autophagosomes autolysosomes were counted in 21 , mean ± s . d . are shown ( right panel ) .

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(b). Electron microscopy analysis of Rubicon knockdown cells. Control (luciferase) or Rubicon shRNA A549 cells grown in nutrient-rich medium was subjected to electron microscopy analysis (left panels). The arrows indicate autophagosomes and the arrowheads indicate autolysosomes. Scale bar, 1 μm. Autophagosomes and autolysosomes were counted in 21 cells, and the mean ± s.d. are shown (right panel).


( c ) EGFR degradation in Rubicon knockdown . Control or Rubicon shRNA A549 were treated with EGF ( 200 ng ml − 1 ) for indicated periods lysates were subjected to western blotting with antibodies indicated ( left panel ) . band was measured in three independent experiments mean ± s . d . are shown ( right panel ) .

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(c) EGFR degradation in Rubicon knockdown cells. Control or Rubicon shRNA A549 cells were treated with EGF (200 ng ml−1) for the indicated periods and the lysates were subjected to western blotting with the antibodies indicated (left panel). The band intensity was measured in three independent experiments and the mean ± s.d. are shown (right panel).


( d ) effect of Rubicon overexpression in turnover of EGF receptor . A549 were transfected with advenovirus - GFP or GFP - Rubicon . After 24 h , EGF was added to expressing GFP - Rubicon or control GFP . At times indicated , were lysed subjected to western blotting with indicated antibodies .

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(d) The effect of Rubicon overexpression in the turnover of EGF receptor. A549 cells were transfected with advenovirus-based GFP or GFP-Rubicon. After 24 h, EGF was added to cells expressing GFP-Rubicon or control GFP. At the times indicated, the cells were lysed and subjected to western blotting with the indicated antibodies.


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D ) SDS - PAGE of purified tubulin from brains of mouse models used in current study , stained with Coomassie Brilliant - Blue . Alpha - β - tubulin are separated using a specific SDS - PAGE

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D) SDS-PAGE analysis of purified tubulin from brains of all mouse models used in the current study, stained with Coomassie Brilliant-Blue. Alpha- and β-tubulin are separated using a specific SDS-PAGE protocol


E ) Immunoblot of purified tubulin variants . molecular in ( D ) ( E ) is indicated in kDa .

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E) Immunoblot analysis of all purified tubulin variants. The molecular weight in (D) and (E) is indicated in kDa.


B ) Representative IRM TIRF microscopy showing microtubule positions Tau - meGFP intensities for different PTM variants ( grey : wild type ; blue : Ttll1 - / - ; yellow : Ttll7 - / - ; green : Ttll1 - / - Ttll7 - / - ; pink : Atat1 ‑ / ‑ ) . In last column , false - coloured TIRF highlight differences of Tau - meGFP intensities on different types of microtubules ( Movie EV1 ) . C ) of integrated of Tau - meGFP on GMPCPP microtubules in three independent sets of experiments performed with independently purified tubulin samples . Single assays ( Fig EV2A ) of one experiment were combined after normalisation to wild - type microtubule values ( A . U . = arbitrary units ) . Each data point represents normalised value of one microtubule . D ) Summary three independent experiments shown in ( C ) . Each point is a normalised mean of triplicates , error bars represent standard deviation .

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B) Representative IRM and TIRF microscopy images showing microtubule positions and Tau-meGFP intensities for different PTM variants (grey: wild type; blue: Ttll1-/-; yellow: Ttll7-/-; green: Ttll1-/-Ttll7-/-; pink: Atat1‑/‑). In the last column, false-coloured TIRF images highlight differences of Tau-meGFP intensities on different types of microtubules (Movie EV1). C) Quantification of integrated intensity of Tau-meGFP on GMPCPP microtubules in three independent sets of experiments performed with independently purified tubulin samples. Single assays (Fig EV2A) of one experiment were combined after normalisation to the wild-type microtubule values (A.U. = arbitrary units). Each data point represents the normalised fluorescence intensity value of one microtubule. D) Summary quantification the three independent experiments shown in (C). Each point is a normalised mean of the triplicates, error bars represent standard deviation.


E ) Saturation curve of increasing Tau - mCherry binding to GMPCPP wild - type Ttll1 - / - Ttll7 - / - microtubules . Each data point is shown as mean ± standard deviation of raw integrated values ( A . U . × 104 ) of three experiments with independently purified tubulin samples ( Fig EV2D ) . Dissociation constants Kd R2 coefficient from non - linear fits of each microtubule type are indicated below plot .

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E) Saturation curve of increasing concentration Tau-mCherry binding to GMPCPP wild-type and Ttll1-/-Ttll7-/- microtubules. Each data point is shown as mean ± standard deviation of the raw integrated intensity values (A.U. ×104) of three experiments with independently purified tubulin samples (Fig EV2D). Dissociation constants Kd and R2 coefficient from non-linear fits of each microtubule type are indicated below the plot.


C ) Decay curves of normalised microtubule plotted against time upon incubation with katanin ( A . U . = arbitrary units ) for single microtubules highlighted by arrow heads with yellow borders in ( B ) . D ) Half - life times ( seconds ) of microtubules from assays of which representative are displayed in ( B ) representative decay curves are shown in ( C ) . Each data point represents half - life of a single microtubule . Medians with interquartile ranges shown . Mann - Whitney , p - values displayed .

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C) Decay curves of normalised microtubule length plotted against time upon incubation with katanin (A.U. = arbitrary units) for single microtubules highlighted by arrow heads with yellow borders in (B). D) Half-life times (seconds) of all microtubules from the assays of which representative images are displayed in (B) and representative decay curves are shown in (C). Each data point represents half-life of a single microtubule. Medians with interquartile ranges shown. Mann-Whitney test, p-values displayed.


E ) of normalised microtubule half - lifetimes from 14 independent experiments ( Fig EV3 ) . Each data point represents half - life of a single microtubule . Medians with interquartile ranges shown , Mann - Whitney , p - values displayed .

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E) Analysis of normalised microtubule half-lifetimes from 14 independent experiments (Fig EV3). Each data point represents half-life of a single microtubule. Medians with interquartile ranges shown, Mann-Whitney test, p-values displayed.


B ) Kymographs showing single Kif5B - EGFP motor molecules moving on one single microtubule of known PTM state ( Movie EV4 ) . Horizontal scale bars 2 µm ; vertical scale bars 5 C ) Scatter plots showing average velocities ( µm / s ) exhibited by single Kif5b - EGFP molecules on different microtubule types ( each data point represents one Kif5b - EGFP molecule ) . Data pooled from 29 independent experiments ( Appendix Fig S2 ) . Medians with interquartile ranges shown , Mann - Whitney , p - values displayed .

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B) Kymographs showing single Kif5B-EGFP motor molecules moving on one single microtubule of known PTM state (Movie EV4). Horizontal scale bars 2 µm; vertical scale bars 5 C) Scatter plots showing the average velocities (µm/s) exhibited by single Kif5b-EGFP molecules on the different microtubule types (each data point represents one Kif5b-EGFP molecule). Data pooled from 29 independent experiments (Appendix Fig S2). Medians with interquartile ranges shown, Mann-Whitney test, p-values displayed.


D , E ) Survival probability ( Kaplan - Meier plots , taking varying microtubule lengths into account ; see Methods ) of interaction time ( D ) run ( E ) of Kif5b - EGFP molecules on different microtubule types . Data in ( C , D , E ) were pooled from experiments ( Appendix Fig S2 ) without normalisation . 50 % probability values are show in plots . Statistical displayed in tables below : hazard ratios with 95 % confidence intervals pairing data from different microtubule types ( * pairs differing with significance level p < 0 . 05 ) .

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D,E) Survival probability (Kaplan-Meier plots, taking varying microtubule lengths into account; see Methods) of the interaction time (D) and run length (E) of Kif5b-EGFP molecules on different microtubule types. Data in (C,D,E) were pooled from individual experiments (Appendix Fig S2) without normalisation. The 50% probability values are show in the plots. Statistical analysis displayed in the tables below: hazard ratios with 95% confidence intervals pairing all data from different microtubule types (*pairs differing with significance level p<0.05).


B : Hippocampal neurons transduced with GFP - TDP - 25 constructs ( DIV5 + 8 ) or GFP - TDP - 43 ( DIV5 + 4 due to higher toxicity ) were sequentially extracted with RIPA buffer followed by 2 % SDS buffer analyzed via immuoblotting . Left panel shows representative immunoblot for GFP loading control Calnexin . Right panel shows of of respective GFP densiometric signal in SDS extract to RIPA extract . Barplots showing mean ± SD from n = 3 independent experiments . TDP - 43 ( 0 . 039 ± 0 . 029 , mean ± CI ) vs TDP - 25 wt ( 1 . 22 ± 0 . 296 ) vs TDP - 25 mut ( 0 . 928 ± 0 . 564 ) : F ( 2 , 6 ) = 51 . 4 , * * * p = 0 . 000168 , η² = 0 . 94 . TDP - 43 vs TDP - 25 wt : * * * p = 1 . 67 * 10 - 4 , TDP - 43 vs TDP - 25 mut : p = 7 . 983 * 10 - 4 , TDP - 25 wt vs TDP - 25 mut : p = 0 . 1182 ,

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B: Hippocampal neurons transduced with GFP-TDP-25 constructs (DIV5+8) or GFP-TDP-43 (DIV5+4 due to higher toxicity) were sequentially extracted with RIPA buffer followed by 2% SDS buffer and analyzed via immuoblotting. Left panel shows representative immunoblot for GFP and loading control Calnexin. Right panel shows quantification of the ratio of the respective GFP densiometric signal in SDS extract to RIPA extract. Barplots showing mean ± SD from n=3 independent experiments. TDP-43 (0.039 ± 0.029, mean ± CI) vs TDP-25 wt (1.22 ± 0.296) vs TDP-25 mut (0.928 ± 0.564): F(2,6)=51.4, ***p=0.000168, η²=0.94. TDP-43 vs TDP-25 wt: ***p=1.67*10-4, TDP-43 vs TDP-25 mut: p=7.983*10-4, TDP-25 wt vs TDP-25 mut: p=0.1182,


( A - D ) rat hippocampal neurons were transduced with GFP - TDP - 25 variants , GFP - NPM1 or Htt97Q - GFP analyzed by recovery after photobleaching ( FRAP ) at DIV5 + 8 , except Htt97Q - GFP which was analyzed at DIV5 + 6 to avoid excessive toxicity . Example with indicated bleach regions ( dashed circles ) are shown ( A , C ) . Scale bar = 1 . 5 µm . Resulting normalized FRAP curves ( relative over time ) with values representing means ± SD are shown in ( B , D ) . ( B ) Comparison of averaged recovery fraction of last six timepoints obtained from three independent experiments . GFP - NPM1 ( 0 . 982 ± 0 . 019 , mean ± CI , n = 37 ) vs GFP - TDP - 25 wt ( 0 . 097 ± 0 . 013 , n = 35 ) vs GFP - TDP - 25 mut ( 0 . 085 ± 0 . 006 , n = 38 ) : H ( 1 ) = 74 . 134 , df = 2 , * * * p < 2 . 2 * 10 - 16 , η² [ H ] = 0 . 674 , Kruskal - Wallis . GFP - NPM1 vs GFP - TDP - 25 wt : * * * p < 2 * 10 - 16 , GFP - NPM1 vs GFP - TDP - 25 mut : p < 2 * 10 - 16 , GFP - TDP - 25 wt vs GFP - TDP - 25 mut : p = 0 . 11 , Pairwise Wilcoxon Rank Sum with Benjamini - Hochberg correction . In ( D ) , recovery fraction averaged at > 20 min timepoints was compared from four independent experiments . Htt97Q - GFP ( 0 . 094 ± 0 . 017 , n = 25 ) vs GFP - TDP - 25 wt ( 0 . 312 ± 0 . 041 , n = 23 ) vs GFP - TDP - 25 mut ( 0 . 271 ± 0 . 029 , n = 24 ) : H ( 1 ) = 46 . 376 , df = 2 , * * * p = 8 . 501 * 10 - 11 , η² [ H ] = 0 . 643 , Kruskal - Wallis . Htt97Q - GFP vs GFP - TDP - 25 wt * * * p = 1 . 9 * 10 - 11 , Htt97Q - GFP vs GFP - TDP - 25 mut * * * p = 4 . 3 * 10 - 12 , GFP - TDP - 25 wt vs GFP - TDP - 25 mut : p = 0 . 12 , Pairwise Wilcoxon Rank Sum with Benjamini - Hochberg correction .

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(A-D) Primary rat hippocampal neurons were transduced with GFP-TDP-25 variants, GFP-NPM1 or Htt97Q-GFP and analyzed by fluorescence recovery after photobleaching (FRAP) at DIV5+8, except Htt97Q-GFP which was analyzed at DIV5+6 to avoid excessive toxicity. Example images with indicated bleach regions (dashed circles) are shown (A, C). Scale bar = 1.5 µm. Resulting normalized FRAP curves (relative fluorescence intensity over time) with values representing means ± SD are shown in (B, D). (B) Comparison of the averaged recovery fraction of the last six timepoints obtained from three independent experiments. GFP-NPM1 (0.982 ± 0.019, mean ± CI, n=37 cells) vs GFP-TDP-25 wt (0.097 ± 0.013, n=35 cells) vs GFP-TDP-25 mut (0.085 ± 0.006, n=38 cells): H(1)=74.134, df=2, ***p<2.2*10-16, η²[H]=0.674, Kruskal-Wallis Test. GFP-NPM1 vs GFP-TDP-25 wt: ***p<2*10-16, GFP-NPM1 vs GFP-TDP-25 mut: p<2*10-16, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.11, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction. In (D), the recovery fraction averaged at >20 min timepoints was compared from four independent experiments. Htt97Q-GFP (0.094 ± 0.017, n=25 cells) vs GFP-TDP-25 wt (0.312 ± 0.041, n=23 cells) vs GFP-TDP-25 mut (0.271 ± 0.029, n=24 cells): H(1)=46.376, df=2, ***p=8.501*10-11, η²[H]=0.643, Kruskal-Wallis Test. Htt97Q-GFP vs GFP-TDP-25 wt ***p=1.9*10-11, Htt97Q-GFP vs GFP-TDP-25 mut ***p=4.3*10-12, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.12, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction.


E : Immunofluorescence of hippocampal neurons transduced with GFP , GFP - TDP - 25 wild - type or mutant treated with MG132 ( 10 µM , 14h , DIV5 + 8 ) . Representative of GFP - TDP - 25 inclusions diffuse . Automated of GFP normalized to number of DAPI stained nuclei ( omitted here for clarity ) in MG132 treated compared to DMSO control . Counterstain to label neuronal cytoskeleton ( MAP2 ) . Scale bar = 10 µm . Box plot as in Fig . 3C . GFP ( 1 . 23 ± 0 . 393 , n = 5 independent experiments ) vs GFP - TDP - 25 wt ( 2 . 11 ± 0 . 804 , n = 5 ) vs GFP - TDP - 25 mut ( 2 . 36 ± 1 . 16 , n = 5 ) : H ( 1 ) = 7 . 34 , df = 2 , p = 0 . 02548 , η² [ H ] = 0 . 445 , Kruskal - Wallis . GFP vs GFP - TDP - 25 wt : * p = 0 . 048 , GFP vs GFP - TDP - 25 mut : * p = 0 . 048 , GFP - TDP - 25 wt vs GFP - TDP - 25 mut : p = 0 . 690 , Pairwise Wilcoxon Rank Sum with Benjamini - Hochberg correction .

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E: Immunofluorescence of hippocampal neurons transduced with GFP, GFP-TDP-25 wild-type or mutant and treated with MG132 (10 µM, 14h, DIV5+8). Representative images of GFP-TDP-25 inclusions and diffuse cytoplasmic staining. Automated quantification of GFP intensity normalized to the number of DAPI stained nuclei (omitted here for clarity) in MG132 treated cells compared to DMSO control. Counterstain to label the neuronal cytoskeleton (MAP2). Scale bar = 10 µm. Box plot as in Fig. 3C. GFP (1.23 ± 0.393, n=5 independent experiments) vs GFP-TDP-25 wt (2.11 ± 0.804, n=5) vs GFP-TDP-25 mut (2.36 ± 1.16, n=5): H(1)=7.34, df=2, p=0.02548, η²[H]=0.445, Kruskal-Wallis test. GFP vs GFP-TDP-25 wt: *p=0.048, GFP vs GFP-TDP-25 mut: *p=0.048, GFP-TDP-25 wt vs GFP-TDP-25 mut: p=0.690, Pairwise Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction.


A , Reconstructed MSOT ( 800 nm ) without with linear unmixing data in neck upper lower abdominal areas , showing ROIs for liver , kidney , iBAT , rpWAT , aorta , SV . Unmixing result : blue for Hb , red for HbO2 , yellow for lipid . colour bar shows colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( maximum value neck / upper abdominal / lower abdominal : Hb : 2 . 5 / 3 . 5 / 0 . 9 ; HbO2 : 2 . 9 / 4 . 9 / 2 . 5 ; Lipid : 24000 / 19000 / 13000 ) Scale bar : 4 mm .

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A, Reconstructed MSOT images (800 nm) without and with linear unmixing data in the neck and the upper and lower abdominal areas, showing the ROIs for the liver, kidney, iBAT, rpWAT, aorta, and SV. Unmixing result: blue for Hb, red for HbO2, yellow for lipid. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (maximum value neck/upper abdominal/lower abdominal: Hb: 2.5/3.5/0.9; HbO2:2.9/4.9/2.5; Lipid: 24000/19000/13000) Scale bar: 4 mm.


A , Reconstructed MSOT image ( 800 nm ) with linear unmixing data of Hb , HbO2 , lipid from chow HFD - fed mice . colour bar shows colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( maximum value chow / HFD : Hb : 1 . 3 / 0 . 6 ; HbO2 : 1 . 8 / 0 . 9 ; Lipid : 27000 / 27000 ) . Scale bar : 4 mm .

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A, Reconstructed MSOT image (800 nm) with linear unmixing data of Hb, HbO2, and lipid from chow and HFD-fed mice. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (maximum value chow/HFD: Hb: 1.3/0.6; HbO2: 1.8/0.9; Lipid: 27000/27000). Scale bar: 4 mm.


D , Tissue oxygenation ( sO2 ) total blood volume ( TBV ) results from liver , kidney , iBAT , rpWAT , Each dot represents data from one , in total 8 ( n = 8 ) . Data represent mean ( + / - 95 % confidence ) . unpaired t was used to verify statistical significance . Data information : In figure , A . U . = arbitrary units .

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D, Tissue oxygenation (sO2) and total blood volume (TBV) results from liver, kidney, iBAT, and rpWAT, Each dot represents data from one animal, in total 8 animals (n = 8). Data represent the mean (+/- 95% confidence). The unpaired t test was used to verify the statistical significance. Data information: In the figure, A.U. = arbitrary units.


A , Oil - red - O of control ( healthy ) steatotic liver . Scale bar : 100 µm .

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A, Oil-red-O staining of control (healthy) and steatotic liver. Scale bar: 100 µm.


B , Reconstructed MSOT image ( 800 nm ) with linear unmixing difference value of lipid in lower abdominal section . Unmixing result : blue for Hb , red for HbO2 , yellow for lipid , jet for 700 nm - 930 nm difference . colour bar shows colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( Maximum value control / steatosis : Hb : 1 . 1 / 0 . 5 ; HbO2 : 1 . 9 / 0 . 8 ; Lipid : 21000 / 24000 ; 700 - 900nm : 3000 / 3000 ) Scale bar : 4 mm .

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B, Reconstructed MSOT image (800 nm) with linear unmixing and difference value of lipid in lower abdominal section. Unmixing result: blue for Hb, red for HbO2, yellow for lipid, jet for 700 nm - 930 nm difference. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value control/steatosis: Hb: 1.1/0.5; HbO2: 1.9/0.8; Lipid: 21000/24000; 700-900nm: 3000/3000) Scale bar: 4 mm.


A , HE of livers with grade 0 - 3 steatosis . Scale bar : 100 µm .

A, HE staining of livers with grade 0 - 3 steatosis. Scale bar: 100 µm.


E , Reconstructed MSOT image ( 800 nm ) with linear unmixing data of Hb , HbO2 , lipid from chow HFD - fed mice . Unmixing result : blue for Hb , red for HbO2 , yellow for lipid . colour bar shows colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( Maximum value chow 1 month / chow 2 month / chow 3 month / HFD 1 month / HFD 2 month / HFD 3 month : Hb : 2 . 5 / 1 . 9 / 2 . 9 / 2 . 7 / 1 . 8 / 1 . 3 ; HbO2 : 2 . 8 / 2 . 6 / 2 . 8 / 3 . 1 / 2 . 1 / 1 . 9 ; Lipid : 27000 / 19000 / 29000 / 32000 / 26000 / 27000 ) . Scale bar : 4 mm .

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E, Reconstructed MSOT image (800 nm) with linear unmixing data of Hb, HbO2, and lipid from chow and HFD-fed mice. Unmixing result: blue for Hb, red for HbO2, yellow for lipid. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value chow 1 month/ chow 2 month/ chow 3 month/HFD 1 month/HFD 2 month/HFD 3 month: Hb: 2.5/1.9/2.9/2.7/1.8/1.3; HbO2: 2.8/2.6/2.8/3.1/2.1/1.9; Lipid: 27000/19000/29000/ 32000/26000/27000). Scale bar: 4 mm.


F , Longitudinal track of lipid in mouse livers . Each represents data from one . Each data point is from a single measurement of each at each time point . have grade 3 steatosis at endpoint confirmed by histology .

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F, Longitudinal track of lipid in mouse livers. Each line represents data from one animal. Each data point is from a single measurement of each animal at each time point. All animals have grade 3 steatosis at the endpoint confirmed by histology.


A , Reconstructed MSOT image ( 800 nm ) with linear unmixing data of ICG from mice with without steatosis . colour bar shows colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( Maximum value control / steatosis : 0 . 07 / 0 . 02 ) Scale bar : 4 mm .

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A, Reconstructed MSOT image (800 nm) with linear unmixing data of ICG from mice with and without steatosis. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value control/steatosis: 0.07/0.02) Scale bar: 4 mm.


E , Representative microscopy HE of control steatotic liver . Scale bar : 100 µm .

E, Representative fluorescence microscopy and HE staining images of control and steatotic liver. Scale bar: 100 µm.


( A ) Ookinetes sandwiched between two hydrogel surfaces .

(A) Ookinetes sandwiched between two hydrogel surfaces.