HuH - 7 ( A ) and Hep3B ( B ) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0 . 1 % FBS in the presence of Dyn2 inhibitors or DMSO as indicated . Representative images of inhibitor - treated and control cells ( stained with Oil Red O ) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments . Pharmacological inhibitors used were : Dynasore ( inhibits Dyn2 GTPase activity ) , MiTMAB ( targets PH domain and interferes with membrane binding ) , Dynole 34 - 2 ( allosteric GTPase inhibitor ) , and Dynole 31 - 2 ( negative control for Dynole 34 - 2 ) . Bars , 20 µM .

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HuH-7 (A) and Hep3B (B) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS in the presence of Dyn2 inhibitors or DMSO as indicated. Representative images of inhibitor-treated and control cells (stained with Oil Red O) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments. Pharmacological inhibitors used were: Dynasore (inhibits Dyn2 GTPase activity), MiTMAB (targets PH domain and interferes with membrane binding), Dynole 34-2 (allosteric GTPase inhibitor), and Dynole 31-2 (negative control for Dynole 34-2). Bars, 20 µM.


( C ) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM Dyn2 for 17 h . Knockout of Dyn2 was induced by treatment with 2 µM 4 - hydroxy - tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 ( top row ) and by immunoblot ( D ) . Bars , 20 µM . ( E and F ) Average LD number ( E ) and area ( F ) per cell from five independent experiments . All data are represented as mean ± SE . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 .

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

(C) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM Dyn2 for 17 h. Knockout of Dyn2 was induced by treatment with 2 µM 4-hydroxy-tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 (top row) and by immunoblot (D). Bars, 20 µM. (E and F) Average LD number (E) and area (F) per cell from five independent experiments. All data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01.


( G ) Whole - cell lysates and LD fractions isolated from HuH - 7 hepatocytes under resting or starved ( 2 h HBSS starvation ) conditions .

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(G) Whole-cell lysates and LD fractions isolated from HuH-7 hepatocytes under resting or starved (2 h HBSS starvation) conditions.


( H ) Primary hepatocyte expressing Dyn2 - GFP , showing an absence of colocalization with the LD surface ( stained with Oil Red O ) . Bar , 20 µM . Inset shows magnification of boxed region ( bar , 2 µM ) .

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(H) Primary hepatocyte expressing Dyn2-GFP, showing an absence of colocalization with the LD surface (stained with Oil Red O). Bar, 20 µM. Inset shows magnification of boxed region (bar, 2 µM).


Hep3B cells treated with either a nontargeting control siRNA ( A and B , siNT ) or an siRNA targeting human Dyn2 ( C and D , siDyn2 ) were fixed and co - stained with antibodies specific for LAMP1 ( red ) and LC3 ( green ) . After Dyn2 knockdown , a juxtanuclear aggregation and enlargement of the LAMP1 - positive compartment is observed ( C and D , arrows ) . Increased labeling of LC3 is also detectable after knockdown of Dyn2 .

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

Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and co-stained with antibodies specific for LAMP1 (red) and LC3 (green). After Dyn2 knockdown, a juxtanuclear aggregation and enlargement of the LAMP1-positive compartment is observed (C and D, arrows). Increased labeling of LC3 is also detectable after knockdown of Dyn2.


( E ) Western blotting of Hep3B lysates after a 3 - d treatment with either the control or Dyn2 - targeted siRNA and further treatment with or without 50 µM leupeptin . Densitometry - based analysis of six independent experiments is shown at the bottom of the figure .

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(E) Western blotting of Hep3B lysates after a 3-d treatment with either the control or Dyn2-targeted siRNA and further treatment with or without 50 µM leupeptin. Densitometry-based analysis of six independent experiments is shown at the bottom of the figure.


( F ) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore , in the presence or absence of 50 µM leupeptin . Quantitation of LC3 - II levels relative to control are shown below the blots . The data are represented as mean ± SE ; * , P ≤ 0 . 05 .

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(F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.


Dyn2 knockdown results in the formation of enlarged autophagic structures . ( A - D ) Transmission electron micrographs ( TEMs ) of oleate - loaded Hep3B hepatocytes treated with nontargeting control ( siNT ) or Dyn2 ( siDyn2 ) siRNA for 72 h . Bars : ( A and B ) 2 µm ; ( A ′ and B ′ ) 1 µm ; ( C and D ) 0 . 5 µm . Insets in A and B show fluorescent micrographs of LAMP1 - stained cells ( bars , 10 µM ) . Control cells ( A ) contain an abundance of small ( < 1 µm ) electron - dense lysosomes and autolysosomes ( arrowheads ) . Under conditions in which Dyn2 expression is suppressed ( B - D ) , far fewer small lysosomes are observed . Instead , the cells are populated by larger autolysosomes ( ≥ 1 µm ) with aberrant morphologies and containing putative LDs ( * ) . ( E ) Quantitative measure of autolysosomal size from cells in which Dyn2 was knocked down as compared with control cells . Values represent the fold - increase in the number of autophagic structures of a given diameter ( measured in microns ) observed over siNT - treated control cells . Data were obtained from a single experiment with n = 6 cells examined by electron microscopy from each condition .

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Dyn2 knockdown results in the formation of enlarged autophagic structures. (A-D) Transmission electron micrographs (TEMs) of oleate-loaded Hep3B hepatocytes treated with nontargeting control (siNT) or Dyn2 (siDyn2) siRNA for 72 h. Bars: (A and B) 2 µm; (A′ and B′) 1 µm; (C and D) 0.5 µm. Insets in A and B show fluorescent micrographs of LAMP1-stained cells (bars, 10 µM). Control cells (A) contain an abundance of small (<1 µm) electron-dense lysosomes and autolysosomes (arrowheads). Under conditions in which Dyn2 expression is suppressed (B-D), far fewer small lysosomes are observed. Instead, the cells are populated by larger autolysosomes (≥1 µm) with aberrant morphologies and containing putative LDs (*). (E) Quantitative measure of autolysosomal size from cells in which Dyn2 was knocked down as compared with control cells. Values represent the fold-increase in the number of autophagic structures of a given diameter (measured in microns) observed over siNT-treated control cells. Data were obtained from a single experiment with n = 6 cells examined by electron microscopy from each condition.


Hep3B cells treated with either a nontargeting control siRNA ( A and B , siNT ) or an siRNA targeting human Dyn2 ( C and D , siDyn2 ) were fixed and stained with an antibody specific for LAMP1 after a 24 - h starvation period in media containing 0 . 1 % FBS . Control cells displayed normal lysosomal compartments , with LAMP1 - stained structures dispersed throughout the cytosol , some containing short reformation tubules ( B ′ ) . After Dyn2 knockdown , many cells contained enlarged autolysosomes with a noticeable increase in persistent and lengthy reformation tubules emanating from LAMP1 - positive compartments ( C ′ and D ′ ) . Bars : ( A - D ) 20 µM ; ( A ′ and B ′ ) 5 µM ; ( C ′ and D ′ ) 10 µM . ( E and F ) Measurements of the length ( E ) and distribution ( F ) of LAMP1 - stained ALR tubules in control ( siNT ) and siDyn2 - treated cells . Data represent the mean derived from measurements of tubules in a minimum of 40 cells for each experimental group over 6 independent experiments . Error bars represent SE ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 .

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

Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and stained with an antibody specific for LAMP1 after a 24-h starvation period in media containing 0.1% FBS. Control cells displayed normal lysosomal compartments, with LAMP1-stained structures dispersed throughout the cytosol, some containing short reformation tubules (B′). After Dyn2 knockdown, many cells contained enlarged autolysosomes with a noticeable increase in persistent and lengthy reformation tubules emanating from LAMP1-positive compartments (C′ and D′). Bars: (A-D) 20 µM; (A′ and B′) 5 µM; (C′ and D′) 10 µM. (E and F) Measurements of the length (E) and distribution (F) of LAMP1-stained ALR tubules in control (siNT) and siDyn2-treated cells. Data represent the mean derived from measurements of tubules in a minimum of 40 cells for each experimental group over 6 independent experiments. Error bars represent SE; *, P ≤ 0.05; **, P ≤ 0.01.


( G ) TEM of siDyn2 - treated Hep3B hepatocyte exhibiting enlarged autolysosomal structures with extensive tubulation ( arrows ) . Bar , 2 µm . ( G ′ ) Inset showing enlargement of autolysosomal tubule ( arrows ) . Bar , 1 µm . ( H ) Tubulating autolysosome with engulfed lipid droplet . Bar , 0 . 5 µm .

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(G) TEM of siDyn2-treated Hep3B hepatocyte exhibiting enlarged autolysosomal structures with extensive tubulation (arrows). Bar, 2 µm. (G′) Inset showing enlargement of autolysosomal tubule (arrows). Bar, 1 µm. (H) Tubulating autolysosome with engulfed lipid droplet. Bar, 0.5 µm.


Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation ( ALR ) and lysosomal tubule scission . ( A - D ) Still frames from time - lapse movies of Hep3B cells expressing LAMP1 - mCherry . Cells were starved for 2 h in HBSS and subsequently treated for 30 min with either DMSO ( A and B ) or 40 µM Dynasore ( C and D ) , which induced extensive tubulation of LAMP1 - positive compartments . Bars ( A - D ) : 20 µM ; ( A ′ - B ′ ) 2 µM ; ( C ′ - D ′ ) 10 µM . ( E - G ) To demonstrate the reversibility of this tubulation , Dynasore - treated cells were washed extensively with drug - free media containing 10 % FBS and monitored by time - lapse microscopy for 45 min . Frequently , after drug washout , LAMP1 - positive tubules exhibited noticeable varicosities ( E and F , arrows ; bars , 10 µM ) along their length . These sites are suggestive of areas of scission and resumed budding of nascent protolysosomes from the reformation tubules ( G ; bars , 10 µM ) . ( H ) Tubules from cells undergoing drug washout were quantified by tracing their lengths at the beginning and end of these movies . Still frames from a representative movie show tubule content at t = 10 and 45 min after drug washout . Bars , 20 µM . ( I ) Analysis of five independent movies showed an average decrease in total tubulation of ∼ 50 % after drug washout . Data represent the average relative change in total tubule length between the first and last frames of the time - lapse movies . Error bars represent SE ; * , P < 0 . 05 .

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

Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. (A-D) Still frames from time-lapse movies of Hep3B cells expressing LAMP1-mCherry. Cells were starved for 2 h in HBSS and subsequently treated for 30 min with either DMSO (A and B) or 40 µM Dynasore (C and D), which induced extensive tubulation of LAMP1-positive compartments. Bars (A-D): 20 µM; (A′-B′) 2 µM; (C′-D′) 10 µM. (E-G) To demonstrate the reversibility of this tubulation, Dynasore-treated cells were washed extensively with drug-free media containing 10% FBS and monitored by time-lapse microscopy for 45 min. Frequently, after drug washout, LAMP1-positive tubules exhibited noticeable varicosities (E and F, arrows; bars, 10 µM) along their length. These sites are suggestive of areas of scission and resumed budding of nascent protolysosomes from the reformation tubules (G; bars, 10 µM). (H) Tubules from cells undergoing drug washout were quantified by tracing their lengths at the beginning and end of these movies. Still frames from a representative movie show tubule content at t = 10 and 45 min after drug washout. Bars, 20 µM. (I) Analysis of five independent movies showed an average decrease in total tubulation of ∼50% after drug washout. Data represent the average relative change in total tubule length between the first and last frames of the time-lapse movies. Error bars represent SE; *, P < 0.05.


( A and B ) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS and treated with 40 µM Dynasore , to induce tubule formation , as in Fig . 5 . Cells were lysed ( WCL ) , and the post - nuclear supernatant ( PNS ) was pelleted by centrifugation to produce a crude lysosomal fraction ( CLF ) and high - speed supernatant ( HSS ) . The CLF was subsequently loaded onto an 8 - 27 % discontinuous iodixanol ( OptiPrep ) gradient for separation by ultracentrifugation . Nine fractions were collected from the top of the gradient and blotted for Dyn2 and the LAMP1 resident protein , LAMP1 . Lysosomal acid phosphatase activity roughly correlates with LAMP1 protein levels in each fraction . Levels of Dyn2 are highest in fraction 2 , corresponding with both the peak levels of LAMP1 and lysosomal activity . ( A ) The data shown are from a single representative experiment out of three repeats . ( B ) Blotting for subcellular components shows that Dyn2 is specific for these same fractions , unlike other organelle markers such as EEA1 ( early endosomes ) and COXIV ( mitochondria ) .

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(A and B) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS and treated with 40 µM Dynasore, to induce tubule formation, as in Fig. 5. Cells were lysed (WCL), and the post-nuclear supernatant (PNS) was pelleted by centrifugation to produce a crude lysosomal fraction (CLF) and high-speed supernatant (HSS). The CLF was subsequently loaded onto an 8-27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected from the top of the gradient and blotted for Dyn2 and the LAMP1 resident protein, LAMP1. Lysosomal acid phosphatase activity roughly correlates with LAMP1 protein levels in each fraction. Levels of Dyn2 are highest in fraction 2, corresponding with both the peak levels of LAMP1 and lysosomal activity. (A) The data shown are from a single representative experiment out of three repeats. (B) Blotting for subcellular components shows that Dyn2 is specific for these same fractions, unlike other organelle markers such as EEA1 (early endosomes) and COXIV (mitochondria).


( C and D ) Dyn2 localizes to the surface of LAMP1 - positive compartments . Fluorescence imaging of Hep3B hepatocytes transfected with Dyn2 - GFP and LAMP1 - mCherry under resting ( C and C ′ ) or starvation ( D ) conditions . ( C ′ ′ ) Dyn2 - GFP localizing to LAMP1 - labeled lysosome structures under starvation conditions . Arrows indicate regions of protein colocalization . Dyn2 ( arrows ) is present at the site of scission of LAMP1 - positive tubules ( arrowhead ) from large autolysosomal structures . Bars : ( C ) 5 µM ; ( C ′ , C ′ ′ , and D ) 3 µM .

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(C and D) Dyn2 localizes to the surface of LAMP1-positive compartments. Fluorescence imaging of Hep3B hepatocytes transfected with Dyn2-GFP and LAMP1-mCherry under resting (C and C′) or starvation (D) conditions. (C′′) Dyn2-GFP localizing to LAMP1-labeled lysosome structures under starvation conditions. Arrows indicate regions of protein colocalization. Dyn2 (arrows) is present at the site of scission of LAMP1-positive tubules (arrowhead) from large autolysosomal structures. Bars: (C) 5 µM; (C′, C′′, and D) 3 µM.


( b ) RT - PCR analysis using the blue primer set ( left ) and green primer set ( right ) from a . Whereas control cDNA samples showed a single product corresponding to the wild - type allele ( WT ) , an apparently longer product was observed in subjects 1 , 2 and 5 , indicating that only the transcripts from the mutant allele were expressed . In subject 3 , both wild - type and mutant alleles were expressed . Template without reverse transcriptase was used as a negative control , RT ( - ) .

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(b) RT-PCR analysis using the blue primer set (left) and green primer set (right) from a. Whereas control cDNA samples showed a single product corresponding to the wild-type allele (WT), an apparently longer product was observed in subjects 1, 2 and 5, indicating that only the transcripts from the mutant allele were expressed. In subject 3, both wild-type and mutant alleles were expressed. Template without reverse transcriptase was used as a negative control, RT(-).


( a - e ) T1 - weighted imaging shows hyperintensity of the substantia nigra with a central band of T1 - weighted hypointensity ( arrowheads ) . Images are shown for subject 1 at 33 years ( a ) , subject 2 at 25 years ( b ) , subject 3 at 39 years ( c ) , subject 4 at 46 years ( d ) and subject 5 at 33 years ( e ) . ( f - h ) T2 - weighted imaging shows marked hypointensity of the globus pallidus ( arrows ) , suggesting iron deposition . Cerebral atrophy and mild cerebellar atrophy are also seen . Images are shown for subject 1 ( f ) , subject 2 ( g ) and subject 3 ( h ) . ( i , j ) The fluid attenuated inversion recovery ( FLAIR ) image of subject 1 ( i ) and the T1 - weighted FLAIR coronal image of subject 2 ( j ) also show cerebral atrophy .

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(a-e) T1-weighted imaging shows hyperintensity of the substantia nigra with a central band of T1-weighted hypointensity (arrowheads). Images are shown for subject 1 at 33 years (a), subject 2 at 25 years (b), subject 3 at 39 years (c), subject 4 at 46 years (d) and subject 5 at 33 years (e). (f-h) T2-weighted imaging shows marked hypointensity of the globus pallidus (arrows), suggesting iron deposition. Cerebral atrophy and mild cerebellar atrophy are also seen. Images are shown for subject 1 (f), subject 2 (g) and subject 3 (h). (i,j) The fluid attenuated inversion recovery (FLAIR) image of subject 1 (i) and the T1-weighted FLAIR coronal image of subject 2 (j) also show cerebral atrophy.


( a ) Immunoblot analysis of the WIPI4 protein ( apparent mobility at ∼ 35 kDa ) in LCLs ( top ) . Truncated forms were not detected ( middle ) . HSP90 was used as a loading control ( bottom ) . An asterisk indicates nonspecific immunoreactive bands .

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(a) Immunoblot analysis of the WIPI4 protein (apparent mobility at ∼35 kDa) in LCLs (top). Truncated forms were not detected (middle). HSP90 was used as a loading control (bottom). An asterisk indicates nonspecific immunoreactive bands.


( b ) Cells were treated with 250 nM Torin1 in the presence or absence of 20 μM chloroquine for 2 h . Cell lysates were analyzed by SDS - PAGE and immunoblotting using antibodies to LC3 , WIPI4 and HSP90 . The positions of LC3 - I ( cytosolic ) and LC3 - II ( membrane bound ) are indicated .

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(b) Cells were treated with 250 nM Torin1 in the presence or absence of 20 μM chloroquine for 2 h. Cell lysates were analyzed by SDS-PAGE and immunoblotting using antibodies to LC3, WIPI4 and HSP90. The positions of LC3-I (cytosolic) and LC3-II (membrane bound) are indicated.


( c - e ) Cells were cultured in the presence of Torin1 for 2 h . ( c ) Cytospun cells were fixed and analyzed by immunofluorescence microscopy using antibodies to LC3 and ATG9A . Abnormal colocalization of LC3 with ATG9A was observed in the LCLs of affected subjects . Scale bars , 10 μm and 1 μm in the inset . ( d , e ) The numbers of LC3 + ( d ) and LC3 + ATG9A + ( e ) foci were quantified from more than 20 images from 3 independent samples ( Online Methods ) . Data are presented as mean ± s . e . m . * P 0 . 05 , ANOVA followed by Bonferroni - Dunn post - hoc test .

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(c-e) Cells were cultured in the presence of Torin1 for 2 h. (c) Cytospun cells were fixed and analyzed by immunofluorescence microscopy using antibodies to LC3 and ATG9A. Abnormal colocalization of LC3 with ATG9A was observed in the LCLs of affected subjects. Scale bars, 10 μm and 1 μm in the inset. (d,e) The numbers of LC3+ (d) and LC3+ATG9A+ (e) foci were quantified from more than 20 images from 3 independent samples (Online Methods). Data are presented as mean ± s.e.m. *P 0.05, ANOVA followed by Bonferroni-Dunn post-hoc test.


B Ub chain interaction of the LUBEL - NZF and UBA2 domains . Immobilized GST - NZF or GST - UBA2 was incubated with Lys ( K ) 48 - , K 63 - linked , or linear di - Ub ( Ub2 ) chains and bound Ub chains were subjected to immunoblotting using anti - Ub antibody or a linear linkage - specific Ub antibody ( anti - linear Ub antibody ) . Loading of GST - fusion proteins was visualized by Ponceau S staining .

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

B Ub chain interaction of the LUBEL-NZF and UBA2 domains. Immobilized GST-NZF or GST-UBA2 was incubated with Lys (K) 48-, K 63-linked, or linear di-Ub (Ub2) chains and bound Ub chains were subjected to immunoblotting using anti-Ub antibody or a linear linkage-specific Ub antibody (anti-linear Ub antibody). Loading of GST-fusion proteins was visualized by Ponceau S staining.


A In vitro ubiquitination assays of LUBEL - RBR - C WT or catalytically dead C2704A in combination with Ube1 and two different Drosophila E2s , UbcD10 or Effete / UbcD1 . Reactions were terminated at indicated times and synthesized Ub chains were detected by immunoblotting using anti - linear Ub antibody . Total protein loading was visualized by Ponceau S staining . * : nonspecific band .

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

A In vitro ubiquitination assays of LUBEL-RBR-C WT or catalytically dead C2704A in combination with Ube1 and two different Drosophila E2s, UbcD10 or Effete/UbcD1. Reactions were terminated at indicated times and synthesized Ub chains were detected by immunoblotting using anti-linear Ub antibody. Total protein loading was visualized by Ponceau S staining. * : nonspecific band.


B In vitro deubiquitination of Ub chains synthesized by HOIP - and LUBEL - RBR - C . Ub chains produced by RBR - C ( Human or Drosophila ) with UbcH7 , UbcD10 or Effete were incubated with a linear linkage - specific DUB , OTULIN ( WT or catalytically dead C129A ( C / A ) mutant ) or a Lys - linkage - specific DUB , vOTU . The DUB - treated samples were subjected to Coomassie staining or immunoblotting using anti - Ub antibody .

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

B In vitro deubiquitination of Ub chains synthesized by HOIP- and LUBEL-RBR-C. Ub chains produced by RBR-C (Human or Drosophila) with UbcH7, UbcD10 or Effete were incubated with a linear linkage-specific DUB, OTULIN (WT or catalytically dead C129A (C/A) mutant) or a Lys-linkage-specific DUB, vOTU. The DUB-treated samples were subjected to Coomassie staining or immunoblotting using anti-Ub antibody.


C Thioester formation assay using Atto647 - labelled Ub . Ubiquitination assay using LUBEL - RBR - C WT ( left panel ) or C2704A ( right panel ) : lanes 1 / 2 Atto - Ub + E1 , lanes 3 / 4 + ATP , lanes 5 / 6 + E2 , lanes 7 / 8 + E3 , and lanes 9 / 10 + Ub - His6 . In the presence of N - terminally tagged Atto - Ub and C - terminally tagged Ub - His6 , only Ub2 product can be synthesized and longer chain formation is restricted . Samples are run without or with DTT in odd or even numbered lanes , respectively . The gels monitor the Atto - labelled Ub .

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

C Thioester formation assay using Atto647-labelled Ub. Ubiquitination assay using LUBEL-RBR-C WT (left panel) or C2704A (right panel): lanes 1/2 Atto-Ub + E1, lanes 3/4 + ATP, lanes 5/6 + E2, lanes 7/8 + E3, and lanes 9/10 + Ub-His6. In the presence of N-terminally tagged Atto-Ub and C-terminally tagged Ub-His6, only Ub2 product can be synthesized and longer chain formation is restricted. Samples are run without or with DTT in odd or even numbered lanes, respectively. The gels monitor the Atto-labelled Ub.


D The linear Ub chain formation activity of LUBEL - LDD mutants . Recombinant proteins of RBR - C WT , C2704A mutant , and LDD mutants ( R2754A and D2755A ) were assessed for their activity by in vitro ubiquitination assay . The samples were resolved on a gel and stained with Coomassie or immunoblotted using anti - linear Ub antibody .

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

D The linear Ub chain formation activity of LUBEL-LDD mutants. Recombinant proteins of RBR-C WT, C2704A mutant, and LDD mutants (R2754A and D2755A) were assessed for their activity by in vitro ubiquitination assay. The samples were resolved on a gel and stained with Coomassie or immunoblotted using anti-linear Ub antibody.


E Linear Ub chain formation by full - length LUBEL transient expression in insect cells . Full - length Myc - LUBEL was transiently expressed in Drosophila Schneider 2 ( S2 ) cells , and Myc - HOIP alone or with HA - HOIL - 1L in HEK293T cells . Total cell lysates ( TCL ) of control and transfected samples were incubated with immobilized GST - Linear - Tandem Ub binding entity ( Linear - TUBE ) containing three tandem repeats of ABIN - 1 - UBAN . Pulldown samples were blotted with anti - linear Ub antibody , while TCL were blotted with anti - Myc antibody for exogenous LUBEL and HOIP , anti - HA antibody for HOIL - 1L , and anti - Tubulin antibody for loading . Input of GST proteins was analyzed by Ponceau S staining . * : nonspecific band .

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

E Linear Ub chain formation by full-length LUBEL transient expression in insect cells. Full-length Myc-LUBEL was transiently expressed in Drosophila Schneider 2 (S2) cells, and Myc-HOIP alone or with HA-HOIL-1L in HEK293T cells. Total cell lysates (TCL) of control and transfected samples were incubated with immobilized GST-Linear-Tandem Ub binding entity (Linear-TUBE) containing three tandem repeats of ABIN-1-UBAN. Pulldown samples were blotted with anti-linear Ub antibody, while TCL were blotted with anti-Myc antibody for exogenous LUBEL and HOIP, anti-HA antibody for HOIL-1L, and anti-Tubulin antibody for loading. Input of GST proteins was analyzed by Ponceau S staining. * : nonspecific band.


A Endogenous linear Ub chains enriched in total protein extracts ( input , lane 1 ) from adult w1118 ( w - ) flies by GST - Linear - TUBE ( lane 3 ) . The enriched samples were further treated with recombinant DUBs , vOTU ( lane 4 ) or OTULIN ( lane 5 ) . Ub chains were visualized by immunoblotting using anti - linear Ub antibody or anti - Ub antibody . Loading of GST proteins was analyzed by Ponceau S staining .

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

A Endogenous linear Ub chains enriched in total protein extracts (input, lane 1) from adult w1118 (w-) flies by GST-Linear-TUBE (lane 3). The enriched samples were further treated with recombinant DUBs, vOTU (lane 4) or OTULIN (lane 5). Ub chains were visualized by immunoblotting using anti-linear Ub antibody or anti-Ub antibody. Loading of GST proteins was analyzed by Ponceau S staining.


B Endogenous linear Ub chains in total cell lysate ( TCL ) of S2 cells expressing human Flag - OTULIN WT or catalytically inactive C129A mutant . TCLs enriched with GST - Linear - TUBE were examined by immunoblotting using anti - linear Ub antibody . Expression of OTULIN was analyzed by anti - Flag antibody and loading of TCL was detected by anti - Tubulin antibody . Input of GST proteins was analyzed by Ponceau S staining . * : nonspecific band .

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

B Endogenous linear Ub chains in total cell lysate (TCL) of S2 cells expressing human Flag-OTULIN WT or catalytically inactive C129A mutant. TCLs enriched with GST-Linear-TUBE were examined by immunoblotting using anti-linear Ub antibody. Expression of OTULIN was analyzed by anti-Flag antibody and loading of TCL was detected by anti-Tubulin antibody. Input of GST proteins was analyzed by Ponceau S staining. * : nonspecific band.


C Mass spectrometry analysis using an identical sample as Figure 3B lane 6 . MS / MS spectra of the prototypic linear Ub chain peptide ( GGMQIFVK ) is shown .

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

C Mass spectrometry analysis using an identical sample as Figure 3B lane 6. MS/MS spectra of the prototypic linear Ub chain peptide (GGMQIFVK) is shown.


C In vitro deubiquitination assay of dCYLD . dCYLD was incubated with all eight linkage types of Ub2 chains . dCYLD catalytically dead C284S mutant was incubated with K 63 - or linear Ub2 chains . All the proteins in the reactions were resolved on Coomassie - stained SDS - PAGE gel . * : nonspecific band .

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

C In vitro deubiquitination assay of dCYLD. dCYLD was incubated with all eight linkage types of Ub2 chains. dCYLD catalytically dead C284S mutant was incubated with K 63- or linear Ub2 chains. All the proteins in the reactions were resolved on Coomassie-stained SDS-PAGE gel. * : nonspecific band.


D Stabilization of linear Ub chains by dCYLD C284S mutant transient expression in S2 cells . Myc - tagged dCYLD WT and C284S were transfected in S2 cells and linear Ub chains were enriched with GST - Linear - TUBE and immunoblotted by anti - linear Ub or anti - Ub antibodies . Input of GST proteins was visualized by Ponceau S staining . Expression of Myc - dCYLD was examined by anti - Myc antibody , and anti - Tubulin antibody was used for the loading control of TCL .

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

D Stabilization of linear Ub chains by dCYLD C284S mutant transient expression in S2 cells. Myc-tagged dCYLD WT and C284S were transfected in S2 cells and linear Ub chains were enriched with GST-Linear-TUBE and immunoblotted by anti-linear Ub or anti-Ub antibodies. Input of GST proteins was visualized by Ponceau S staining. Expression of Myc-dCYLD was examined by anti-Myc antibody, and anti-Tubulin antibody was used for the loading control of TCL.


E Linear Ub chains in dCYLD mutant flies . Endogenous levels of linear Ub chains in dCYLD mutant flies were compared with a control w - fly strain by performing GST - Linear - TUBE pulldown . Enriched linear Ub chains were immunoblotted with anti - linear Ub antibody . Input of GST proteins was visualized by Ponceau S staining . Tubulin was used for the loading control of the input .

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

E Linear Ub chains in dCYLD mutant flies. Endogenous levels of linear Ub chains in dCYLD mutant flies were compared with a control w- fly strain by performing GST-Linear-TUBE pulldown. Enriched linear Ub chains were immunoblotted with anti-linear Ub antibody. Input of GST proteins was visualized by Ponceau S staining. Tubulin was used for the loading control of the input.


B Linear Ub chains in w - and LUBEL mutant fly lysates . w - , CC / SS and delR2 flies were analyzed for the level of endogenous linear Ub chains by enriching the linear ubiquitin chains by GST - Linear - TUBE . Pulldown samples were immunoblotted by anti - linear Ub antibody , while GST - proteins were analyzed by Ponceau S staining . Anti - Tubulin antibody was used for the loading control of the input . * : nonspecific band .

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

B Linear Ub chains in w- and LUBEL mutant fly lysates. w-, CC/SS and delR2 flies were analyzed for the level of endogenous linear Ub chains by enriching the linear ubiquitin chains by GST-Linear-TUBE. Pulldown samples were immunoblotted by anti-linear Ub antibody, while GST-proteins were analyzed by Ponceau S staining. Anti-Tubulin antibody was used for the loading control of the input. * : nonspecific band.


C The lifespans of female w - and catalytically dead LUBEL mutant flies ( two clones of CC / SS , indicated as # 1 and # 2 , and delR2 ) . Survival of three independent cohorts with approximately 80 flies each was monitored over time . Median survival time ( days ) : w - = 78 , CC / SS # 1 = 68 , CC / SS # 2 = 65 , and delR2 = 71 . Total sample sizes are as follow ; w - = 215 , CC / SS # 1 = 242 , CC / SS # 2 = 233 , delR2 = 273 . Log - rank ( Mantel - cox ) test between fly lines : w - and CC / SS # 1 > 0 . 0001 , w - and CC / SS # 2 > 0 . 0001 , w - and delR2 > 0 . 0001 , CC / SS # 1 and CC / SS # 2 = 0 . 0007 , CC / SS # 1 and delR2 = not significant , CC / SS # 2 and delR2 = not significant .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The lifespans of female w- and catalytically dead LUBEL mutant flies (two clones of CC/SS, indicated as #1 and #2, and delR2). Survival of three independent cohorts with approximately 80 flies each was monitored over time. Median survival time (days): w- = 78, CC/SS #1 = 68, CC/SS #2 = 65, and delR2 = 71. Total sample sizes are as follow; w- = 215, CC/SS #1 = 242, CC/SS #2 = 233, delR2 = 273. Log-rank (Mantel-cox) test between fly lines: w- and CC/SS #1 > 0.0001, w- and CC/SS #2 > 0.0001, w- and delR2 > 0.0001, CC/SS #1 and CC/SS #2 = 0.0007, CC/SS #1 and delR2 = not significant, CC/SS #2 and delR2 = not significant.


D Histological analysis of muscle in LUBEL mutant flies . Hematoxylin and eosin ( H & amp ; amp ; E ) ( top panels ) and actin immunofluorescent staining ( Actin - IF ) ( bottom panels ) of thorax muscles from young ( Day 3 ) or aged ( Day 60 ) w - or CC / SS female flies . Scale bars : 50 μm .

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

D Histological analysis of muscle in LUBEL mutant flies. Hematoxylin and eosin (H&amp;E) (top panels) and actin immunofluorescent staining (Actin-IF) (bottom panels) of thorax muscles from young (Day 3) or aged (Day 60) w- or CC/SS female flies. Scale bars: 50 μm.


A The level of poly - Ub chains in w - flies after heat shock . w - male flies were incubated in a 36 ° C water bath for indicated times and the levels of poly - Ub chains in total protein extracts were compared by Ub using antibodies against linear , K 48 - , and K 63 - linked Ub chains . Ponceau S staining was used to visualize the protein loading .

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

A The level of poly-Ub chains in w- flies after heat shock. w- male flies were incubated in a 36 °C water bath for indicated times and the levels of poly-Ub chains in total protein extracts were compared by Ub using antibodies against linear, K 48-, and K 63-linked Ub chains. Ponceau S staining was used to visualize the protein loading.


B Survival of catalytically dead LUBEL mutant flies upon heat shock . Young adult flies ( 15 males and 15 females ) were incubated in a 36 ° C water bath and immobilized flies were counted over the time indicated . Median survival time ( hours ) : w - = 9 . 25 , CC / SS # 1 = 5 . 5 , CC / SS # 2 = 5 , delR2 = 5 . 75 , CC / SS # 1 / + = 8 . 5 , CC / SS # 2 / + = 10 , delR2 / + = 8 . p - values calculated by Gehan - Breslow - Wilcoxon test between fly lines : w - and CC / SS # 1 , CC / SS # 2 , or delR2 > 0 . 0001 , w - and CC / SS # 1 / + = 0 . 0134 , w - and CC / SS # 2 / + = ns , w - and delR2 / + = 0 . 0002 , CC / SS # 1 and CC / SS # 1 / + > 0 . 0001 , CC / SS # 2 and CC / SS # 2 / + > 0 . 0001 , delR2 and delR2 / + > 0 . 0001 . Representative data are shown from six independent experiments .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Survival of catalytically dead LUBEL mutant flies upon heat shock. Young adult flies (15 males and 15 females) were incubated in a 36 °C water bath and immobilized flies were counted over the time indicated. Median survival time (hours): w- = 9.25, CC/SS #1 = 5.5, CC/SS #2 = 5, delR2 = 5.75, CC/SS #1/+ = 8.5, CC/SS #2/+ = 10, delR2/+ = 8. p-values calculated by Gehan-Breslow-Wilcoxon test between fly lines: w- and CC/SS #1, CC/SS#2, or delR2 > 0.0001, w- and CC/SS #1/+ = 0.0134, w- and CC/SS #2/+ = ns, w- and delR2/+ = 0.0002, CC/SS #1 and CC/SS #1/+ > 0.0001, CC/SS #2 and CC/SS #2/+ > 0.0001, delR2 and delR2/+ > 0.0001. Representative data are shown from six independent experiments.


C Survival of whole body or muscle - specific LUBEL knockdown flies upon heat shock . shRNA - based knockdown ( KD ) of LUBEL were driven by Tub - Gal4 or Mef2 - Gal4 flies . Control fly lines ( Tub - Gal4 / + and Mef2 - Gal4 / + ) were used to compare with the KD flies . 15 males and 15 females per each line were used in this assay . Median survival time ( hours ) : Tub - Gal4 / + = 7 , Tub - Gal4 > LUBEL = 3 . 5 , Mef2 - Gal4 / + = 7 . 5 , Mef2 - Gal4 > LUBEL = 7 . p - values calculated by Gehan - Breslow - Wilcoxon test : Tub - Gal4 < 0 . 0001 ( * * * * ) , Mef2 - Gal4 = 0 . 0029 ( * * ) . Representative data are shown from three independent experiments .

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

C Survival of whole body or muscle-specific LUBEL knockdown flies upon heat shock. shRNA-based knockdown (KD) of LUBEL were driven by Tub-Gal4 or Mef2-Gal4 flies. Control fly lines (Tub-Gal4/+ and Mef2-Gal4/+) were used to compare with the KD flies. 15 males and 15 females per each line were used in this assay. Median survival time (hours): Tub-Gal4/+ = 7, Tub-Gal4>LUBEL = 3.5, Mef2-Gal4/+ = 7.5, Mef2-Gal4>LUBEL= 7. p-values calculated by Gehan-Breslow-Wilcoxon test: Tub-Gal4 <0.0001 (****), Mef2-Gal4 = 0.0029 (**). Representative data are shown from three independent experiments.


D Heat - induced mRNA expression of HSP70 . w - , CC / SS # 2 and delR2 flies were heat treated for 60 min and recovered for 1 hour , and mRNA HSP70 was quantified by qPCR . Rp49 was used as a reference and w - untreated sample was used as calibrator to calculate the expression ratio . Data are presented as meanSD ( * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 ) . Representative data are shown from three independent experiments .

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 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

D Heat-induced mRNA expression of HSP70. w-, CC/SS #2 and delR2 flies were heat treated for 60 min and recovered for 1 hour, and mRNA HSP70 was quantified by qPCR. Rp49 was used as a reference and w- untreated sample was used as calibrator to calculate the expression ratio. Data are presented as meanSD (*<0.01, ***<0.001, ****<0.0001). Representative data are shown from three independent experiments.


Representative still images of control , Rod depleted cells and Rod depleted cells supplemented with RNAi resistant Venus - Rod treated with nocodazole and time in minutes indicated . Scale bar , 5 μm .

O O O O O O B-CONTROLLED_VAR 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

Representative still images of control, Rod depleted cells and Rod depleted cells supplemented with RNAi resistant Venus-Rod treated with nocodazole and time in minutes indicated. Scale bar, 5 μm.


Time from NEBD to mitotic exit for the indicated conditions with each circle representing a single cell analyzed and mean time indicated by red line and standard error of mean shown . The number of cells counted and mean time indicated above .

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

Time from NEBD to mitotic exit for the indicated conditions with each circle representing a single cell analyzed and mean time indicated by red line and standard error of mean shown. The number of cells counted and mean time indicated above.


Immunofluorescence analysis of ZW10 and Mad1 upon depletion of Rod . Scale bar , 5 μm .

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

Immunofluorescence analysis of ZW10 and Mad1 upon depletion of Rod. Scale bar, 5 μm.


Quantification of kinetochore intensities of the indicated proteins in control or Rod depleted cells with the signal normalized to CREST levels and Bub1 pSpT normalized to Bub1 . Bar indicates mean and standard error of mean is shown by line . At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments 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 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

Quantification of kinetochore intensities of the indicated proteins in control or Rod depleted cells with the signal normalized to CREST levels and Bub1 pSpT normalized to Bub1. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.


Time from NEBD to mitotic exit in control depleted , Rod depleted , Bub1 depleted and Rod and Bub1 co - depleted HeLa , U2OS and RPE1 cells . Cells were treated with nocodazole .

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

Time from NEBD to mitotic exit in control depleted, Rod depleted, Bub1 depleted and Rod and Bub1 co-depleted HeLa, U2OS and RPE1 cells. Cells were treated with nocodazole.


Mad1 kinetochore levels in the indicated conditions normalized to level of CREST in HeLa cells . Cells were fixed at 45 min after releasing from RO3306 arrest into nocodazole . Bar indicates mean and standard error of mean is shown by line . At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown .

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

Mad1 kinetochore levels in the indicated conditions normalized to level of CREST in HeLa cells. Cells were fixed at 45 min after releasing from RO3306 arrest into nocodazole. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.


Western blot analysis of whole cell extract for the indicated conditions . Quantitative analysis of Rod levels using LiCor technology is indicated below . The asterisk indicates an unspecific band .

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Western blot analysis of whole cell extract for the indicated conditions. Quantitative analysis of Rod levels using LiCor technology is indicated below. The asterisk indicates an unspecific band.


Analysis of ZW10 and Mad1 kinetochore levels in the indicated cells and conditions . Kinetochore signal is normalized to CREST signal . Bar indicates mean and standard error of mean is shown by line . At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown .

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Analysis of ZW10 and Mad1 kinetochore levels in the indicated cells and conditions. Kinetochore signal is normalized to CREST signal. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown.


Representative still images of unperturbed mitosis for parental cells , Rod C cells and Rod CR cells with time in minutes indicated . CFP - Histone 3 was used here as the chromosome marker . Scale bar , 5 μm .

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Representative still images of unperturbed mitosis for parental cells, Rod C cells and Rod CR cells with time in minutes indicated. CFP-Histone 3 was used here as the chromosome marker. Scale bar, 5 μm.


Time from NEBD to exit of indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped .

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

Time from NEBD to exit of indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped.


Time from NEBD to exit of indicated conditions with cells treated with taxol . Red circles represent cells still arrested in mitosis when the filming stopped .

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

Time from NEBD to exit of indicated conditions with cells treated with taxol. Red circles represent cells still arrested in mitosis when the filming stopped.


Time from NEBD to exit in indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped .

Time from NEBD to exit in indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped.


Time from NEBD to exit in indicated conditions with cells treated with taxol . Red circles represent cells still arrested in mitosis when the filming stopped .

Time from NEBD to exit in indicated conditions with cells treated with taxol. Red circles represent cells still arrested in mitosis when the filming stopped.


Mass spectrometry analysis of BubR1 IPs from the indicated cell lines . Relative protein quantification values ( MaxLFQ , log10 ) are plotted across conditions . Data from analysis of 3 technical repeats of BubR1 purifications with standard deviation indicated .

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Mass spectrometry analysis of BubR1 IPs from the indicated cell lines. Relative protein quantification values (MaxLFQ, log10) are plotted across conditions. Data from analysis of 3 technical repeats of BubR1 purifications with standard deviation indicated.


Estimated protein levels of Bub1 in the indicated conditions relative to parental cell lines . In Bub1 CR we only detected one peptide from three purifications . Standard deviation indicated .

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Estimated protein levels of Bub1 in the indicated conditions relative to parental cell lines. In Bub1 CR we only detected one peptide from three purifications. Standard deviation indicated.


Representative still images of unperturbed mitosis for parental cells , Bub1 C cells and Bub1 CR cells with time in minutes indicated . CFP - Histone 3 was used as the chromosome marker . Scale bar , 5 μm .

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Representative still images of unperturbed mitosis for parental cells, Bub1 C cells and Bub1 CR cells with time in minutes indicated. CFP-Histone 3 was used as the chromosome marker. Scale bar, 5 μm.


Tethering of Mad1 ( Mad1 485 - 718 ) to Ndc80 and the time from NEBD to exit was measured in each condition . Red circles represent cells still arrested in mitosis when the filming stopped .

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Tethering of Mad1 (Mad1 485-718) to Ndc80 and the time from NEBD to exit was measured in each condition. Red circles represent cells still arrested in mitosis when the filming stopped.


Tethering of Mad1 ( Mad1 485 - 718 ) to Ndc80 , KNL1 ( Zhang et al , 2017 ) or Bub1 1 - 553ΔCD1 and the time from NEBD to exit was measured in each condition . Red circles represent cells still arrested in mitosis when the filming stopped .

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Tethering of Mad1 (Mad1 485-718) to Ndc80, KNL1 (Zhang et al, 2017) or Bub1 1-553ΔCD1 and the time from NEBD to exit was measured in each condition. Red circles represent cells still arrested in mitosis when the filming stopped.


Localization of Venus - tagged Mad1 in parental HeLa cells , Bub1 CR cells and Rod CR cells in the presence of nocodazole . Scale bar , 5 μm .

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Localization of Venus-tagged Mad1 in parental HeLa cells, Bub1 CR cells and Rod CR cells in the presence of nocodazole. Scale bar, 5 μm.


Still images from FRAP experiments using Venus - tagged Mad1 in the indicated conditions . Image before bleach is shown and then time following bleach . Arrowheads indicate kinetochore pairs . Scale bar , 5 μm for whole cell image and 1 μm for the zoom in on kinetochores .

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Still images from FRAP experiments using Venus-tagged Mad1 in the indicated conditions. Image before bleach is shown and then time following bleach. Arrowheads indicate kinetochore pairs. Scale bar, 5 μm for whole cell image and 1 μm for the zoom in on kinetochores.


Analysis of Rod levels in cells used for FRAP experiment .

Analysis of Rod levels in cells used for FRAP experiment.


Venus - tagged Mad1 kinetochore intensity versus time from bleaching shortly after NEBD in unperturbed cells ( left ) or in cells arrested with nocodazole ( right ) . Data combined from 3 independent experiments and standard deviation indicated .

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Venus-tagged Mad1 kinetochore intensity versus time from bleaching shortly after NEBD in unperturbed cells (left) or in cells arrested with nocodazole (right). Data combined from 3 independent experiments and standard deviation indicated.


A HeLa cell line stably expressing Mad1 - BirA was depleted of Rod and synchronized in mitosis using thymidine and nocodazole . Biotin was added to media where indicated and biotinylated proteins were purified and analyzed by western blot with the indicated antibodies . Representative of 2 independent experiments .

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A HeLa cell line stably expressing Mad1-BirA was depleted of Rod and synchronized in mitosis using thymidine and nocodazole. Biotin was added to media where indicated and biotinylated proteins were purified and analyzed by western blot with the indicated antibodies. Representative of 2 independent experiments.


The duration from NEBD to exit in each indicated conditions was recorded by time - lapse microscopy .

The duration from NEBD to exit in each indicated conditions was recorded by time-lapse microscopy.


Mitotic duration in the indicated conditions expressing Mad1 - Bub1 ( Mad1 485 - 718 fused to Bub1 1 - 553ΔCD1 ) fusion protein . The time from NEBD to exit is indicated . Red circles represent cells still arrested in mitosis when the filming stopped .

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Mitotic duration in the indicated conditions expressing Mad1-Bub1 (Mad1 485-718 fused to Bub1 1-553ΔCD1) fusion protein. The time from NEBD to exit is indicated. Red circles represent cells still arrested in mitosis when the filming stopped.


Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs . ( D ) Representative immunofluorescence images of cells stained for YFP and Mad1 in each condition . Scale bar , 5 μm .

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Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs. (D) Representative immunofluorescence images of cells stained for YFP and Mad1 in each condition. Scale bar, 5 μm.


Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs . Mad1 kinetochore levels were measured and normalized to Bub1 ( YFP ) level . Bar indicates mean and standard error of mean is shown by line . At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown .

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Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs. Mad1 kinetochore levels were measured and normalized to Bub1 (YFP) level. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.


Bub1 CR cells complemented with the indicated Bub1 constructs and time from NEBD to exit in nocodazole measured by time - lapse microscopy . Mean ( red line ) and standard error of mean ( black bar ) indicated .

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Bub1 CR cells complemented with the indicated Bub1 constructs and time from NEBD to exit in nocodazole measured by time-lapse microscopy. Mean (red line) and standard error of mean (black bar) indicated.


Kinetochore levels of Mad1 in Bub1 CR cells complemented with the indicated YFP tagged Bub1 constructs . Mad1 levels are normalized to YFP signal . Bar indicates mean and standard error of mean is shown by line . At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown .

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Kinetochore levels of Mad1 in Bub1 CR cells complemented with the indicated YFP tagged Bub1 constructs. Mad1 levels are normalized to YFP signal. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown.


Still images of unperturbed mitosis of HeLa cells transfected with Venus - Bub1 4xCD1 . CFP - Histone 3 was used as the chromosome marker ( the middle panel ) and Venus - Bub1 4xCD1 was shown in the bottom panel . Scale bar , 5 μm .

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Still images of unperturbed mitosis of HeLa cells transfected with Venus-Bub1 4xCD1. CFP-Histone 3 was used as the chromosome marker (the middle panel) and Venus-Bub1 4xCD1 was shown in the bottom panel. Scale bar, 5 μm.


HeLa cells transfected with the indicated Bub1 constructs and time from NEBD to exit determined during an unperturbed mitosis . The time from NEBD to exit was analyzed in each condition . Mean ( red line ) and standard error of mean ( black bar ) indicated . ( Mann Whitney test , ns : non - significant ) . A representative result from at least 3 independent experiments is shown .

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HeLa cells transfected with the indicated Bub1 constructs and time from NEBD to exit determined during an unperturbed mitosis. The time from NEBD to exit was analyzed in each condition. Mean (red line) and standard error of mean (black bar) indicated. (Mann Whitney test, ns: non-significant). A representative result from at least 3 independent experiments is shown.


Localization of Venus - Rod during unperturbed mitosis in control depleted or Bub1 depleted cells .

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Localization of Venus-Rod during unperturbed mitosis in control depleted or Bub1 depleted cells.


HeLa cells were depleted of Bub1 using RNAi and complemented with indicated Venus - Bub1 constructs . Immunofluorescence images were shown for Bub1 and ZW10 localization . Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions .

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HeLa cells were depleted of Bub1 using RNAi and complemented with indicated Venus-Bub1 constructs. Immunofluorescence images were shown for Bub1 and ZW10 localization. Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions.


HeLa cells were depleted of Bub1 using RNAi Immunofluorescence images were shown for Bub1 and ZW10 localization . Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions . with the indicated Bub1 fusion

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HeLa cells were depleted of Bub1 using RNAi Immunofluorescence images were shown for Bub1 and ZW10 localization. Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions. with the indicated Bub1 fusion proteins


( B ) Representative images of β - Gal staining ( left panel ) and quantification of β - Gal positive cells ( right panel ) of BMSCs isolated from 2 ‑ month - old and 24 - month - old mice . Scale bar : 100 μm . β - Gal , beta ‑ galactosidase .

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(B) Representative images of β-Gal staining (left panel) and quantification of β-Gal positive cells (right panel) of BMSCs isolated from 2‑month-old and 24-month-old mice. Scale bar: 100 μm. β-Gal, beta‑galactosidase.


( C ) Representative images of Alizarin Red staining at 21 days of osteogenic induction ( left panel ) and quantification of calcification ( right panel ) of BMSCs isolated from 2 - month - old and 24 - month - old mice by detecting the amount of Alizarin Red extracted from the matrix .

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(C) Representative images of Alizarin Red staining at 21 days of osteogenic induction (left panel) and quantification of calcification (right panel) of BMSCs isolated from 2-month-old and 24-month-old mice by detecting the amount of Alizarin Red extracted from the matrix.


( A - B ) Representative images ( A ) of immunofluorescence staining and quantification ( B ) of YBX1 ( green ) in primary BMSCs .

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(A-B) Representative images (A) of immunofluorescence staining and quantification (B) of YBX1 (green) in primary BMSCs.


( C - D ) Representative immunohistochemical staining images of YBX1 ( red ) and LEPR ( green ) ( C ) and quantification of YBX1 + LEPR + cell numbers ( D ) in femoral bone marrow . MP , metaphysis ; T Ar , Tissue area . Arrows point to YBX1 + LEPR + cells .

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(C-D) Representative immunohistochemical staining images of YBX1 (red) and LEPR (green) (C) and quantification of YBX1+ LEPR + cell numbers (D) in femoral bone marrow. MP, metaphysis; T Ar, Tissue area. Arrows point to YBX1+ LEPR + cells.


( E ) Age ‑ associated changes in YBX1 levels in human BMSCs from 30 males ( left panel ) and 30 females ( right panel ) .

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(E) Age‑associated changes in YBX1 levels in human BMSCs from 30 males (left panel) and 30 females (right panel).


( F and G ) Representative images of Alizarin Red staining ( F ) and quantification of calcification ( G ) by detecting the amount of Alizarin Red extracted from the matrix in BMSCs transfected with adenovirus driven control and Ybx1 shRNA at 21 days of osteogenic induction .

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(F and G) Representative images of Alizarin Red staining (F) and quantification of calcification (G) by detecting the amount of Alizarin Red extracted from the matrix in BMSCs transfected with adenovirus driven control and Ybx1 shRNA at 21 days of osteogenic induction.


( H and I ) Representative images of Oil Red O staining ( H ) and quantification of lipid formation by detecting the amount of Oil Red O extracted from the matrix ( I ) in BMSCs after 10 days of adipogenic induction .

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(H and I) Representative images of Oil Red O staining (H) and quantification of lipid formation by detecting the amount of Oil Red O extracted from the matrix (I) in BMSCs after 10 days of adipogenic induction.


( J and K ) Representative images of β - Gal staining ( J ) and quantification ( K ) of β - Gal positive cells among BMSCs .

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(J and K) Representative images of β-Gal staining (J) and quantification (K) of β-Gal positive cells among BMSCs.


( L - M ) Relative mRNA levels of osteogenic differentiation related genes ( L ) , adipogenic differentiation and senescence related genes ( M ) between BMSCs transfected with adenovirus driven control and Ybx1 shRNA .

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(L-M) Relative mRNA levels of osteogenic differentiation related genes (L), adipogenic differentiation and senescence related genes (M) between BMSCs transfected with adenovirus driven control and Ybx1 shRNA.


( B - F ) Representative μCT images , Scale bar : 1mm . ( B ) and quantitative μCT analysis of trabecular bone microarchitecture ( C - F ) in distal femora from 3 - and 12 - month - old male Ybx1Prx1 - CKO mice and Ybx1flox / flox mice ( BV / TV , bone volume per tissue volume ; Tb . N , trabecular number ; Tb . Th , trabecular thickness ; Tb . Sp , trabecular separation ) .

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(B-F) Representative μCT images, Scale bar: 1mm. (B) and quantitative μCT analysis of trabecular bone microarchitecture (C-F) in distal femora from 3- and 12-month-old male Ybx1Prx1-CKO mice and Ybx1flox/flox mice (BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation).


( G ) Representative images of H & E staining ( Hematoxylin - Eosin Staining ) in distal femora . Scale bar : 100μm . ( H ) Quantification of the number of adipocytes related to the tissue area ( N . adipocytes / T . Ar ) . ( I ) Representative images of osteocalcin ( OCN ) immunohistochemical staining in distal femora . Arrows point to osteocalcin positive cells . Scale bar : 50 μ ( J ) Quantification of osteocalcin positive cells on the bone surface . Number of OCN + cells per bone perimeter ( N . Ocn + / B . Pm ) . ( K ) Representative images of TRAP staining in distal femora . Scale bar : 100 μm . ( L ) Quantification of TRAP positive cells on the bone surface . Number of TRAP + cells per bone perimeter ( N . Trap + / B . Pm ) .

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(G) Representative images of H&E staining (Hematoxylin-Eosin Staining) in distal femora. Scale bar: 100μm. (H) Quantification of the number of adipocytes related to the tissue area (N. adipocytes/T.Ar). (I) Representative images of osteocalcin (OCN) immunohistochemical staining in distal femora. Arrows point to osteocalcin positive cells. Scale bar: 50 μ (J) Quantification of osteocalcin positive cells on the bone surface. Number of OCN+ cells per bone perimeter (N. Ocn+/B.Pm). (K) Representative images of TRAP staining in distal femora. Scale bar: 100 μm. (L) Quantification of TRAP positive cells on the bone surface. Number of TRAP+ cells per bone perimeter (N. Trap+/B.Pm).


( M ) Representative images of calcein double labeling of trabecular bone . Scale bar : 50 μm . ( N ) Quantification of the bone formation rate ( BFR ) based on calcein double labeling . ( O ) Quantification of the mineral apposition rate ( MAR ) based on calcein double labeling .

(M) Representative images of calcein double labeling of trabecular bone. Scale bar: 50 μm. (N) Quantification of the bone formation rate (BFR) based on calcein double labeling. (O) Quantification of the mineral apposition rate (MAR) based on calcein double labeling.


( E - F ) Representative μCT images , Scale bar : 1mm . ( E ) and quantitative μCT analysis of trabecular bone microarchitecture ( F ) in distal femora from 24 - month - old mice with rAAV8 - GFP - Ybx1 or rAAV8 - GFP transfection ( BV / TV , bone volume per tissue volume ; Tb . N , trabecular number ; Tb . Th , trabecular thickness ; Tb . Sp , trabecular separation ) .

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(E-F) Representative μCT images, Scale bar: 1mm. (E) and quantitative μCT analysis of trabecular bone microarchitecture (F) in distal femora from 24-month-old mice with rAAV8-GFP-Ybx1 or rAAV8-GFP transfection (BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation).


( G ) Representative images of H & E staining in distal femora . ( H ) Quantification of the number of adipocytes related to the tissue area ( N . adipocytes / T . Ar ) . ( I ) Representative images of osteocalcin immunohistochemical staining in distal femora . Arrows point to osteocalcin positive cells . ( J ) Quantification of osteocalcin positive cells in bone surface . Number of OCN + cells per bone perimeter ( N . Ocn + / B . Pm ) . ( K ) Representative images of TRAP staining in distal femora . ( L ) Quantification of TRAP positive cells on the bone surface . Number of TRAP + cells per bone perimeter ( N . Trap + / B . Pm ) . ( M ) Representative images of calcein double labeling of trabecular bone . ( N ) Quantification of the bone formation rate ( BFR ) and mineral apposition rate ( MAR ) based on calcein double labeling .

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(G) Representative images of H&E staining in distal femora. (H) Quantification of the number of adipocytes related to the tissue area (N. adipocytes/T.Ar). (I) Representative images of osteocalcin immunohistochemical staining in distal femora. Arrows point to osteocalcin positive cells. (J) Quantification of osteocalcin positive cells in bone surface . Number of OCN+ cells per bone perimeter (N. Ocn+/B.Pm). (K) Representative images of TRAP staining in distal femora. (L) Quantification of TRAP positive cells on the bone surface. Number of TRAP+ cells per bone perimeter (N. Trap+/B.Pm). (M) Representative images of calcein double labeling of trabecular bone. (N) Quantification of the bone formation rate (BFR) and mineral apposition rate (MAR) based on calcein double labeling.


( C ) Semi - quantitative PCR showing the different isoforms of Fn1 , Nrp2 , Sp7 , and Sirt2 from BMSCs of Ybx1Prx1 - CKO mice and Ybx1flox / flox mice .

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(C) Semi-quantitative PCR showing the different isoforms of Fn1, Nrp2, Sp7, and Sirt2 from BMSCs of Ybx1Prx1-CKO mice and Ybx1flox/flox mice.


( D - G ) Representative images of Alizarin Red staining ( D and F ) and quantification of calcification by detecting the amount of Alizarin Red extracted from the matrix ( E and G ) in BMSCs transfected with different isoforms of Sp7 or different isoforms of Spp1 .

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(D-G) Representative images of Alizarin Red staining (D and F) and quantification of calcification by detecting the amount of Alizarin Red extracted from the matrix (E and G) in BMSCs transfected with different isoforms of Sp7 or different isoforms of Spp1.


( H - I ) Representative images ( H ) and quantification of Oil Red O staining ( I ) in BMSCs transfected with different isoforms of Spp1 with 10 days of adipogenic induction .

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(H-I) Representative images (H) and quantification of Oil Red O staining (I) in BMSCs transfected with different isoforms of Spp1 with 10 days of adipogenic induction.


( J - K ) Representative images of β - Gal staining ( J ) and quantification ( K ) of β - Gal positive cells in BMSCs transfected with different isoforms of Sirt2 .

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(J-K) Representative images of β-Gal staining (J) and quantification (K) of β-Gal positive cells in BMSCs transfected with different isoforms of Sirt2.


( L - T ) BMSCs were isolated from Ybx1Prx1 - CKO mice and Ybx1flox / flox mice , among them , the BMSCs from Ybx1flox / flox mice were transfected with the blank control , BMSCs from Ybx1Prx1 - CKO mice were transfected with the blank control or different isoforms of the target genes . ( L - P ) Representative images of Alizarin Red staining ( L and O ) and quantification of calcification by detecting the amount of Alizarin Red extracted from the matrix ( M and P ) . ( Q - R ) Representative images ( Q ) and quantification of Oil Red O staining ( R ) in BMSCs after 10 days of adipogenic induction . ( S - T ) Representative images of β - Gal staining ( S ) and quantification ( T ) of β - Gal positive cells among BMSCs .

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(L-T) BMSCs were isolated from Ybx1Prx1-CKO mice and Ybx1flox/flox mice, among them, the BMSCs from Ybx1flox/flox mice were transfected with the blank control, BMSCs from Ybx1Prx1-CKO mice were transfected with the blank control or different isoforms of the target genes. (L-P) Representative images of Alizarin Red staining (L and O) and quantification of calcification by detecting the amount of Alizarin Red extracted from the matrix (M and P). (Q-R) Representative images (Q) and quantification of Oil Red O staining (R) in BMSCs after 10 days of adipogenic induction. (S-T) Representative images of β-Gal staining (S) and quantification (T) of β-Gal positive cells among BMSCs.


( D ) Representative images of Alizarin Red staining ( up panel ) , Oil Red O staining ( middle panel , scale bar : 100 μm ) and β - Gal staining ( bottom panel , scale bar : 100 μm ) in BMSCs treated with different compounds . ( E - G ) Quantification of calcium mineralization based on Alizarin Red staining ( E ) , quantification of Oil Red O based on Oil Red O staining ( F ) , and quantification of β - Gal positive cells based on β ‑ Gal staining in BMSCs treated with different compounds ( G ) .

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(D) Representative images of Alizarin Red staining (up panel), Oil Red O staining (middle panel, scale bar: 100 μm) and β-Gal staining (bottom panel, scale bar: 100 μm) in BMSCs treated with different compounds. (E-G) Quantification of calcium mineralization based on Alizarin Red staining (E), quantification of Oil Red O based on Oil Red O staining (F), and quantification of β-Gal positive cells based on β‑Gal staining in BMSCs treated with different compounds (G).


( K ) Co - IP of His - FBXO33 with HA - YBX1 and a series of mutant HA - YBX1 proteins following transfection into BMSCs .

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(K) Co-IP of His-FBXO33 with HA-YBX1 and a series of mutant HA-YBX1 proteins following transfection into BMSCs.


( M ) Western blotting analysis of FBXO33 and YBX1 levels in BMSCs treated with different concentrations of sciadopitysin .

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(M) Western blotting analysis of FBXO33 and YBX1 levels in BMSCs treated with different concentrations of sciadopitysin.


( N ) Semi - quantitative PCR showed the isoforms of Fn1 , Nrp2 and Sirt2 in cultured BMSCs isolated from 2 - month - old or 24 - month - old mice then treated with or without sciadopitysin .

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(N) Semi-quantitative PCR showed the isoforms of Fn1, Nrp2 and Sirt2 in cultured BMSCs isolated from 2-month-old or 24-month-old mice then treated with or without sciadopitysin.


( B - F ) Representative μCT images , Scale bar : 1mm . ( B ) and quantitative analysis of trabecular bone microarchitecture ( C - F ) in distal femora of 25 - month - old mice administered with sciadopitysin or vehicle ( BV / TV , bone volume per tissue volume ; Tb . N , trabecular number ; Tb . Th , trabecular thickness ; Tb . Sp , trabecular separation ) .

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(B-F) Representative μCT images, Scale bar: 1mm. (B) and quantitative analysis of trabecular bone microarchitecture (C-F) in distal femora of 25-month-old mice administered with sciadopitysin or vehicle (BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation).


( G , H ) Representative images ( G ) and quantification ( H ) of osteocalcin ( OCN ) positive cells in distal femora of 25 - month - old mice administered with sciadopitysin or vehicle . Number of Ocn + cells per bone perimeter ( N . Ocn + / B . Pm ) . Arrows point to osteocalcin positive cells . Scale bar : 50

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(G, H) Representative images (G) and quantification (H) of osteocalcin (OCN) positive cells in distal femora of 25-month-old mice administered with sciadopitysin or vehicle. Number of Ocn+ cells per bone perimeter (N. Ocn+/B.Pm). Arrows point to osteocalcin positive cells. Scale bar: 50 μ


( I , J ) Representative images ( I ) of H & E staining in distal femora and quantification ( J ) of the number of adipocytes related to the tissue area ( right panel , N . adipocytes / T . Ar ) in distal femora of 25 - month - old mice administered with sciadopitysin or vehicle . Scale bar : 100 μm .

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(I, J) Representative images (I) of H&E staining in distal femora and quantification (J) of the number of adipocytes related to the tissue area (right panel, N. adipocytes/T.Ar) in distal femora of 25-month-old mice administered with sciadopitysin or vehicle. Scale bar: 100 μm.


( K , L ) Representative images ( K ) and quantification ( L ) of TRAP positive cells in distal femora of 25 - month - old mice administered with sciadopitysin or vehicle . Number of TRAP + cells per bone perimeter ( N . Trap + / B . Pm ) . Scale bar : 50 μm .

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(K, L) Representative images (K) and quantification (L) of TRAP positive cells in distal femora of 25-month-old mice administered with sciadopitysin or vehicle. Number of TRAP+ cells per bone perimeter (N. Trap+/B.Pm). Scale bar: 50 μm.


( M ) Representative images of calcein double labeling of trabecular bone . Scale bar : 50μm . ( N , O ) Quantification of the bone formation rate ( BFR , N ) and mineral apposition rate ( MAR , O ) based on calcein double labeling .

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(M) Representative images of calcein double labeling of trabecular bone. Scale bar: 50μm. (N, O) Quantification of the bone formation rate (BFR, N) and mineral apposition rate (MAR, O) based on calcein double labeling.


Representative density plots of Ca2 + flux from conjugated OptoCAR ‑ T cells over time at 37ºC . Conjugated cells were stimulated with dimerizer addition for 0 ( F ) , 5 ( G ) , 10 ( H ) , 15 ( I ) or 30 ( J ) minutes prior to disrupting signaling by illuminating cells .

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Representative density plots of Ca2+ flux from conjugated OptoCAR‑T cells over time at 37ºC. Conjugated cells were stimulated with dimerizer addition for 0 (F), 5 (G), 10 (H), 15 (I) or 30 (J) minutes prior to disrupting signaling by illuminating cells.


OptoCAR ‑ T cells were conjugated with ligand - presenting cells and activated as in the upper regime shown in ( A ) . The median of ppERK intensity distribution was plotted with time , after subtraction of background intensity . Bars show mean ± SEM ( n = 3 ) . An equivalent experiment was performed as in ( B ) but following the middle regime shown in ( A ) , with sample illumination after 5 minutes for the remainder of the dataset . Indicated half - life was calculated from the time taken for the median ppERK to decrease to 50 % of maximum . Bars show mean ± SEM ( n = 4 ) . An equivalent experiment was performed as in ( B ) but following the lower regime shown in ( A ) , with sample illumination after 5 minutes for 3 minutes . Bars show mean ± SEM ( n = 3 ) .

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OptoCAR‑T cells were conjugated with ligand-presenting cells and activated as in the upper regime shown in (A). The median of ppERK intensity distribution was plotted with time, after subtraction of background intensity. Bars show mean ± SEM (n=3). An equivalent experiment was performed as in (B) but following the middle regime shown in (A), with sample illumination after 5 minutes for the remainder of the dataset. Indicated half-life was calculated from the time taken for the median ppERK to decrease to 50% of maximum. Bars show mean ± SEM (n=4). An equivalent experiment was performed as in (B) but following the lower regime shown in (A), with sample illumination after 5 minutes for 3 minutes. Bars show mean ± SEM (n=3).


Representative fluorescent Western blot showing the dynamics of FOS phosphorylation ( p - FOSS32 ) on light - modulated control . Conjugated OptoCAR ‑ T cells were stimulated for a defined period after dimerizer addition ( denoted above blots ) before either being left in the dark ( dark line ) or illuminated ( blue line ) . Full blot image with total protein normalization control shown in Figure EV3A . Representative fluorescent Western blot showing the dynamics of FOS expression ( FOSTotal ) on light - modulated control , from the same dataset as in ( A ) . Full blot image with total protein normalization control shown in Figure EV3B .

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Representative fluorescent Western blot showing the dynamics of FOS phosphorylation (p-FOSS32) on light-modulated control. Conjugated OptoCAR‑T cells were stimulated for a defined period after dimerizer addition (denoted above blots) before either being left in the dark (dark line) or illuminated (blue line). Full blot image with total protein normalization control shown in Figure EV3A. Representative fluorescent Western blot showing the dynamics of FOS expression (FOSTotal) on light-modulated control, from the same dataset as in (A). Full blot image with total protein normalization control shown in Figure EV3B.


RT - qPCR analysis of relative mRNA levels for four representative genes following OptoCAR - mediated T cell activation . OptoCAR ‑ T cell conjugates were activated in the dark ( signaling competent ) state for a period of 3 hours before light - mediated disruption of signaling ( open circles ) or maintained for a further 60 minutes in the dark ( filled circles ) . The relative mRNA values were calculated by subtracting baseline value before scaling to mean output between 3 - 4 hours . Individual datasets are presented as gray lines , with bars showing mean ± SEM ( n = 3 ) . Raw cRQ plots for all datasets are provided in Appendix Fig S2 and RQ values can be found in Dataset EV1 .

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RT-qPCR analysis of relative mRNA levels for four representative genes following OptoCAR-mediated T cell activation. OptoCAR‑T cell conjugates were activated in the dark (signaling competent) state for a period of 3 hours before light-mediated disruption of signaling (open circles) or maintained for a further 60 minutes in the dark (filled circles). The relative mRNA values were calculated by subtracting baseline value before scaling to mean output between 3-4 hours. Individual datasets are presented as gray lines, with bars showing mean ± SEM (n=3). Raw cRQ plots for all datasets are provided in Appendix Fig S2 and RQ values can be found in Dataset EV1.


Single pulses of varying duration were applied to OptoCAR ‑ T cells and the geometric mean of GFP expression was measured for all pulse lengths 24 hours after initiation of signaling , with illumination started once the signal pulse had ended . All values have been corrected for background GFP fluorescence from non - activated OptoCAR ‑ T cells and shown relative to the output at 24 hours . A representative dataset is provided in Figure EV4F . Bars show mean ± SEM ( n = 3 ) Equivalent datasets as in ( B ) for the geometric mean of CD69 upregulation . A representative dataset is provided in Figure EV4F . Bars show mean ± SEM ( n = 3 )

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Single pulses of varying duration were applied to OptoCAR‑T cells and the geometric mean of GFP expression was measured for all pulse lengths 24 hours after initiation of signaling, with illumination started once the signal pulse had ended. All values have been corrected for background GFP fluorescence from non-activated OptoCAR‑T cells and shown relative to the output at 24 hours. A representative dataset is provided in Figure EV4F. Bars show mean ± SEM (n=3) Equivalent datasets as in (B) for the geometric mean of CD69 upregulation. A representative dataset is provided in Figure EV4F. Bars show mean ± SEM (n=3)


The cRQ values from RT - qPCR of indicated cells . Total RNA was extracted and CD137 mRNA levels measured relative to that from HEK293T cells . Conjugated OptoCARCD19 ‑ T cells were also assayed either before or after 24 hours stimulation in the dark state . Individual values from three biological replicates are shown , with mean depicted by bar plot .

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The cRQ values from RT-qPCR of indicated cells. Total RNA was extracted and CD137 mRNA levels measured relative to that from HEK293T cells. Conjugated OptoCARCD19‑T cells were also assayed either before or after 24 hours stimulation in the dark state. Individual values from three biological replicates are shown, with mean depicted by bar plot.


A . In vivo genetic complementation of the E . coli MC4100 wild - type ( WT ) and secB or / and tig knock - out derivatives by either an empty vector or one carrying the secB or tig ( encoding TF ) genes or derivatives ( see " Plasmid " table in Appendix ) , as indicated . Serial dilutions of a culture ( OD600 = 0 . 5 ) were spotted ( 12μl ) on LB - Ampicillin plates containing or not anhydrotetracycline ( AHT ; 5 ng / ml ) and grown at 16 ° C or 30 ° C ( as indicated ) . n = 3 - 7 biological replicates .

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A. In vivo genetic complementation of the E. coli MC4100 wild-type (WT) and secB or/and tig knock-out derivatives by either an empty vector or one carrying the secB or tig (encoding TF) genes or derivatives (see "Plasmid" table in Appendix), as indicated. Serial dilutions of a culture (OD600= 0.5) were spotted (12μl) on LB-Ampicillin plates containing or not anhydrotetracycline (AHT; 5 ng/ml) and grown at 16°C or 30°C (as indicated). n= 3-7 biological replicates.


C . SecA ATPase activity determination in solution ( basal ; B ) , or plus SecYEG IMVs ; ( membrane ; M ) or plus proOmpA ( pre - treated with 10 mM DTT ; 5 mM EDTA ; 20 min ; 4 ° C ; translocation ; T ) . n = 21 biological replicates . Mean values ( ± SEM ) shown .

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C. SecA ATPase activity determination in solution (basal; B), or plus SecYEG IMVs; (membrane; M) or plus proOmpA (pre-treated with 10 mM DTT; 5 mM EDTA; 20 min; 4°C; translocation; T). n= 21 biological replicates. Mean values (± SEM) shown.


D . proOmpA translocation ATPase activity stimulation of SecA ( as in C ) in the absence or presence of TF2 or / and SecB4 , added at the indicated molar excess over proOmpA1 . The T / M ratios were calculated ; the one in the absence of chaperones was considered as 100 % , all other values were expressed as % of this . n = 2 - 4 biological replicates . Mean values ( ± SEM ) shown . Unpaired parametric t - test , 95 % confidence interval : ns : not significant ( p = 0 . 1032 ) ; * * * * : p < 0 . 0001 ; * * : p = 0 . 0037 .

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D. proOmpA translocation ATPase activity stimulation of SecA (as in C) in the absence or presence of TF2 or/and SecB4, added at the indicated molar excess over proOmpA1. The T/M ratios were calculated; the one in the absence of chaperones was considered as 100%, all other values were expressed as % of this. n= 2-4 biological replicates. Mean values (± SEM) shown. Unpaired parametric t-test, 95% confidence interval: ns: not significant (p = 0.1032); ****: p < 0.0001; **: p = 0.0037.