D . Immunoblots with the indicated antibodies ( top ) and their quantifications . The densitometry measurements were normalized relative to the signal of the samples transduced by non - targeting shRNA ( Ctrl ) . Note that the same β - actin immunoblot was used twice because the Jam - C and E - cadherin samples were immunoblotted on the same membrane ( mean ± SD , n = 3 ) .

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D. Immunoblots with the indicated antibodies (top) and their quantifications. The densitometry measurements were normalized relative to the signal of the samples transduced by non-targeting shRNA (Ctrl). Note that the same β-actin immunoblot was used twice because the Jam-C and E-cadherin samples were immunoblotted on the same membrane (mean ± SD, n=3).


A . TEM images of longitudinal sections of lateral ventricle CP villi of P18 - P21 Mpdz + / + and Mpdz - / - mice . Scale bar , 2 μm . B . A large number of voids of varying sizes was evident in the CPECs of Mpdz - / - mice . Higher magnification images of the areas in the rectangles are shown underneath the panels . Adherens junctions are denoted by horizontal lines and arrows ; voids are indicated by arrows . Scale bar , 1 μm ; insets , 200 nm .

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A. TEM images of longitudinal sections of lateral ventricle CP villi of P18-P21 Mpdz+/+ and Mpdz-/- mice. Scale bar, 2 μm. B. A large number of voids of varying sizes was evident in the CPECs of Mpdz-/- mice. Higher magnification images of the areas in the rectangles are shown underneath the panels. Adherens junctions are denoted by horizontal lines and arrows; voids are indicated by arrows. Scale bar, 1 μm; insets, 200 nm.


C . Triplicate images of tight junctions proximal to the apical faces of CPECs in Mpdz + / + and Mpdz - / - mice . Scale bar , 100

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C. Triplicate images of tight junctions proximal to the apical faces of CPECs in Mpdz+/+ and Mpdz-/- mice. Scale bar, 100 nm


D . Mitochondria were smaller and frequently lacked large portions of their cristae . Autophagosomes are present in some of them ( arrows ) . Scale bar , 200 nm . The images are representative of 2 Mpdz + / + mice and 2 Mpdz - / - mice .

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D. Mitochondria were smaller and frequently lacked large portions of their cristae. Autophagosomes are present in some of them (arrows). Scale bar, 200 nm. The images are representative of 2 Mpdz+/+ mice and 2 Mpdz-/- mice.


E . Time course and standard deviations of the impedance of confluent hpCPEC monolayers that were transduced by either MPDZ or non - targeting ( Ctrl ) shRNA . Each record represents 4 wells . MPDZ immunoblot of each cell group is shown in the inset .

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E. Time course and standard deviations of the impedance of confluent hpCPEC monolayers that were transduced by either MPDZ or non-targeting (Ctrl) shRNA. Each record represents 4 wells. MPDZ immunoblot of each cell group is shown in the inset.


A . TEM images of CPEC sections from lateral ventricle villi of two Mpdz + / + and two Mpdz - / - P14 - P16 mice injected with HRP . The CPs were reacted with hydrogen peroxide and DAB ex - vivo . The dark particles are DAB deposits internalized by micropinocytosis . The magnified fields to the right show individual particles engulfed in macropinosomes . Note the layered structure of the particles , and the macropinosome that is open to the ventricular space in the Mpdz - / - section . Scale bars , 1 μm ; insets , 100 nm . B . Mean number of engulfed DAB particles per cell in Mpdz + / + and Mpdz - / - mice ( mean ± SD , n = 22 ) .

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A. TEM images of CPEC sections from lateral ventricle villi of two Mpdz+/+ and two Mpdz-/- P14-P16 mice injected with HRP. The CPs were reacted with hydrogen peroxide and DAB ex-vivo. The dark particles are DAB deposits internalized by micropinocytosis. The magnified fields to the right show individual particles engulfed in macropinosomes. Note the layered structure of the particles, and the macropinosome that is open to the ventricular space in the Mpdz-/- section. Scale bars, 1 μm; insets, 100 nm. B. Mean number of engulfed DAB particles per cell in Mpdz+/+ and Mpdz-/- mice (mean ± SD, n=22).


C . A CPEC section showing the engulfment of a DAB particle by its basal ruffles in the magnified field . Scale bars in panels C and D , 1 μm ; insets , 200 nm . D . A CPEC section showing a preponderance of macropinosomes close to the apical face of the cell , and a magnified field that contains several macropinosomes . E . Mean numbers of DAB - containing macropinosomes close to the apical or basal sides of CPECs from Mpdz + / + or Mpdz - / - mice ( mean ± SD , n = 20 ) .

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C. A CPEC section showing the engulfment of a DAB particle by its basal ruffles in the magnified field. Scale bars in panels C and D, 1 μm; insets, 200 nm. D. A CPEC section showing a preponderance of macropinosomes close to the apical face of the cell, and a magnified field that contains several macropinosomes. E. Mean numbers of DAB-containing macropinosomes close to the apical or basal sides of CPECs from Mpdz+/+ or Mpdz-/- mice (mean ± SD, n=20).


A . Immunofluorescence images of 10 μm - thick sections from lateral ventricle CP villi of Mpdz + / + and Mpdz - / - P14 - P16 mice immunolabeled by anti - LDLR . Scale bars , 50 ( top ) and 25 ( bottom ) μm . B . Mean fluorescence intensities ( normalized relative to the highest recorded intensity ) per cell in several LDLR - immunolabeled CP sections ( mean ± SD , n = 101 ) .

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A. Immunofluorescence images of 10 μm-thick sections from lateral ventricle CP villi of Mpdz+/+ and Mpdz-/- P14-P16 mice immunolabeled by anti-LDLR. Scale bars, 50 (top) and 25 (bottom) μm. B. Mean fluorescence intensities (normalized relative to the highest recorded intensity) per cell in several LDLR-immunolabeled CP sections (mean ± SD, n=101).


C . Immunoblots showing the abundances of LDLR and MPDZ in hCPECs transduced by MPDZ - targeting or non - targeting shRNA . Mean abundances are quantified by densitometry of the LDLR bands , normalized relative to the β - actin bands ( mean ± SD , n = 3 ) .

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C. Immunoblots showing the abundances of LDLR and MPDZ in hCPECs transduced by MPDZ-targeting or non-targeting shRNA. Mean abundances are quantified by densitometry of the LDLR bands, normalized relative to the β-actin bands (mean ± SD, n=3).


D . Fluorescence images of hCPECs transduced by either MPDZ or non - targeting ( Ctrl ) shRNA and immunolabeled by anti - LDLR either before ( 0 min ) or after 8 min of constitutive endocytosis of LDLR . The histograms below show the mean fluorescence intensities per hCPEC in each cell group , normalized relative to the highest recorded intensity ( mean ± SD , n = 41 ÷ 53 ) .

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D. Fluorescence images of hCPECs transduced by either MPDZ or non-targeting (Ctrl) shRNA and immunolabeled by anti-LDLR either before (0 min) or after 8 min of constitutive endocytosis of LDLR. The histograms below show the mean fluorescence intensities per hCPEC in each cell group, normalized relative to the highest recorded intensity (mean ± SD, n=41÷53).


A . Mean protein concentration in the CSF of Mpdz + / + and Mpdz - / - P14 - P21 mice ( mean ± SD , n = 2 ÷ 3 ) .

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A. Mean protein concentration in the CSF of Mpdz+/+ and Mpdz-/- P14-P21 mice (mean ± SD, n=2÷3).


B . Volcano plot showing that the protein contents in sera of P15 - P21 Mpdz + / + and Mpdz - / - mice are similar to each other .

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B. Volcano plot showing that the protein contents in sera of P15-P21 Mpdz+/+ and Mpdz-/- mice are similar to each other.


C . A volcano plot showing that 23 proteins are at least two - fold significantly more abundant in the CSF of P15 - 21 Mpdz - / - mice ( red circles , protein names are indicated ) whereas only two proteins were more abundant in the CSF of Mpdz + / + mice ( green circles ) . The CSF composition was analyzed in 3 Mpdz - / - and 3 Mpdz + / + mice .

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C. A volcano plot showing that 23 proteins are at least two-fold significantly more abundant in the CSF of P15-21 Mpdz-/- mice (red circles, protein names are indicated) whereas only two proteins were more abundant in the CSF of Mpdz+/+ mice (green circles). The CSF composition was analyzed in 3 Mpdz-/- and 3 Mpdz+/+ mice.


D . Images of 10 μm - thick sections from lateral ventricle CP villi of Mpdz + / + and Mpdz - / - mice , immunolabeled for Nkcc1 . The areas in the marked squares are magnified below . Scale bars , top , 50 μm , bottom 20 μm .

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D. Images of 10 μm-thick sections from lateral ventricle CP villi of Mpdz+/+ and Mpdz-/- mice, immunolabeled for Nkcc1. The areas in the marked squares are magnified below. Scale bars, top, 50 μm, bottom 20 μm.


E . Immunoblots of LDLR and Mpdz , with β - actin as loading control . The MPDZ immunoblot shows the efficiency of the shRNA - mediated knockdown . Bands from three immunoblots were quantified by normalization to β - actin bands ( mean ± SD ) .

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E. Immunoblots of LDLR and Mpdz, with β-actin as loading control. The MPDZ immunoblot shows the efficiency of the shRNA-mediated knockdown. Bands from three immunoblots were quantified by normalization to β-actin bands (mean ± SD).


( A ) Immunoblots of mitochondrial fractions from siRNA ‐ transfected HEK293T cells ± CCCP .

(A) Immunoblots of mitochondrial fractions from siRNA‐transfected HEK293T cells±CCCP.


( B ) Immunoblots against indicated proteases in siRNA ‐ transfected cells .

(B) Immunoblots against indicated proteases in siRNA‐transfected cells.


( C ) Quantification of full ‐ length PINK1 ( n = 4 ) and knockdown efficiency ( n = 2 ) compared with non ‐ targeting ( NT ) siRNA from experiments performed as in B . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 . CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase and tensin homologue ‐ induced kinase 1 ; siRNA , short interfering RNA .

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(C) Quantification of full‐length PINK1 (n=4) and knockdown efficiency (n=2) compared with non‐targeting (NT) siRNA from experiments performed as in B. *P0.05; **P0.01; ***P0.001. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.


( A ) Live confocal micrographs of siRNA ‐ transfected HEK293T cells ± CCCP stained with MitoTracker ( MTR ) Green and Deep Red ( DR ) ; Scale bar , 5 μm .

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(A) Live confocal micrographs of siRNA‐transfected HEK293T cells±CCCP stained with MitoTracker (MTR) Green and Deep Red (DR); Scale bar, 5 μm.


( B ) MTR DR / Green ratio from images as in A . N = 3 ; * P0 . 05 compared with NT siRNA .

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(B) MTR DR/Green ratio from images as in A. N=3; *P0.05 compared with NT siRNA.


( C ) Mitochondrial interconnectivity from images as in A , N = 3 .

(C) Mitochondrial interconnectivity from images as in A, N=3.


( D ) Oxygen consumption in state 3 respiration in mitochondrial fractions from siRNA ‐ transfected cells . Antimycin A with NT siRNA was used as a control . A . U . , arbitrary unit ; CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; siRNA , short interfering RNA .

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(D) Oxygen consumption in state 3 respiration in mitochondrial fractions from siRNA‐transfected cells. Antimycin A with NT siRNA was used as a control. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; siRNA, short interfering RNA.


( A ) Immunoblots of mitochondrial ( M ) and cytosolic ( C ) fractions from siRNA ‐ transfected HEK293T cells ± CCCP for 3 . 5 h , ± MG132 for the final hour .

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(A) Immunoblots of mitochondrial (M) and cytosolic (C) fractions from siRNA‐transfected HEK293T cells±CCCP for 3.5 h, ±MG132 for the final hour.


( B ) Quantification of 52 ‐ kDa PINK1 from A ( with MG132 ) , n = 3 ; * P0 . 05 ; * * P0 . 001 compared with NT siRNA .

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(B) Quantification of 52‐kDa PINK1 from A (with MG132), n=3; *P0.05; **P0.001 compared with NT siRNA.


( C ) Mitochondria from siRNA ‐ transfected cells treated with 10 μM CCCP or dimethylsulphoxide were incubated with or without respiration substrate ( Resp . mix ) for 30 min at 37 ° C . al hour .

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(C) Mitochondria from siRNA‐transfected cells treated with 10 μM CCCP or dimethylsulphoxide were incubated with or without respiration substrate (Resp. mix) for 30 min at 37°C.al hour.


( A ) Mitochondria from CCCP ‐ treated or siRNA ‐ transfected HEK293T cells were incubated with increasing concentrations of proteinase K ( PK ) .

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(A) Mitochondria from CCCP‐treated or siRNA‐transfected HEK293T cells were incubated with increasing concentrations of proteinase K (PK).


( B ) Mitochondria from CCCP ‐ treated or MPPβ knockdown cells were incubated ± 0 . 1 M Na2CO3 before supernatant ( S ) and pellet ( P ) were separated by centrifugation . CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase and tensin homologue ‐ induced kinase 1 ; siRNA , short interfering RNA ; WB , western blot .

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(B) Mitochondria from CCCP‐treated or MPPβ knockdown cells were incubated ±0.1 M Na2CO3 before supernatant (S) and pellet (P) were separated by centrifugation. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA; WB, western blot.


( A ) Live confocal micrographs of siRNA ‐ , GFP ‐ Parkin ‐ and OCT ‐ DsRed ‐ transfected HEK293T cells ± CCCP ; Scale bars , 20 μm ( low magnification ) and 5 μm ( high magnification ) .

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(A) Live confocal micrographs of siRNA‐, GFP‐Parkin‐ and OCT‐DsRed‐transfected HEK293T cells ±CCCP; Scale bars, 20 μm (low magnification) and 5 μm (high magnification).


( B ) Percentage of transfected cells displaying a punctate mitochondrial pattern of GFP ‐ Parkin distribution , n = 3 ; * P0 . 05 ; * * P0 . 01 compared with NT siRNA .

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(B) Percentage of transfected cells displaying a punctate mitochondrial pattern of GFP‐Parkin distribution, n=3; *P0.05; **P0.01 compared with NT siRNA.


( C ) Immunoblots of endogenous Parkin in mitochondrial fractions from siRNA ‐ transfected cells ± CCCP .

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(C) Immunoblots of endogenous Parkin in mitochondrial fractions from siRNA‐transfected cells ±CCCP.


( D ) Mitochondrial mass measured by flow cytometry of MTR Green fluorescence in siRNA ‐ transfected cells treated ± CCCP for 24 h , n = 3 . A . U . , arbitrary unit ; CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; GFP , green fluorescent protein ; MPP , mitochondrial processing peptidase ; MTR , MitoTracker ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase and tensin homologue ‐ induced kinase 1 ; siRNA , short interfering RNA .

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(D) Mitochondrial mass measured by flow cytometry of MTR Green fluorescence in siRNA‐transfected cells treated ±CCCP for 24 h, n=3. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; GFP, green fluorescent protein; MPP, mitochondrial processing peptidase; MTR, MitoTracker; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.


( a ) Transverse slice of live E10 . 5 Venus : : HES5 homozygous knock - in mouse showing the ventral HES5 domain in spinal cord ex vivo ( left panel ) ; Draq5 live nuclear stain with nuclear segmentation overlay ( right panel ) ; scale bar 30 μm .

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(a) Transverse slice of live E10.5 Venus::HES5 homozygous knock-in mouse showing the ventral HES5 domain in spinal cord ex vivo (left panel); Draq5 live nuclear stain with nuclear segmentation overlay (right panel); scale bar 30 μm.


( a ) 20x snapshot of an ex - vivo slice culture of E10 . 5 spinal cord from Venus : : HES5 heterozygous knock - in mouse , transverse section ; delineated region ( blue ) correspond to data shown in ( b - c ) . D - dorsal , V - ventral . ( b ) Spatial profile of Venus : : HES5 intensity averaged over 2 . 5hrs with 0 distance representing the ventral end of kymograph ; black line represents the trend in Venus : : HES5 data across the domain produced using an polynomial order 6 ( see Materials and Methods ) . ( c ) Detrended spatial profile of Venus : : HES5 corresponding to data shown in ( b ) .

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(a) 20x snapshot of an ex-vivo slice culture of E10.5 spinal cord from Venus::HES5 heterozygous knock-in mouse, transverse section; delineated region (blue) correspond to data shown in (b-c). D- dorsal, V- ventral. (b) Spatial profile of Venus::HES5 intensity averaged over 2.5hrs with 0 distance representing the ventral end of kymograph; black line represents the trend in Venus::HES5 data across the domain produced using an polynomial order 6 (see Materials and Methods). (c) Detrended spatial profile of Venus::HES5 corresponding to data shown in (b).


( a ) Start : Finish Venus : : HES5 intensity ratio in E10 . 5 Venus : : HES5 heterozygous spinal cord slices treated with control ( DMSO ) and Notch inhibitor DBZ ( 2 μM ) observed over 16h ; bars indicate mean and standard deviation of DMS0 ( n = 3 experiments ) and DBZ ( n = 4 experiments ) ; 2 - tailed t - test * * * * p = 0 . 0001 .

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(a) Start:Finish Venus::HES5 intensity ratio in E10.5 Venus::HES5 heterozygous spinal cord slices treated with control (DMSO) and Notch inhibitor DBZ (2 μM) observed over 16h; bars indicate mean and standard deviation of DMS0 (n=3 experiments) and DBZ (n=4 experiments); 2-tailed t-test ****p=0.0001.


( a ) Transverse cryosection of E10 . 5 Venus : : HES5 spinal cord . Venus : : HES5 endogenous signal , OLIG2 - motorneuron progenitor marker , NGN2 - early marker of neuronal commitment ; scale bar 20 μm . ( b ) Relative nuclear intensities of Venus : : HES5 and NGN2 in motorneuron and interneuron progenitors ; bars show mean and SD of at least 494 cells per domain from 5 slices in 2 experiments ; Kruskal - Wallis with Dunn ' s multiple comparison test adjusted p - values * * p = 0 . 0032 , * * * p < 0 . 001 .

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(a) Transverse cryosection of E10.5 Venus::HES5 spinal cord. Venus::HES5 endogenous signal, OLIG2 - motorneuron progenitor marker, NGN2 - early marker of neuronal commitment; scale bar 20 μm. (b) Relative nuclear intensities of Venus::HES5 and NGN2 in motorneuron and interneuron progenitors; bars show mean and SD of at least 494 cells per domain from 5 slices in 2 experiments; Kruskal-Wallis with Dunn's multiple comparison test adjusted p-values **p=0.0032, ***p<0.001.


( a ) Detrended spatial profile of NGN2 : : mScarlet - I intensity from transverse slice of E10 . 5 spinal cord from heterozygous knock - in mouse in ventral - dorsal direction ; red indicates motorneuron ( MN ) domain , blue interneuron domain ( IN ) .

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(a) Detrended spatial profile of NGN2::mScarlet-I intensity from transverse slice of E10.5 spinal cord from heterozygous knock-in mouse in ventral-dorsal direction;red indicates motorneuron (MN) domain, blue interneuron domain (IN).


A Combined IF and DNA FISH analysis of RAD54 ( IF ) and telomeres ( FISH ) in ALT and non - ALT cell lines . White arrows indicate RAD54 foci that colocalize with telomeres . Scale bars = 10 µm B Quantification of data in A . A cell was counted positive if it contained 1 or more colocalization event between RAD54 and the telomere . At least 100 cells were counted per cell line per repeat . For SaOS2 , NOS , SJSA1 , HeLa LT n = 3 . For Cal72 , U2OS , HuO9 n = 4 . Values shown are mean ± sd

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A Combined IF and DNA FISH analysis of RAD54 (IF) and telomeres (FISH) in ALT and non-ALT cell lines. White arrows indicate RAD54 foci that colocalize with telomeres. Scale bars = 10 µm B Quantification of data in A. A cell was counted positive if it contained 1 or more colocalization event between RAD54 and the telomere. At least 100 cells were counted per cell line per repeat. For SaOS2, NOS, SJSA1, HeLa LT n = 3. For Cal72, U2OS, HuO9 n = 4. Values shown are mean±sd


Combined IF and DNA FISH analysis of RAD54 ( IF ) , PML ( IF ) and telomeres ( FISH ) in ALT cell lines SaOS2 and HuO9 . Scale bars = 10

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Combined IF and DNA FISH analysis of RAD54 (IF), PML (IF) and telomeres (FISH) in ALT cell lines SaOS2 and HuO9. Scale bars = 10 µm


Western blot showing expression of TRF1 - FOK1 - mCherry fusion protein following doxycycline induction , both WT and D450A ( nuclease dead ) . Cells were treated with 40 ng / mL doxycycline for 16 hrs and then 1µM Shield - 1 and 1µM 4 - OHT for 4

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Western blot showing expression of TRF1-FOK1-mCherry fusion protein following doxycycline induction, both WT and D450A (nuclease dead). Cells were treated with 40 ng/mL doxycycline for 16 hrs and then 1µM Shield-1 and 1µM 4-OHT for 4 hrs


Combined IF and DNA FISH for RAD54 ( IF ) and telomeres ( FISH ) in U2OS - TRF1 - FOK1 - WT or U2OS - TRF1 - FOK1 - D450A ( nuclease dead ) cells . Cells were treated as in E immediately prior to staining . Scale bars = 10 µm Quantification of data from F . RAD54 foci were detected using particle analysis in Fiji . At least a total of 300 cells were counted from 3 repeated experiments . Values shown are mean ± sem . Values were compared using Kruskal - Wallis test followed by Dunn ' s multiple comparison test . Quantification of data from F . RAD54 foci were detected using particle analysis in Fiji . Cells were counted as positive if they counted 8 or more RAD54 foci that colocalized with telomeres . Values shown are mean ± sem of n = 3 biological replicates . Values were compared using standard two - way ANOVA followed by Sidak ' s multiple comparison test .

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Combined IF and DNA FISH for RAD54 (IF) and telomeres (FISH) in U2OS-TRF1-FOK1-WT or U2OS-TRF1-FOK1-D450A (nuclease dead) cells. Cells were treated as in E immediately prior to staining. Scale bars = 10 µm Quantification of data from F. RAD54 foci were detected using particle analysis in Fiji. At least a total of 300 cells were counted from 3 repeated experiments. Values shown are mean±sem. Values were compared using Kruskal-Wallis test followed by Dunn's multiple comparison test. Quantification of data from F. RAD54 foci were detected using particle analysis in Fiji. Cells were counted as positive if they counted 8 or more RAD54 foci that colocalized with telomeres. Values shown are mean±sem of n=3 biological replicates. Values were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.


Combined IF and DNA FISH for RAD51 ( IF ) and telomeres ( FISH ) in SaOS2 or HuO9 cells . Cells were transfected with 20nM siRAD54 # 2 for 48 hours immediately prior to staining . Scale bars = 10 µm Quantification of A . A cell was counted as positive it is contained at least 1 colocalization event between RAD51 and telomeres . At least 100 cells were counted per condition per repeat , n = 3 . Values shown are mean ± sd . Data were compared using standard two - way ANOVA followed by Sidak ' s multiple comparison test .

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Combined IF and DNA FISH for RAD51 (IF) and telomeres (FISH) in SaOS2 or HuO9 cells. Cells were transfected with 20nM siRAD54#2 for 48 hours immediately prior to staining. Scale bars = 10 µm Quantification of A. A cell was counted as positive it is contained at least 1 colocalization event between RAD51 and telomeres. At least 100 cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.


ChIP for telomeric DNA associated with RAD51 . DNA dot blot probed with DIG labeled telomere probe or Alu probe . HuO9 cells were transfected with 20nM siRAD54 # 2 , or 15µg myc - TRF2 for 72 hours prior to immunoprecipitation with RAD51 antibody ( Mock , siRAD54 conditions ) or myc antibody ( myc - TRF2 positive control ) . Quantification of ( CCCTAA ) 4 dot blot performed in C . IP values were normalized to loading ( IP / input ) and then normalized to mock control . Values shown are mean ± sd of n = 3 biological replicates . Values were compared using two - tailed unpaired student ' s t - test .

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

ChIP for telomeric DNA associated with RAD51. DNA dot blot probed with DIG labeled telomere probe or Alu probe. HuO9 cells were transfected with 20nM siRAD54 #2, or 15µg myc-TRF2 for 72 hours prior to immunoprecipitation with RAD51 antibody (Mock, siRAD54 conditions) or myc antibody (myc-TRF2 positive control). Quantification of (CCCTAA)4 dot blot performed in C. IP values were normalized to loading (IP/input) and then normalized to mock control. Values shown are mean±sd of n=3 biological replicates. Values were compared using two-tailed unpaired student's t-test.


Western blot of samples used for ChIP in C - D .

Western blot of samples used for ChIP in C-D.


Combined IF and DNA FISH for GFP ( IF ) and telomeres ( FISH ) in SaOS2 and HuO9 cells . Cells were forward transfected with Polη - GFP , and then after 24 hours transfected with 20nM siRAD54 # 2 . Cells were stained for GFP and telomeres 48 hours after siRNA transfection . Scale bars = 10 µm Quantification of data in F . GFP negative cells were excluded from analysis . Cells were considered positive if they showed 1 or more colocalization events between GFP ( Polη ) and telomeres . At least 50 GFP positive cells were counted per condition per repeat , n = 3 . Values shown are mean ± sd . Values were compared using standard two - way ANOVA followed by Sidak ' s multiple comparison test .

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Combined IF and DNA FISH for GFP (IF) and telomeres (FISH) in SaOS2 and HuO9 cells. Cells were forward transfected with Polη-GFP, and then after 24 hours transfected with 20nM siRAD54#2. Cells were stained for GFP and telomeres 48 hours after siRNA transfection. Scale bars = 10 µm Quantification of data in F. GFP negative cells were excluded from analysis. Cells were considered positive if they showed 1 or more colocalization events between GFP (Polη) and telomeres. At least 50 GFP positive cells were counted per condition per repeat, n=3. Values shown are mean±sd. Values were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.


Representative images of combined EdU staining ( Click - it ) and DNA FISH for telomeres . HuO9 or SaOS2 cells were transfected with 20nM siRAD54 # 2 ( representative images for siRAD54 # 1 not shown ) for 48 hours followed by a 1 . 5 hr pulse of EdU . Pan - nuclear EdU stain represents S - phase cells . White arrows indicate non - S - phase cells containing EdU colocalizing with telomeres . Scale bars = 10 µm Quantification of data shown in A . HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54 # 1 or 20nM siRAD54 # 2 for 48 hours followed by a 1 . 5 hr pulse of EdU . Cells with pan - nuclear EdU signal or greater than 8 EdU foci were excluded from analysis as S - phase cells . Non - S - phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres . Data were normalized to mock condition for each repeat . At least 100 non - S - phase cells were counted per condition per repeat , n = 3 . Values shown are mean ± sd . Data were analyzed using two - way ANOVA followed by Dunnett ' s multiple comparison test .

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Representative images of combined EdU staining (Click-it) and DNA FISH for telomeres. HuO9 or SaOS2 cells were transfected with 20nM siRAD54#2 (representative images for siRAD54#1 not shown) for 48 hours followed by a 1.5 hr pulse of EdU. Pan-nuclear EdU stain represents S-phase cells. White arrows indicate non-S-phase cells containing EdU colocalizing with telomeres. Scale bars = 10 µm Quantification of data shown in A. HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#1 or 20nM siRAD54#2 for 48 hours followed by a 1.5 hr pulse of EdU. Cells with pan-nuclear EdU signal or greater than 8 EdU foci were excluded from analysis as S-phase cells. Non-S-phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres. Data were normalized to mock condition for each repeat. At least 100 non-S-phase cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Dunnett's multiple comparison test.


Representative DNA dot blot from C - circle assay on HuO9 cells , either mock transfected or transfected with 20nM siRAD54 # 2 for 48 hours . Quantification of C - circle assay in C . Signal was quantified with densitometry , background ( - Φ29 ) was subtracted and signal was normalized to mock . N = 5 biological replicates values shown are mean ± sd . Conditions were analyzed using unpaired two - tailed student ' s t - test .

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Representative DNA dot blot from C-circle assay on HuO9 cells, either mock transfected or transfected with 20nM siRAD54#2 for 48 hours. Quantification of C-circle assay in C. Signal was quantified with densitometry, background (- Φ29) was subtracted and signal was normalized to mock. N=5 biological replicates values shown are mean±sd. Conditions were analyzed using unpaired two-tailed student's t-test.


Representative images of combined IF and DNA FISH for PML ( IF ) and telomeres ( FISH ) . HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54 # 2 for 48 hours . White arrows indicate large colocalization events . Blue arrows indicate small colocalization events . Scale bars = 10 µm Quantification HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54 # 1 or 20nM siRAD54 # 2 for 48 hours . Cells were considered positive if they contained 1 large or 3 small colocalization events . At least 100 cells were counted per condition per repeat , n = 3 . Values shown are mean ± sd . Data were analyzed using two - way ANOVA followed by Dunnett ' s multiple comparison test .

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Representative images of combined IF and DNA FISH for PML (IF) and telomeres (FISH). HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#2 for 48 hours. White arrows indicate large colocalization events. Blue arrows indicate small colocalization events. Scale bars = 10 µm Quantification HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#1 or 20nM siRAD54#2 for 48 hours. Cells were considered positive if they contained 1 large or 3 small colocalization events. At least 100 cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Dunnett's multiple comparison test.


Representative images of combined EdU staining ( Click - it ) and DNA FISH for telomeric DNA . HuO9 cells were forward transfected with 2µg empty vector ( EV ) , RAD54 - WT , RAD54 - K189R , or S49E construct . After 24 hours , cells were transfected with 20 nM siRAD54 # 2 for 48 hours followed by a 1 . 5 hr pulse of EdU . White arrows indicate non - S - phase cells containing EdU colocalizing with telomeres . Scale bars = 10 µm Quantification of data shown in G . Non - S - phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres . Data were normalized to EV - Mock condition for each repeat . At least 100 non - S - phase cells were counted per condition per repeat , n = 4 for EV , WT , S49E , n = 3 for K189R . Values shown are mean ± sd . Data were analyzed using two - way ANOVA followed by Sidak ' s multiple comparison test .

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Representative images of combined EdU staining (Click-it) and DNA FISH for telomeric DNA. HuO9 cells were forward transfected with 2µg empty vector (EV), RAD54-WT, RAD54-K189R, or S49E construct. After 24 hours, cells were transfected with 20 nM siRAD54#2 for 48 hours followed by a 1.5 hr pulse of EdU. White arrows indicate non-S-phase cells containing EdU colocalizing with telomeres. Scale bars = 10 µm Quantification of data shown in G. Non-S-phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres. Data were normalized to EV-Mock condition for each repeat. At least 100 non-S-phase cells were counted per condition per repeat, n=4 for EV, WT, S49E, n=3 for K189R. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Sidak's multiple comparison test.


Representative images of combined IF and DNA FISH for BLM ( IF ) and telomeres ( FISH ) . HuO9 cells were transfected with 20nM siRAD54 # 2 for 48 hours . Scale bars = 10 µm Quantification of data shown in A . Cells were counted positive if they contained at least 1 colocalization event between BLM and telomeres . Data were normalized to mock condition of each repeat . At least 100 cells per condition per repeat were counted , n = 3 . Values shown are mean ± sd . Conditions were analyzed using unpaired two - tailed student ' s t - test .

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Representative images of combined IF and DNA FISH for BLM (IF) and telomeres (FISH). HuO9 cells were transfected with 20nM siRAD54#2 for 48 hours. Scale bars = 10 µm Quantification of data shown in A. Cells were counted positive if they contained at least 1 colocalization event between BLM and telomeres. Data were normalized to mock condition of each repeat. At least 100 cells per condition per repeat were counted, n=3. Values shown are mean±sd. Conditions were analyzed using unpaired two-tailed student's t-test.


Representative images of IF - DNA FISH for MUS81 and telomeres . HuO9 cells were transfected with 20nM siRAD54 # 2 for 48 hours prior to staining . Scale bars = 10 µm Quantification of data shown in C . Cells were counted positive if they contained at least 1 colocalization event between MUS81 and telomeres . At least 100 cells per condition were counted per repeat . n = 5 , values shown are mean ± sd . Conditions were analyzed using unpaired two - tailed student ' s t - test .

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Representative images of IF-DNA FISH for MUS81 and telomeres. HuO9 cells were transfected with 20nM siRAD54 #2 for 48 hours prior to staining. Scale bars = 10 µm Quantification of data shown in C. Cells were counted positive if they contained at least 1 colocalization event between MUS81 and telomeres. At least 100 cells per condition were counted per repeat. n=5, values shown are mean±sd. Conditions were analyzed using unpaired two-tailed student's t-test.


Representative images of HuO9 and SaOS2 chromosomes with or without an exchange event . Colocalization of green and red signal indicates an exchange event .

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Representative images of HuO9 and SaOS2 chromosomes with or without an exchange event. Colocalization of green and red signal indicates an exchange event.


Quantification of telomere sister chromatid exchange events . Cells were transfected with 20nM of siRNA for 48 hours prior to the addition of BrdU / BrdC . Metaphases were imaged by confocal microscopy and each data point represents a metaphase . For each condition , there were 3 biological repeats with a total of at least 1300 chromosome ends quantified . Values shown are mean ± sem . Conditions were analyzed using two - way ANOVA followed by Sidak ' s test .

Quantification of telomere sister chromatid exchange events. Cells were transfected with 20nM of siRNA for 48 hours prior to the addition of BrdU/BrdC. Metaphases were imaged by confocal microscopy and each data point represents a metaphase. For each condition, there were 3 biological repeats with a total of at least 1300 chromosome ends quantified. Values shown are mean±sem. Conditions were analyzed using two-way ANOVA followed by Sidak's test.


Representative images of UFB detection staining for PICH in HuO9 cells . Cells were transfected with 20nM of noted siRNAs for 72 hrs . Scale bars = 10

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Representative images of UFB detection staining for PICH in HuO9 cells. Cells were transfected with 20nM of noted siRNAs for 72 hrs. Scale bars = 10 µm


Quantification of data shown in G . Anaphases were scored from 4 independent experiments ( mock ) , or 3 independent experiments ( remaining conditions ) . Anaphases were considered positive for UFB if there was PICH staining between DAPI bodies . Single knockdown conditions had > 50 anaphases scored . Combined knockdown conditions had > 88 anaphases scored . Values shown are mean ± sd . Conditions were compared using one - way ANOVA followed by Dunnett ' s multiple comparison test . Quantification of data shown in G . Number of PICH stained UFBs counted per anaphase . Values shown are total counts over 3 independent experiments , shown mean ± sem . Total number of anaphases per condition were as follows : Mock n = 134 , siBLM / siSLX4 n = 90 , siRAD54 / siSLX4 n = 96 , siRAD54 / siBLM / siSLX4 n = 88 . Conditions were compared using Kruskal - Wallis test followed by Dunn ' s multiple comparison test .

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Quantification of data shown in G. Anaphases were scored from 4 independent experiments (mock), or 3 independent experiments (remaining conditions). Anaphases were considered positive for UFB if there was PICH staining between DAPI bodies. Single knockdown conditions had >50 anaphases scored. Combined knockdown conditions had >88 anaphases scored. Values shown are mean±sd. Conditions were compared using one-way ANOVA followed by Dunnett's multiple comparison test. Quantification of data shown in G. Number of PICH stained UFBs counted per anaphase. Values shown are total counts over 3 independent experiments, shown mean±sem. Total number of anaphases per condition were as follows: Mock n=134, siBLM/siSLX4 n=90, siRAD54/siSLX4 n=96, siRAD54/siBLM/siSLX4 n=88. Conditions were compared using Kruskal-Wallis test followed by Dunn's multiple comparison test.


Representative images of UFB staining for TRF2 in HuO9 cells . Cells were transfected with 20nM siRAD54 # 2 alone or cotransfected siRAD54 # 2 and siSLX4 for 72 hours prior to staining . Scale bars = 10

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Representative images of UFB staining for TRF2 in HuO9 cells. Cells were transfected with 20nM siRAD54#2 alone or cotransfected siRAD54#2 and siSLX4 for 72 hours prior to staining. Scale bars = 10 µm


Quantification of data shown in J . Anaphases were scored from 3 independent experiments . A total of 90 anaphases were scored per condition . Values shown are mean ± sd . Conditions were compared using one - way ANOVA followed by Dunnett ' s multiple comparison test . Quantification of micronuclei from HuO9 cells transfected with siRAD54 # 2 alone or siRAD54 # 2 cotransfected with siSLX4 for 48 hours . Micronuclei containing telomeric DNA were identified using DAPI and telomere FISH . Micronuclei were normalized to the number of nuclei counted . Values shown are mean ± sd of n = 3 biological replicates . Conditions were compared using one - way ANOVA followed by Dunnett ' s multiple comparison test .

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Quantification of data shown in J. Anaphases were scored from 3 independent experiments. A total of 90 anaphases were scored per condition. Values shown are mean±sd. Conditions were compared using one-way ANOVA followed by Dunnett's multiple comparison test. Quantification of micronuclei from HuO9 cells transfected with siRAD54#2 alone or siRAD54#2 cotransfected with siSLX4 for 48 hours. Micronuclei containing telomeric DNA were identified using DAPI and telomere FISH. Micronuclei were normalized to the number of nuclei counted. Values shown are mean±sd of n=3 biological replicates. Conditions were compared using one-way ANOVA followed by Dunnett's multiple comparison test.


( A ) Immunostaining of API in the vacuole of SEY6210 ( wild - type ) cell showing a dispersed pattern .

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(A) Immunostaining of API in the vacuole of SEY6210 (wild-type) cell showing a dispersed pattern.


( B - D ) Immunostaining of TVY1 ( pep4 ) cells showing API in the Cvt complex . The arrow points to a Cvt complex . Higher magnification images are shown in C and D .

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(B-D) Immunostaining of TVY1 (pep4) cells showing API in the Cvt complex. The arrow points to a Cvt complex. Higher magnification images are shown in C and D.


E ) Freeze - substitution fixation image of Cvt complex in TVY1 cell .

E) Freeze-substitution fixation image of Cvt complex in TVY1 cell.


( F ) Freeze - substitution fixation image of a Cvt complex in strain SEY6210 transformed with a 2μ plasmid encoding API grown in SD medium .

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(F) Freeze-substitution fixation image of a Cvt complex in strain SEY6210 transformed with a 2μ plasmid encoding API grown in SD medium.


( G ) Immunostaining of strain SEY6210 containing a 2μ APE1 plasmid grown in SD medium . N , Nucleus ; V , vacuole .

(G) Immunostaining of strain SEY6210 containing a 2μ APE1 plasmid grown in SD medium. N, Nucleus; V, vacuole.


( A and B ) Immunostaining image showing Cvt vesicles ( marked with arrows in A ) in the cytosol . Arrowhead and double arrowheads show the inner and outer membrane of Cvt vesicle , respectively .

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(A and B) Immunostaining image showing Cvt vesicles (marked with arrows in A) in the cytosol. Arrowhead and double arrowheads show the inner and outer membrane of Cvt vesicle, respectively.


( C ) Freeze - substitution fixation image of Cvt vesicles ( marked with arrows ) .

(C) Freeze-substitution fixation image of Cvt vesicles (marked with arrows).


( D ) Immunostaining of Cvt vesicle ( marked with arrows ) in the cytosol and Cvt body ( marked with double arrows ) in the vacuole .

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(D) Immunostaining of Cvt vesicle (marked with arrows) in the cytosol and Cvt body (marked with double arrows) in the vacuole.


( E and F ) Freeze - substitution fixation image of Cvt bodies in the vacuole ( marked with arrows ) . A Cvt vesicle is marked with an arrowhead .

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(E and F) Freeze-substitution fixation image of Cvt bodies in the vacuole (marked with arrows). A Cvt vesicle is marked with an arrowhead.


( G and H ) Freeze - substitution fixation image of Cvt vesicle contacting and fusing to a vacuole . V , Vacuole ; VM , vacuolar membrane .

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(G and H) Freeze-substitution fixation image of Cvt vesicle contacting and fusing to a vacuole. V, Vacuole; VM, vacuolar membrane.


Cvt complexes in TVY1 ( pep4 ) cells under nitrogen starvation conditions . Logarithmically growing cells in YEPD were transferred to nitrogen starvation medium ( SD [ - N ] ) at 30 ° C for 30 min ( A and B ) or 60 min ( C and D ) and prepared for electron microscopy as described in Fig . 1 . ( A ) Freeze - substitution fixation image showing a Cvt complex in an autophagosome ( marked by an arrow ) in the cytosol . ( B ) Freeze - substitution fixation image showing a Cvt complex in an autophagic body in the vacuole . ( C ) Immunostaining of a Cvt complex in an autophagic body in the vacuole . ( D ) Immunostaining of Cvt vesicles ( marked with arrows ) in the vacuole . AB , Autophagic body ; AP , autophagosome ; V , vacuole .

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

Cvt complexes in TVY1 (pep4) cells under nitrogen starvation conditions. Logarithmically growing cells in YEPD were transferred to nitrogen starvation medium (SD[-N]) at 30°C for 30 min (A and B) or 60 min (C and D) and prepared for electron microscopy as described in Fig. 1. (A) Freeze-substitution fixation image showing a Cvt complex in an autophagosome (marked by an arrow) in the cytosol. (B) Freeze-substitution fixation image showing a Cvt complex in an autophagic body in the vacuole. (C) Immunostaining of a Cvt complex in an autophagic body in the vacuole. (D) Immunostaining of Cvt vesicles (marked with arrows) in the vacuole. AB, Autophagic body; AP, autophagosome; V, vacuole.


Transport of Cvt complex in SEY6210 ( wild - type ) harboring 2μ plasmid encoding API under growing and nitrogen starvation conditions . Vegetative cells grown in SD medium were observed ( A - E ) . For starvation , logarithmically growing cells in SD were transferred to SD ( - N ) for 25 min ( F - H ) or 1 h ( I - K ) in the presence of 1 mM PMSF . Samples were prepared for electron microscopy as described in Fig . 1 . ( A ) Freeze - substitution fixation image of Cvt vesicle ( marked with an arrow ) . ( B ) Immunostaining of a Cvt vesicle ( marked with an arrow ) . ( C and D ) Higher magnification image of Cvt vesicle . Arrowhead and double arrowheads show the inner and outer membrane of a Cvt vesicle , respectively . ( E ) Freeze - substitution fixation image of a membrane sac enclosing small portion of a large Cvt complex ( marked with an arrow ) . ( F ) Freeze - substitution image of the wrapping of a Cvt complex by the isolation membrane ( marked with an arrow ) . ( G ) Freeze - substitution image of a Cvt vesicle ( marked with an arrow ) fusing to a vacuole . ( H ) Freeze - substitution image of autophagic body containing a Cvt complex . ( I ) Immunostaining image depicting the wrapping of a Cvt complex . The arrow marks the enwrapping membrane . ( J ) Immunostaining of an autophagosome containing a Cvt complex . ( K ) Immunostaining of an autophagic body containing a Cvt complex in the vacuole . V , vacuole .

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

Transport of Cvt complex in SEY6210 (wild-type) harboring 2μ plasmid encoding API under growing and nitrogen starvation conditions. Vegetative cells grown in SD medium were observed (A-E). For starvation, logarithmically growing cells in SD were transferred to SD(-N) for 25 min (F-H) or 1 h (I-K) in the presence of 1 mM PMSF. Samples were prepared for electron microscopy as described in Fig. 1. (A) Freeze-substitution fixation image of Cvt vesicle (marked with an arrow). (B) Immunostaining of a Cvt vesicle (marked with an arrow). (C and D) Higher magnification image of Cvt vesicle. Arrowhead and double arrowheads show the inner and outer membrane of a Cvt vesicle, respectively. (E) Freeze-substitution fixation image of a membrane sac enclosing small portion of a large Cvt complex (marked with an arrow). (F) Freeze-substitution image of the wrapping of a Cvt complex by the isolation membrane (marked with an arrow). (G) Freeze-substitution image of a Cvt vesicle (marked with an arrow) fusing to a vacuole. (H) Freeze-substitution image of autophagic body containing a Cvt complex. (I) Immunostaining image depicting the wrapping of a Cvt complex. The arrow marks the enwrapping membrane. (J) Immunostaining of an autophagosome containing a Cvt complex. (K) Immunostaining of an autophagic body containing a Cvt complex in the vacuole. V, vacuole.


Mutant analysis allows for differentiation between vacuolar delivery by Cvt vesicles and autophagosomes . ( A ) SEY6210 ( wild - type ) , cvt3 , and cvt7 yeast were grown in SD to 1 OD600 U / ml and transferred to SD ( - N ) . Aliquots were collected after 0 h ( + ) and 4 h ( − ) incubation and subjected to immunoblotting . The positions of precursor ( pro ) and mature ( m ) API are indicated . ( B ) cvt3 and cvt7 cells were pulse labeled for 10 min . After addition of cold cysteine and methionine , the cells were harvested , washed , and resuspended in either SD or SD ( - N ) and chased for the indicated times . API was recovered by immunoprecipitation , resolved on SDS polyacrylamide gels , and quantified using a phosphorimager ( STORM ; Molecular Dynamics , Sunnyvale , CA ) .

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

Mutant analysis allows for differentiation between vacuolar delivery by Cvt vesicles and autophagosomes. (A) SEY6210 (wild-type), cvt3, and cvt7 yeast were grown in SD to 1 OD600 U/ml and transferred to SD (-N). Aliquots were collected after 0 h (+) and 4 h (−) incubation and subjected to immunoblotting. The positions of precursor (pro) and mature (m) API are indicated. (B) cvt3 and cvt7 cells were pulse labeled for 10 min. After addition of cold cysteine and methionine, the cells were harvested, washed, and resuspended in either SD or SD(-N) and chased for the indicated times. API was recovered by immunoprecipitation, resolved on SDS polyacrylamide gels, and quantified using a phosphorimager (STORM; Molecular Dynamics, Sunnyvale, CA).


Analysis of the Cvt complex in the cvt3 mutant under vegetative and starvation conditions . Vegetative cells were grown in SD medium at 30 ° C to log phase . For starvation , logarithmically growing cells in SD medium were transferred to SD ( - N ) for 5 h . Samples were prepared for electron microscopy as described in Fig . 1 . ( A ) Immunostaining image depicting Cvt complex in the cytosol . Cvt vesicles are not detected in this strain . ( B ) Freeze - substitution fixation image showing Cvt complex in autophagosome ( marked with an arrow ) under starvation conditions , indicating that autophagy can still proceed . AP , Autophagosome ; N , nucleus ; V , vacuole .

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

Analysis of the Cvt complex in the cvt3 mutant under vegetative and starvation conditions. Vegetative cells were grown in SD medium at 30°C to log phase. For starvation, logarithmically growing cells in SD medium were transferred to SD(-N) for 5 h. Samples were prepared for electron microscopy as described in Fig. 1. (A) Immunostaining image depicting Cvt complex in the cytosol. Cvt vesicles are not detected in this strain. (B) Freeze-substitution fixation image showing Cvt complex in autophagosome (marked with an arrow) under starvation conditions, indicating that autophagy can still proceed. AP, Autophagosome; N, nucleus; V, vacuole.


Representative images of combined IF for H3K27me3 or H2AK119ub ( green ) with RNA FISH for Xist ( red ) in Xist - TetOP lines ( for clone 1 of each mutant type ) upon D2 in DOX conditions ; Blue - DAPI staining ; Scale bar : 10 µm .

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

Representative images of combined IF for H3K27me3 or H2AK119ub (green) with RNA FISH for Xist (red) in Xist-TetOP lines (for clone 1 of each mutant type) upon D2 in DOX conditions; Blue - DAPI staining; Scale bar: 10 µm.


Graph represents the mean % + S . E . M . of Xist - coated X chromosomes enriched for H3K27me3 or H2AK119ub in the different Xist - TetOP mutants ( for clone 1 of each mutant type ) from 2 - to - 4 independent experiments ; A minimum of 50 Xist - coated X chromosomes were counted per experiment ; Only p - values corresponding to significant differences from unpaired Student ' s t - test comparing mutants to Xist FL are indicated as * ( p - value < 0 . 05 ) .

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

Graph represents the mean % + S.E.M. of Xist-coated X chromosomes enriched for H3K27me3 or H2AK119ub in the different Xist-TetOP mutants (for clone 1 of each mutant type) from 2-to-4 independent experiments; A minimum of 50 Xist-coated X chromosomes were counted per experiment; Only p-values corresponding to significant differences from unpaired Student's t-test comparing mutants to Xist FL are indicated as * (p-value < 0.05).


Top 20 protein hits from the ChIRP - MS of Xist FL ; The ranking was based on fold - enrichment of Xist FL DOX versus Xist FL noDOX ; Weakly annotated protein isoforms with an Annotation score in UniprotKB < 3 ( out of 5 ) were excluded ; Fold - enrichment for Xist ΔB + C is also displayed for comparison ; Light green boxes correspond to proteins as Xist interactors [ 13 ] ; protein in red ( RNF2 / RING1B ) represents a protein not found in the Xist ΔB + C interactome ; Protein in light brown ( TRIM71 ) is less enriched in Xist ΔB + C than in Xist FL .

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

Top 20 protein hits from the ChIRP-MS of Xist FL; The ranking was based on fold-enrichment of Xist FL DOX versus Xist FL noDOX; Weakly annotated protein isoforms with an Annotation score in UniprotKB < 3 (out of 5) were excluded; Fold-enrichment for Xist ΔB+C is also displayed for comparison; Light green boxes correspond to proteins as Xist interactors [13]; protein in red (RNF2/RING1B) represents a protein not found in the Xist ΔB+C interactome; Protein in light brown (TRIM71) is less enriched in Xist ΔB+C than in Xist FL.


Scatter plot displaying the differences in peptide counts between Xist FL and Xist ΔB + C for the 74 out of 81 Xist - interactors with a minimum of fold - change of 2 . 5 in Xist FL or Xist ΔB + C ; Shown is the log2 fold change of peptide counts of each mutant in DOX conditions compared with the Xist FL in noDOX conditions ; proteins retrieved by both Xist FL and Xist ΔB + C ChIRPs with a proposed role in XCI such as SPEN , RBM15 , WTAP , YTHDC1 and hnRNPU are indicated ; light brown dots mark proteins more represented in Xist FL than in Xist ΔB + C ChIRPs , while red dots display proteins which are only retrieved by Xist FL ChIRP .

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

Scatter plot displaying the differences in peptide counts between Xist FL and Xist ΔB+C for the 74 out of 81 Xist-interactors with a minimum of fold-change of 2.5 in Xist FL or Xist ΔB+C; Shown is the log2 fold change of peptide counts of each mutant in DOX conditions compared with the Xist FL in noDOX conditions; proteins retrieved by both Xist FL and Xist ΔB+C ChIRPs with a proposed role in XCI such as SPEN, RBM15, WTAP, YTHDC1 and hnRNPU are indicated; light brown dots mark proteins more represented in Xist FL than in Xist ΔB+C ChIRPs, while red dots display proteins which are only retrieved by Xist FL ChIRP.


Plots showing H3K27me3 and H2AK119ub accumulation over the X chromosome in Xist FL and Xist ΔB + C with upon DOX induction at day 2 ( D2 ) of differentiation ; Each dot represents a single 10Kb window and its enrichment relative to noDOX condition ; Black line is a loess regression on all windows ; Xist locus is represented by a blue long line , active genes by green lines .

O O B-MEASURED_VAR O B-MEASURED_VAR 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 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

Plots showing H3K27me3 and H2AK119ub accumulation over the X chromosome in Xist FL and Xist ΔB+C with upon DOX induction at day 2 (D2) of differentiation; Each dot represents a single 10Kb window and its enrichment relative to noDOX condition; Black line is a loess regression on all windows; Xist locus is represented by a blue long line, active genes by green lines.


Violin plots quantifying H3K27me3 and H2AK119ub enrichment over intergenic regions , active promoters and active gene bodies in the X chromosome in Xist FL and Xist ΔB + C cell lines at D2 upon DOX induction ; Shown is the log2 fold change of DOX vs noDOX conditions ; violin plots represent the distribution of the values , the horizontal band is the median , the lower and upper hinges correspond to the 25th and 75th percentiles ; n = indicates the number of regions / genes analyzed ; p - values were calculated using paired Wilcoxon test , comparing Xist FL and Xist ΔB + C cell lines .

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

Violin plots quantifying H3K27me3 and H2AK119ub enrichment over intergenic regions, active promoters and active gene bodies in the X chromosome in Xist FL and Xist ΔB+C cell lines at D2 upon DOX induction; Shown is the log2 fold change of DOX vs noDOX conditions; violin plots represent the distribution of the values, the horizontal band is the median, the lower and upper hinges correspond to the 25th and 75th percentiles; n = indicates the number of regions/genes analyzed; p-values were calculated using paired Wilcoxon test, comparing Xist FL and Xist ΔB+C cell lines.


Average plots showing the mean enrichment of H3K27me3 ( top ) and H2AK119ub ( bottom ) over all X - linked initially active transcriptional start sites ( TSS ) ; Shown is the mean of normalized log2 enrichment of DOX vs noDOX in both Xist FL and Xist ΔB + C cell lines .

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

Average plots showing the mean enrichment of H3K27me3 (top) and H2AK119ub (bottom) over all X-linked initially active transcriptional start sites (TSS); Shown is the mean of normalized log2 enrichment of DOX vs noDOX in both Xist FL and Xist ΔB+C cell lines.


Genome browser plots showing H3K27me3 ( top ) and H2AK119ub ( bottom ) enrichments in a region encompassing the inactive Atp1b4 and the initially active Lamp2 genes within the XqA3 . 3 region and the initially active Rlim / Rnf12 gene at the XqD region ; Region around the promoter of Rlim / Rnf12 is highlighted in yellow .

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

Genome browser plots showing H3K27me3 (top) and H2AK119ub (bottom) enrichments in a region encompassing the inactive Atp1b4 and the initially active Lamp2 genes within the XqA3.3 region and the initially active Rlim/Rnf12 gene at the XqD region; Region around the promoter of Rlim/Rnf12 is highlighted in yellow.


Graph represents the mean % + S . E . M . of Xist - coated chromosomes presenting an active Pgk1 or Lamp2 gene as determined by RNA FISH ( as represented in B ) at D2 in the presence of DOX ( Xist FL was also used in noDOX conditions ) in the different Xist - TetOP mutants ; each bar represents the mean from to 2 - to - 4 independent experiments ; A minimum of 59 Xist - coated chromosomes were counted per experiment ; For Xist FL noDOX a minimum of 100 cells ( which do not have Xist - coated chromosome ) were counted ; Only p - values corresponding to significant differences comparing mutants ( or Xist FL noDOX ) to Xist FL DOX are indicated as * ( p - value < 0 . 05 ) or * * * ( p - value < 0 . 01 ) , unpaired Student ' s t - test ; dashed line marks the mean percentage of silencing for the Lamp2 gene in Xist FL DOX .

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

Graph represents the mean % + S.E.M. of Xist-coated chromosomes presenting an active Pgk1 or Lamp2 gene as determined by RNA FISH (as represented in B) at D2 in the presence of DOX (Xist FL was also used in noDOX conditions) in the different Xist-TetOP mutants; each bar represents the mean from to 2-to-4 independent experiments; A minimum of 59 Xist-coated chromosomes were counted per experiment; For Xist FL noDOX a minimum of 100 cells (which do not have Xist-coated chromosome) were counted; Only p-values corresponding to significant differences comparing mutants (or Xist FL noDOX) to Xist FL DOX are indicated as * (p-value < 0.05) or *** (p-value < 0.01), unpaired Student's t-test; dashed line marks the mean percentage of silencing for the Lamp2 gene in Xist FL DOX.


Representative RNA FISH images for Xist ( red ) and nascent - transcript of Pgk1 ( green ) in Xist - TetOP lines at day 4 of differentiation in the presence of DOX ( Xist FL is also shown in noDOX conditions ) ; DNA stained in blue by DAPI ; Numbers represent % of Xist - coated X - chromosomes ± S . E . M . with active Pgk1 gene ( except for Xist FL noDOX , where numbers represent % of cells with Pgk1 active gene ) ; The values represent 2 - to - 4 independent experiments , where a minimum of 50 Xist - coated chromosomes were counted per experiment ; Significant differences compared with Xist FL ( DOX ) are indicated as * ( p - value < 0 . 05 ) or * * * ( p - value < 0 . 01 ) , unpaired Student ' s t - test .

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

Representative RNA FISH images for Xist (red) and nascent-transcript of Pgk1 (green) in Xist-TetOP lines at day 4 of differentiation in the presence of DOX (Xist FL is also shown in noDOX conditions); DNA stained in blue by DAPI; Numbers represent % of Xist-coated X-chromosomes ± S.E.M. with active Pgk1 gene (except for Xist FL noDOX, where numbers represent % of cells with Pgk1 active gene); The values represent 2-to-4 independent experiments, where a minimum of 50 Xist-coated chromosomes were counted per experiment; Significant differences compared with Xist FL (DOX) are indicated as * (p-value < 0.05) or *** (p-value < 0.01), unpaired Student's t-test.


Clustering analysis of the normalized RNA - seq counts on the X chromosome ( chrX ) for all the duplicates of Xist FL , Xist ΔA and Xist ΔB + C in DOX and noDOX conditions .

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

Clustering analysis of the normalized RNA-seq counts on the X chromosome (chrX) for all the duplicates of Xist FL, Xist ΔA and Xist ΔB+C in DOX and noDOX conditions.


Plots displays the log2 ( fold - change ) in the expression of X - linked genes along the chrX comparing DOX versus noDOX samples for Xist FL , Xist ΔA and Xist ΔB + C at day 2 of differentiation ; red dots correspond to genes which are differently expressed in DOX vs noDOX ( p < 0 . 05 , Limma t - test ) , while black dots represent genes which are not differentially expressed between the two conditions ( 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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O

Plots displays the log2(fold-change) in the expression of X-linked genes along the chrX comparing DOX versus noDOX samples for Xist FL, Xist ΔA and Xist ΔB+C at day 2 of differentiation; red dots correspond to genes which are differently expressed in DOX vs noDOX (p < 0.05, Limma t-test), while black dots represent genes which are not differentially expressed between the two conditions (p ≥ 0.05).


Violin plots displaying the average log2 ( fold - change ) in gene expression between DOX and noDOX conditions on the chrX in Xist FL , Xist ΔA and Xist ΔB + C at day 2 of differentiation ; violin plots represent the distribution of the values , the horizontal band is the median , the lower and upper hinges correspond to the 25th and 75th percentiles ; p - values were calculated using paired Wilcoxon test ; n = indicates the number of genes analyzed .

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 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

Violin plots displaying the average log2(fold-change) in gene expression between DOX and noDOX conditions on the chrX in Xist FL, Xist ΔA and Xist ΔB+C at day 2 of differentiation; violin plots represent the distribution of the values, the horizontal band is the median, the lower and upper hinges correspond to the 25th and 75th percentiles; p-values were calculated using paired Wilcoxon test; n = indicates the number of genes analyzed.


Box plots displaying the log2 ( DOX / noDOX ) fold - change in expression of X - linked genes in Xist FL and Xist ΔB + C categorized according to the enrichment of H3K27me3 and H2AK119ub marks at promoters in Xist ΔB + C upon DOX induction ( with no or little accumulation vs accumulation ) ; the horizontal band of the boxplot is the median of the values , the lower and upper hinges correspond to the 25th and 75th percentiles , the upper whisker extends from the hinge to the largest value not further than 1 . 5 interquartile range from the hinge , the lower whisker extends from the hinge to the smallest value at most 1 . 5 interquartile range of the hinge ; p - values between samples were calculated using paired Wilcoxon test ; n = indicates the number of genes analyzed .

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

Box plots displaying the log2(DOX/noDOX) fold-change in expression of X-linked genes in Xist FL and Xist ΔB+C categorized according to the enrichment of H3K27me3 and H2AK119ub marks at promoters in Xist ΔB+C upon DOX induction (with no or little accumulation vs accumulation); the horizontal band of the boxplot is the median of the values, the lower and upper hinges correspond to the 25th and 75th percentiles, the upper whisker extends from the hinge to the largest value not further than 1.5 interquartile range from the hinge, the lower whisker extends from the hinge to the smallest value at most 1.5 interquartile range of the hinge; p-values between samples were calculated using paired Wilcoxon test; n = indicates the number of genes analyzed.


Genome browser plots showing RNA - seq reads , H3K27me3 and H2AK119ub nChIP reads around the Abcb7 gene for Xist FL ( left ) and Xist ΔB + C ( right ) at day 2 of differentiation in both DOX and noDOX conditions .

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Genome browser plots showing RNA-seq reads, H3K27me3 and H2AK119ub nChIP reads around the Abcb7 gene for Xist FL (left) and Xist ΔB+C (right) at day 2 of differentiation in both DOX and noDOX conditions.


Boxplots showing differences of the APTT time ( left ) and ME1 - enriched protein expressions ( right ) between mild ( n = 13 ) and severe ( n = 18 ) COVID - 19 patients . Differences between groups were estimated using Mann - Whitney - Wilcoxon test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The horizontal box lines in the boxplots represent the first quartile , the median , and the third quartile . Whiskers denote the range of points within the first quartile − 1 . 5 × the interquartile range and the third quartile + 1 . 5 × the interquartile range .

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Boxplots showing differences of the APTT time (left) and ME1-enriched protein expressions (right) between mild (n=13) and severe (n=18) COVID-19 patients. Differences between groups were estimated using Mann-Whitney-Wilcoxon test. * p <0.05, ** p <0.01, *** p <0.001. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.


Boxplots showing differences of IL - 6 level , IL - 10 level ( left ) and ME15 - enriched protein expressions ( right ) between mild ( n = 13 ) and severe ( n = 18 ) COVID - 19 patients . Differences between groups were estimated using Mann - Whitney - Wilcoxon test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The horizontal box lines in the boxplots represent the first quartile , the median , and the third quartile . Whiskers denote the range of points within the first quartile − 1 . 5 × the interquartile range and the third quartile + 1 . 5 × the interquartile range .

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Boxplots showing differences of IL-6 level, IL-10 level (left) and ME15-enriched protein expressions (right) between mild (n=13) and severe (n=18) COVID-19 patients. Differences between groups were estimated using Mann-Whitney-Wilcoxon test. * p <0.05, ** p <0.01, *** p <0.001. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.


Rose plots indicating the number of all detected and differently expressed tissues - enhanced proteins in mild ( left ) and severe patients ( right ) compared with healthy controls . Tissues labeled in orange represented tissues with the largest proportions ( 100 % of all detected tissue - enhanced proteins ) of altered tissues - enhanced proteins .

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Rose plots indicating the number of all detected and differently expressed tissues-enhanced proteins in mild (left) and severe patients (right) compared with healthy controls. Tissues labeled in orange represented tissues with the largest proportions (100% of all detected tissue-enhanced proteins) of altered tissues-enhanced proteins.


Boxplots indicating the expression level of known brain dysfunctional biomarkers in control ( n = 12 ) , mild ( n = 13 ) and severe ( n = 18 ) patients . Differences between groups were estimated using Kruskal - Wallis test . The horizontal box lines in the boxplots represent the first quartile , the median , and the third quartile . Whiskers denote the range of points within the first quartile − 1 . 5 × the interquartile range and the third quartile + 1 . 5 × the interquartile range .

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Boxplots indicating the expression level of known brain dysfunctional biomarkers in control (n=12), mild (n=13) and severe (n=18) patients. Differences between groups were estimated using Kruskal-Wallis test. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.


Boxplot showing the selected cell type specific proteins among control ( n = 12 ) , mild ( n = 13 ) and severe ( n = 18 ) COVID - 19 patients . Differences between groups were estimated using Kruskal - Wallis test . The horizontal box lines in the boxplots represent the first quartile , the median , and the third quartile . Whiskers denote the range of points within the first quartile − 1 . 5 × the interquartile range and the third quartile + 1 . 5 × the interquartile range .

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Boxplot showing the selected cell type specific proteins among control (n=12), mild (n=13) and severe (n=18) COVID-19 patients. Differences between groups were estimated using Kruskal-Wallis test. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.


Heatmap of IFNG , GZMB and PRF1 gene expression in COVID - 19 patients . Average expression values were centered and scaled . Red indicates a higher expression and blue indicates a lower expression .

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Heatmap of IFNG, GZMB and PRF1 gene expression in COVID-19 patients. Average expression values were centered and scaled. Red indicates a higher expression and blue indicates a lower expression.


Heatmap of the expression of genes enriched in IL - 17 signaling pathway between healthy control and COVID - 19 patients . Average expression values were centered and scaled . Red indicates a higher expression and blue indicates a lower expression .

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Heatmap of the expression of genes enriched in IL-17 signaling pathway between healthy control and COVID-19 patients. Average expression values were centered and scaled. Red indicates a higher expression and blue indicates a lower expression.


COVID - 19 severity is associated with significant changes in lipoprotein subclasses including high - density lipoprotein subclass - 1 ( HDL1 ) , HDL4 , low - density lipoprotein subclasses ( LDL1 , LDL4 , LDL5 ) , very low - density lipoprotein subclass - 5 ( VLDL5 ) and their compositional components ( ApoA1 , triglycerides , cholesterol ) . TG : triglycerides ; FC : free cholesterol ; CE : cholesteryl esters ; CH : total cholesterol ( i . e . , FC + CE ) ; PL : total phospholipids ; A1 : ApoA1 ; A2 : ApoA2 ; L1TG : TG in LDL1 ; L1TG % : percentages of L1TG in total lipids of LDL1 ; L1 % : percentage of LDL1 in all LDL ; L - TG % : percentages of L - TG ( TG in LDL ) in total lipids of LDL ; V5FC % , V5CE % : percentages of V5FC ( FC in VLDL5 ) and V5CE ( CE in VLDL5 ) in total lipids of VLDL5 ; L5CE % , L5CH % : percentages of L5CE ( CE in LDL5 ) and L5CH ( CH in LDL5 ) in total lipids of LDL5 ; H1FC % : percentages of H1FC ( FC in HDL1 ) in total lipids of HDL1 ; H - A2 : ApoA2 in both HDL and nascent HDL ; H4A1 , H4A2 , H4CE , H4CH , H4FC , H4PL : ApoA1 , ApoA2 , CE , CH , FC and PL in HDL4 ;

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COVID-19 severity is associated with significant changes in lipoprotein subclasses including high-density lipoprotein subclass-1 (HDL1), HDL4, low-density lipoprotein subclasses (LDL1, LDL4, LDL5), very low-density lipoprotein subclass-5 (VLDL5) and their compositional components (ApoA1, triglycerides, cholesterol). TG: triglycerides; FC: free cholesterol; CE: cholesteryl esters; CH: total cholesterol (i.e., FC + CE); PL: total phospholipids; A1: ApoA1; A2: ApoA2; L1TG: TG in LDL1; L1TG%: percentages of L1TG in total lipids of LDL1; L1%: percentage of LDL1 in all LDL; L-TG%: percentages of L-TG(TG in LDL) in total lipids of LDL; V5FC%, V5CE%: percentages of V5FC(FC in VLDL5) and V5CE(CE in VLDL5) in total lipids of VLDL5; L5CE%, L5CH%: percentages of L5CE(CE in LDL5) and L5CH(CH in LDL5) in total lipids of LDL5; H1FC%: percentages of H1FC (FC in HDL1) in total lipids of HDL1; H-A2: ApoA2 in both HDL and nascent HDL; H4A1, H4A2, H4CE, H4CH, H4FC, H4PL: ApoA1, ApoA2, CE, CH, FC and PL in HDL4;


Plasma levels of key enzymes and proteins directly involving lipoprotein metabolism are indicators for COVID - 19 severity . Data were represented as means ± SD and differences between groups were estimated using a Student ' s t test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Control , n = 12 ; mild , n = 29 ; severe , n = 17 ; discharge , n = 16 ; sLDLR : soluble low - density lipoprotein receptor ; LCAT : lecithin - cholesterol acyltransferase ; CEPT : cholesteryl - ester transfer protein .

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Plasma levels of key enzymes and proteins directly involving lipoprotein metabolism are indicators for COVID-19 severity. Data were represented as means ± SD and differences between groups were estimated using a Student's t test. * p < 0.05; ** p < 0.01; *** p < 0.001. Control, n=12; mild, n=29; severe, n=17; discharge, n=16; sLDLR: soluble low-density lipoprotein receptor; LCAT: lecithin-cholesterol acyltransferase; CEPT: cholesteryl-ester transfer protein.


TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress . ( A ) ROS levels in U2OS cells stably over - expressing Flag - tagged - TIGAR ( clones TIGAR # 5 and TIGAR # 7 ) or control cells ( clones Cont # 1 and Cont # 3 ) left untreated , after 6 h of nutrient starvation or 24 h of metabolic stress . ROS levels were measured by flow cytometry after DCF treatment . The results are expressed as the mean DCF fluorescence ( and standard deviation ) , from three independent experiments .

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TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress. (A) ROS levels in U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) left untreated, after 6 h of nutrient starvation or 24 h of metabolic stress. ROS levels were measured by flow cytometry after DCF treatment. The results are expressed as the mean DCF fluorescence (and standard deviation), from three independent experiments.


TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress . ( B ) Basal , nutrient starvation - induced ( 5 h ) or metabolic stress - induced ( 18 h ) ROS levels in U2OS cells in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 , measured by flow cytometry after DCF treatment . The results are expressed as the mean intensity of cell fluorescence ( and standard deviation ) . * represents significant difference from control conditions ( P < 0 . 05 ) .

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TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress.(B) Basal, nutrient starvation-induced (5 h) or metabolic stress-induced (18 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, measured by flow cytometry after DCF treatment. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05).


TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress . ( A ) ( Left panel ) confocal microscopy images of the fluorescence in U2OS cells stably over - expressing Flag - tagged - TIGAR ( clone TIGAR # 7 ) or control cells ( clone Cont # 1 ) and infected with an adenovirus expressing GFP - LC3 for 16 h . Cells were then left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h . ( Right panel ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta from three independent experiments . The mean values with standard deviation are presented .

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TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress. (A) (Left panel) confocal microscopy images of the fluorescence in U2OS cells stably over-expressing Flag-tagged-TIGAR (clone TIGAR#7) or control cells (clone Cont#1) and infected with an adenovirus expressing GFP-LC3 for 16 h. Cells were then left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. (Right panel) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented.


TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress . ( B ) ( Left panel ) Confocal microscopy images of the fluorescence in U2OS cells stably expressing GFP - LC3 and transfected with scrambled or TIGAR siRNAs . After 48 h transfection , cells were then left untreated , exposed to nutrient starvation for 5 h or to metabolic stress for 18 h . ( Right panel ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta from three independent experiments . The mean values with standard deviation are presented .

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TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress.(B) (Left panel) Confocal microscopy images of the fluorescence in U2OS cells stably expressing GFP-LC3 and transfected with scrambled or TIGAR siRNAs. After 48 h transfection, cells were then left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h. (Right panel) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented.


TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress . ( C ) ( Left panel ) Western blot showing the expression levels of endogenous LC3 - I , LC3 - II and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs , and 48 h later exposed to nutrient starvation for 0 , 2 . 5 and 6 h . ( Middle panel ) Western blot showing the expression levels of endogenous LC3 - I , LC3 - II and TIGAR in U2OS stably over - expressing Flag - tagged - TIGAR ( clones TIGAR # 5 and TIGAR # 7 ) or control cells ( clones Cont # 1 and Cont # 3 ) and left untreated . ( Right panel ) Western blot showing the expression levels of p62 , COX - IV and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs and left untreated . Actin expression was examined as a loading control .

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TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress. (C) (Left panel) Western blot showing the expression levels of endogenous LC3-I, LC3-II and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs, and 48 h later exposed to nutrient starvation for 0, 2.5 and 6 h. (Middle panel) Western blot showing the expression levels of endogenous LC3-I, LC3-II and TIGAR in U2OS stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) and left untreated. (Right panel) Western blot showing the expression levels of p62, COX-IV and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs and left untreated. Actin expression was examined as a loading control.


TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress . ( D ) Western blot showing the expression levels of endogenous TIGAR in U2OS cells after exposure to nutrient starvation or metabolic stress for 0 , 1 , 3 , 5 and 8 h ; * represents significant difference from control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

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TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress.(D) Western blot showing the expression levels of endogenous TIGAR in U2OS cells after exposure to nutrient starvation or metabolic stress for 0, 1, 3, 5 and 8 h; * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).