Figure 3Representative 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 . 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 .

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

Figure 3Representative 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.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.


Figure 4Representative 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 . 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 . 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 µmQuantification 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 µmQuantification 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 .

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

Figure 4Representative 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.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.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 µmQuantification 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 µmQuantification 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.


Figure 1 ( 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 . 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 . ( G ) Immunostaining of strain SEY6210 containing a 2μ APE1 plasmid grown in SD medium . N , Nucleus ; V , vacuole .

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

Figure 1(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.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.(G) Immunostaining of strain SEY6210 containing a 2μ APE1 plasmid grown in SD medium. N, Nucleus; V, vacuole.


Figure 2 ( 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 ) . ( 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 . ( G and H ) Freeze - substitution fixation image of Cvt vesicle contacting and fusing to a vacuole . V , Vacuole ; VM , vacuolar membrane .

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

Figure 2(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).(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.(G and H) Freeze-substitution fixation image of Cvt vesicle contacting and fusing to a vacuole. V, Vacuole; VM, vacuolar membrane.


Figure 3Cvt 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 .

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Figure 3Cvt 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.


Figure 4Transport 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Transport 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.


Figure 5Mutant 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 ) .

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Figure 5Mutant 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).


Figure 6Analysis 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 .

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Figure 6Analysis 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.


Figure 1Representative 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Representative 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).


Figure 2Top 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Top 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.


Figure 3Plots 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Plots 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.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.


Figure 4Graph 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 . 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 ) . 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 . 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 .

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

Figure 4Graph 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.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).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.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.


Figure 3Boxplots 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 .

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

Figure 3Boxplots 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.


Figure 4Rose 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 .

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

Figure 4Rose 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.


Figure 5Boxplot 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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Figure 5Boxplot 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.


Figure 6Heatmap 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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Figure 6Heatmap 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.


Figure 7COVID - 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 .

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

Figure 7COVID-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.


Figure 1TIGAR 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 ) .

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

Figure 1TIGAR 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).


Figure 2TIGAR 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 . 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 ) .

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

Figure 2TIGAR 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.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).


Figure 3TIGAR expression modulates autophagy independently of p53 . ( A ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta . U2OS cells stably expressing GFP - LC3 were transfected with scrambled or TIGAR siRNAs , and 48 h after transfection , cells were left untreated ( t0 ) or treated with Bafilomycin A1 ( 100 nM ) for 1 or 2 h ( t1 and t2 ) . The percentage of cells with GFP - LC3 puncta was calculated at the indicated time points . Data are shown as the mean and standard deviation from three independent experiments . ( B ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta . U2OS cells stably expressing GFP - LC3 were cotransfected with scrambled or TIGAR siRNAs , and scrambled or p53 siRNA . After 48 h transfection , cells were left untreated or exposed for 5 h to nutrient starvation . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments . ( C ) Western blot showing the expression levels of endogenous p53 and TIGAR in U2OS cells cotransfected with scrambled or TIGAR siRNAs , and scrambled or p53 siRNA , and 48 h later exposed to nutrient starvation for 5 h . Actin expression was examined as a loading control . ( D ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta . U2OS cells stably expressing GFP - LC3 were cotransfected with scrambled or TIGAR siRNAs , and scrambled or DRAM siRNA1 / 2 . After 48 h transfection , cells were left untreated or exposed for 5 h to nutrient starvation . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments ; * represents significant difference from starved control conditions ( P < 0 . 05 ) ; @ represents significant difference from untreated control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

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

Figure 3TIGAR expression modulates autophagy independently of p53. (A) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were transfected with scrambled or TIGAR siRNAs, and 48 h after transfection, cells were left untreated (t0) or treated with Bafilomycin A1 (100 nM) for 1 or 2 h (t1 and t2). The percentage of cells with GFP-LC3 puncta was calculated at the indicated time points. Data are shown as the mean and standard deviation from three independent experiments.(B) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were cotransfected with scrambled or TIGAR siRNAs, and scrambled or p53 siRNA. After 48 h transfection, cells were left untreated or exposed for 5 h to nutrient starvation. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.(C) Western blot showing the expression levels of endogenous p53 and TIGAR in U2OS cells cotransfected with scrambled or TIGAR siRNAs, and scrambled or p53 siRNA, and 48 h later exposed to nutrient starvation for 5 h. Actin expression was examined as a loading control.(D) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were cotransfected with scrambled or TIGAR siRNAs, and scrambled or DRAM siRNA1/2. After 48 h transfection, cells were left untreated or exposed for 5 h to nutrient starvation. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments; * represents significant difference from starved control conditions (P<0.05); @ represents significant difference from untreated control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


Figure 4 ( A ) U2OS cells were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h , with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) for 24 h . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments . ( B ) ( Left panel ) ROS levels in U2OS cells left untreated or treated with 0 . 5 or 1 mM of H2O2 for 24 h . 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 . ( Right panel ) Quantitation of the percentage of GFP - LC3 puncta positive cells for cells treated as described above . Data are shown as the mean and standard deviation from three independent experiments . ( C ) Quantitation of the percentage of GFP - LC3 puncta positive cells . U2OS cells stably expressing GFP - LC3 were transfected with vector pCHER1A expressing the mCherry gene as control , or expression plasmids for Flag - tagged - TIGAR or HA - tagged - FBPase - 2 . After 48 h transfection , cells were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h , with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) for 24 h . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments . ( D ) Quantitation of the percentage of GFP - LC3 puncta positive cells . Cells were left untreated , exposed to nutrient starvation for 6 h with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) , AO2 ( glutathione ethyl ester ( 4 mM ) ) or AO3 ( ethyl pyruvate ( 4 mM ) ) for 24 h . U2OS cells stably expressing GFP - LC3 were transfected with vector pCHER1A expressing the mCherry gene as control , or expression plasmid for Flag - tagged - TIGAR . After 48 h transfection , cells were treated . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments . ( E ) ( Left panel ) Quantitation of the percentage of GFP - LC3 puncta positive cells . Cells were left untreated , exposed to nutrient starvation for 5 h or to metabolic stress for 18 h , with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) for 24 h . U2OS cells stably over - expressing GFP - LC3 in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 . ( Right panel ) Basal or nutrient starvation - induced ( 5 h ) ROS levels in U2OS cells in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) for 24 h , 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 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

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

Figure 4(A) U2OS cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.(B) (Left panel) ROS levels in U2OS cells left untreated or treated with 0.5 or 1 mM of H2O2 for 24 h. 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. (Right panel) Quantitation of the percentage of GFP-LC3 puncta positive cells for cells treated as described above. Data are shown as the mean and standard deviation from three independent experiments.(C) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably expressing GFP-LC3 were transfected with vector pCHER1A expressing the mCherry gene as control, or expression plasmids for Flag-tagged-TIGAR or HA-tagged-FBPase-2. After 48 h transfection, cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.(D) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 6 h with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)), AO2 (glutathione ethyl ester (4 mM)) or AO3 (ethyl pyruvate (4 mM)) for 24 h. U2OS cells stably expressing GFP-LC3 were transfected with vector pCHER1A expressing the mCherry gene as control, or expression plasmid for Flag-tagged-TIGAR. After 48 h transfection, cells were treated. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.(E) (Left panel) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2. (Right panel) Basal or nutrient starvation-induced (5 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2 with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h, 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); # represents a lack of significant difference from control conditions (P>0.05).


Figure 5 ( A ) Western blot showing knockdown of ATG5 protein expression by ATG5 siRNA . ( B ) RT - PCR showing knockdown of ATG10 mRNA expression by ATG10 siRNA . ( C ) Quantitation of the percentage of GFP - LC3 puncta positive cells . U2OS cells stably over - expressing GFP - LC3 in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 , and either scrambled , ATG5 siRNA or ATG10 siRNA . After 48 h , cells were left untreated or exposed to nutrient starvation for 5 h . ( D ) ROS levels in U2OS cells in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 , and scrambled , ATG5 siRNA or ATG10 siRNA , measured by flow cytometry after DCF treatment . After 48 h , cells were left untreated or exposed to nutrient starvation for 5 h . The results are expressed as the mean intensity of cell fluorescence ( and standard deviation ) . * represents significant difference from control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

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

Figure 5(A) Western blot showing knockdown of ATG5 protein expression by ATG5 siRNA.(B) RT-PCR showing knockdown of ATG10 mRNA expression by ATG10 siRNA.(C) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, and either scrambled, ATG5 siRNA or ATG10 siRNA. After 48 h, cells were left untreated or exposed to nutrient starvation for 5 h.(D) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, and scrambled, ATG5 siRNA or ATG10 siRNA, measured by flow cytometry after DCF treatment. After 48 h, cells were left untreated or exposed to nutrient starvation for 5 h. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


Figure 6 ( A ) Quantitation of the percentage of GFP - LC3 puncta positive cells . U2OS cells stably over - expressing Flag - tagged - TIGAR ( clones TIGAR # 5 and TIGAR # 7 ) or control cells ( clones Cont # 1 and Cont # 3 ) were left untreated or exposed to nutrient starvation for 6 h , with or without treatment with Z - VAD - FMK for 24 h . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation of the mean from three independent experiments . ( B ) Apoptosis in U2OS cells stably over - expressing Flag - tagged - TIGAR ( clones TIGAR # 5 and TIGAR # 7 ) or control cells ( clones Cont # 1 and Cont # 3 ) , as measured by cells with a sub - G1 DNA content . Cells were transfected with either scrambled , TIGAR siRNA1 or TIGAR siRNA2 . Cells were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h . ( C ) Apoptosis in U2OS cells cotransfected with scrambled or TIGAR siRNAs , and scrambled , ATG5 siRNA or ATG10 siRNA , as measured by cells with a sub - G1 DNA content . After 48 h , cells were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h . Data are shown as the mean and standard deviation from three independent experiments . In each case , the increase in apoptosis after knockdown of ATG5 or ATG10 , compared with the matched control , was statistically significant ; * 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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Figure 6(A) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) were left untreated or exposed to nutrient starvation for 6 h, with or without treatment with Z-VAD-FMK for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation of the mean from three independent experiments.(B) Apoptosis in U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3), as measured by cells with a sub-G1 DNA content. Cells were transfected with either scrambled, TIGAR siRNA1 or TIGAR siRNA2. Cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h.(C) Apoptosis in U2OS cells cotransfected with scrambled or TIGAR siRNAs, and scrambled, ATG5 siRNA or ATG10 siRNA, as measured by cells with a sub-G1 DNA content. After 48 h, cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. Data are shown as the mean and standard deviation from three independent experiments. In each case, the increase in apoptosis after knockdown of ATG5 or ATG10, compared with the matched control, was statistically significant; * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


Figure 1 ( G ) Liver proteins were resolved by PAGE and the binding of serum antibodies from 4 individual mice from each group ( numbered 1 - 4 ) was detected by anti - IgG secondary antibody .

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Figure 1(G) Liver proteins were resolved by PAGE and the binding of serum antibodies from 4 individual mice from each group (numbered 1-4) was detected by anti-IgG secondary antibody .


Figure 3Male C57BL / 6 Nr4a2 cKO or wildtype littermate C57BL / 6 mice were immunised twice with NP - KLH emulsified in alum . After 2 weeks , 7 μm spleen sections were stained with Red : Alexa - Fluor - 594 - CD4 and Green : FITC - B220 ( D ) or FITC - GL7 ( E ) . Scale bar represent 100 nm . Male C57BL / 6 Nr4a2 cKO or wildtype littermate C57BL / 6 mice were immunised twice with NP - KLH emulsified in alum . Serum was assessed for level of IgG antibodies binding NP - 31 and NP - 4 ( F ) .

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Figure 3Male C57BL/6 Nr4a2 cKO or wildtype littermate C57BL/6 mice were immunised twice with NP-KLH emulsified in alum. After 2 weeks, 7 μm spleen sections were stained with Red:Alexa-Fluor-594-CD4 and Green: FITC-B220 (D) or FITC-GL7 (E). Scale bar represent 100 nm.Male C57BL/6 Nr4a2 cKO or wildtype littermate C57BL/6 mice were immunised twice with NP-KLH emulsified in alum. Serum was assessed for level of IgG antibodies binding NP-31 and NP-4 (F).


Figure 8 ( A ) PBMC were isolated from healthy volunteers and stained for flow cytometry to identify NRP1 expression in CD3 + CD4 + Th cells against CD127 ( left ) and intracellular Foxp3 ( right ) NRP1 - expressing cells amongst CD4 + CD3 + Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers ( healthy controls , HC ) and 10 SLE patients . Representative staining is shown for CD4 + CD3 + Th cells ( B ) , and summary data for CD3 + CD4 + Th cells ( C ) Plots show individual subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * p < 0 . 001 , * * * * p < 0 . 0001 Unpaired Student ' s T test . NRP1 - expressing cells amongst CD4 + CD3 + Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers ( healthy controls , HC ) and 10 SLE patients . summary data for CD3 + CD4 + CD127hi conventional cells ( D ) . Plots show individual subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * p < 0 . 001 , * * * * p < 0 . 0001 Unpaired Student ' s T test . ( E ) Individuals were divided into NRP1 - high subjects ( NRP1 + cells > 1 . 3 % in Th cells ) and NRP1 - low subjects ( NRP1 + cells ≤ 1 . 35 % in Th cells ) . Numbers of HC and SLE falling into each group are shown . * * * p < 0 . 001 Chi squared test with Yates ' correction . ( F , G ) NRP1 - expressing cells amongst CD3 + CD4 + CD45RO - cells were measured , representative staining ( F ) and summary data ( G ) . The plot shows individual subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * * p < 0 . 0001 Unpaired Student ' s T test .

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Figure 8(A) PBMC were isolated from healthy volunteers and stained for flow cytometry to identify NRP1 expression in CD3+CD4+ Th cells against CD127 (left) and intracellular Foxp3 (right)NRP1-expressing cells amongst CD4+CD3+ Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers (healthy controls, HC) and 10 SLE patients. Representative staining is shown for CD4+CD3+ Th cells (B), and summary data for CD3+CD4+ Th cells (C) Plots show individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ***p<0.001, ****p<0.0001 Unpaired Student's T test.NRP1-expressing cells amongst CD4+CD3+ Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers (healthy controls, HC) and 10 SLE patients. summary data for CD3+CD4+CD127hi conventional cells (D). Plots show individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ***p<0.001, ****p<0.0001 Unpaired Student's T test.(E) Individuals were divided into NRP1-high subjects (NRP1+ cells >1.3% in Th cells) and NRP1-low subjects (NRP1+ cells≤1.35% in Th cells). Numbers of HC and SLE falling into each group are shown. *** p<0.001 Chi squared test with Yates' correction.(F, G) NRP1-expressing cells amongst CD3+CD4+CD45RO- cells were measured, representative staining (F) and summary data (G). The plot shows individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ****p<0.0001 Unpaired Student's T test.


Figure 1B . Bright - field picture of a 0B K8 - eYFP embryo ( Left ) and time series of two - photon live imaging of ExE region ( Right ) . Time interval is 5 min ( n = 3 ) . C . Bright - field picture of EB K8 - eYFP embryo ( Left ) and time series of two - photon live imaging of ExE region ( Right ) . Time interval is 5 min ( n = 4 ) . D . Bright - field pictures of K8 - eYFP LB embryos ( n = 30 ) , and high magnification spectral acquisition of areas of ExE mesoderm corresponding to dashed white areas . K8 - eYFP and DAPI appear in yellow and cyan , respectively .

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Figure 1B. Bright-field picture of a 0B K8-eYFP embryo (Left) and time series of two-photon live imaging of ExE region (Right). Time interval is 5 min (n=3). C. Bright-field picture of EB K8-eYFP embryo (Left) and time series of two-photon live imaging of ExE region (Right). Time interval is 5 min (n=4). D. Bright-field pictures of K8-eYFP LB embryos (n=30), and high magnification spectral acquisition of areas of ExE mesoderm corresponding to dashed white areas. K8-eYFP and DAPI appear in yellow and cyan, respectively. Da


Figure 3A - H . SEM images of an opened embryo ( A ) , the ExE cavity wall ( B ) where blood islands are indicated by white arrow , the allantois surface ( C - C ' ) , an opened allantois ( D ) and its zooms ( E - E ' ) where filaments are indicated by white arrows , the epiblast - derived side of the amnion ( F ) , the mesoderm - derived side of the amnion ( G ) and its zoom ( G ' ) .

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Figure 3A-H. SEM images of an opened embryo (A), the ExE cavity wall (B) where blood islands are indicated by white arrow, the allantois surface (C-C'), an opened allantois (D) and its zooms (E-E') where filaments are indicated by white arrows, the epiblast-derived side of the amnion (F), the mesoderm-derived side of the amnion (G) and its zoom (G').


Figure 4B . Sagittal sections of C57BL / 6J wild - type and KtyII - / - E7 . 5 embryos immunostained for Krt8 ( n = 15 and n = 5 , respectively ) , F - actin ( n = 11 and 2 ) , Vimentin ( n = 5 and 1 ) , E - cadherin ( n = 1 and 1 ) , T ( n = 4 and 3 ) , Dsg2 ( n = 15 and 5 ) and Pecam - 1 ( n = 4 and 3 ) . Data information : White arrows point toward allantois and scale bars represent 25 µm . Quantification of embryo morphology at LB stage in C57BL / 6J wild - type ( white boxes / black dots , n = 22 including 16 in which allantois could be measured ) , and KtyII - / - embryos ( grey boxes / black triangles , n = 9 including 8 in which allantois could be measured ) . ( D ) Embryonic region length . Ratios of whole embryo and ExE cavity lengths ( E ) , over embryonic region length . I - I ' ' . SEM images of opened C57BL / 6J wild - type ( n = 3 ) and KtyII - / - ( n = 2 ) LB stage embryos with zooms on the allantois region ( I ' - I ' ' ) . Scale bars represent 200 ( I ) , 20 ( I ' ) and 10 ( I ' ' ) µm .

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Figure 4B. Sagittal sections of C57BL/6J wild-type and KtyII-/- E7.5 embryos immunostained for Krt8 (n= 15 and n=5, respectively), F-actin (n=11 and 2), Vimentin (n=5 and 1), E-cadherin (n=1 and 1), T (n=4 and 3), Dsg2 (n=15 and 5) and Pecam-1 (n=4 and 3). Data information: White arrows point toward allantois and scale bars represent 25 µm.Quantification of embryo morphology at LB stage in C57BL/6J wild-type (white boxes/ black dots, n=22 including 16 in which allantois could be measured), and KtyII-/- embryos (grey boxes/ black triangles, n=9 including 8 in which allantois could be measured). (D) Embryonic region length. Ratios of whole embryo and ExE cavity lengths (E) , over embryonic region length.I-I''. SEM images of opened C57BL/6J wild-type (n=3) and KtyII-/- (n=2) LB stage embryos with zooms on the allantois region (I'-I''). Scale bars represent 200 (I), 20 (I') and 10 (I'') µm.


Figure 5F . Dot plot of specific gene expression in amnion clusters . G . Immunostaining for Cdx2 ( n = 20 ) . H . Immunostaining for Nrp1 ( n = 8 ) and Col1a1 ( n = 7 ) , primarily expressed in Amnion Mesenchyme . I . Immunostaining for Epcam ( n = 12 ) , Cldn7 ( n = 7 ) , and Lrp2 ( n = 6 ) , primarily expressed in Amnion Ectoderm . J - K . Sections of C57BL / 6J wild - type and KtyII - / - E7 . 5 embryos immunostained for Nrp1 ( K ) ( n = 8 and 2 , respectively ) , and Lrp2 ( L ) ( n = 3 and 2 ) .

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Figure 5F. Dot plot of specific gene expression in amnion clusters.G. Immunostaining for Cdx2 (n=20). H. Immunostaining for Nrp1 (n=8) and Col1a1 (n=7), primarily expressed in Amnion Mesenchyme. I. Immunostaining for Epcam (n=12), Cldn7 (n=7), and Lrp2 (n=6), primarily expressed in Amnion Ectoderm.J-K. Sections of C57BL/6J wild-type and KtyII-/- E7.5 embryos immunostained for Nrp1 (K) (n=8 and 2, respectively), and Lrp2 (L) (n=3 and 2).


Figure 6D . Dot plot of specific gene expression in chorion clusters . E . Immunostaining for Cdx2 ( n = 20 ) . F . Immunostaining for DAPI ( cyan , left ) , Sox2 ( middle ) , and Gata2 ( right ) . Sox2 is primarily expressed in Trophoblast Progenitors and Gata2 in Chorion Ectoderm . G . Immunostaining for Cebpb , detectable in most cells with variable intensity . H . Immunostaining for Fn1 ( n = 22 ) , Dlk1 ( n = 8 ) , and Acta2 ( n = 12 ) , primarily expressed in Chorion Mesenchyme . I . Sections from C57BL / 6J wild - type and KtyII - / - LB embryos immunostained for Dlk - 1 ( n = 8 and 4 , respectively ) and Acta2 ( n = 6 and 3 ) .

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Figure 6D. Dot plot of specific gene expression in chorion clusters.E. Immunostaining for Cdx2 (n=20). F. Immunostaining for DAPI (cyan, left), Sox2 (middle), and Gata2 (right). Sox2 is primarily expressed in Trophoblast Progenitors and Gata2 in Chorion Ectoderm. G. Immunostaining for Cebpb, detectable in most cells with variable intensity. H. Immunostaining for Fn1 (n=22), Dlk1 (n=8), and Acta2 (n=12), primarily expressed in Chorion Mesenchyme. I. Sections from C57BL/6J wild-type and KtyII-/- LB embryos immunostained for Dlk-1 (n=8 and 4, respectively) and Acta2 (n=6 and 3). Data


Figure 7D . Dot plot of specific gene expression in allantois clusters . F . Immunostaining for Ifitm3 ( n = 7 ) ( Allantois Base ) . G . Immunostaining for Cdh11 ( n = 9 ) , Stard8 ( n = 4 ) and Nrp1 ( n = 8 ) ( Allantois Cortex ) . H . Immunostaining for Pitx1 ( n = 4 ) ( Allantois Mesenchyme ) . I . Hhex - GFP ( n = 5 ) and immunostaining for Kdr ( n = 8 ) ( Endothelial Progenitors ) . J . Immunostaining for Runx1 ( n = 3 ) ( Erythroid Progenitors ) . K - L . K - L . Sections from C57BL / 6J wild - type and KtyII - / - LB embryos immunostained for Cdh11 ( Top ) and Krt8 ( Bottom ) ( K , n = 8 and 4 , respectively ) and Ifitm3 ( L , n = 2 and 2 ) . Data information : White arrows point toward signal in allantois . Sagittal sections with anterior to the left .

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Figure 7D. Dot plot of specific gene expression in allantois clusters.F. Immunostaining for Ifitm3 (n=7) (Allantois Base). G. Immunostaining for Cdh11 (n=9), Stard8 (n=4) and Nrp1 (n=8) (Allantois Cortex). H. Immunostaining for Pitx1 (n=4) (Allantois Mesenchyme). I. Hhex-GFP (n=5) and immunostaining for Kdr (n=8) (Endothelial Progenitors). J. Immunostaining for Runx1 (n=3) (Erythroid Progenitors). K-L.K-L. Sections from C57BL/6J wild-type and KtyII-/- LB embryos immunostained for Cdh11 (Top) and Krt8 (Bottom) (K, n=8 and 4, respectively) and Ifitm3 (L, n=2 and 2). Data information: White arrows point toward signal in allantois. Sagittal sections with anterior to the left.