( C ) Representative TUNEL / CD31 / LYVE1 / DAPI immunostaining images of distal jejunums for the analysis of apoptotic BECs / LECs in intestinal paraffin sections ( 15 µm ) . High magnification images are from the dotted line boxes ( 1 ~ 4 ) . Arrow heads show the apoptotic BECs ( B in 1 , 2 ) and LECs ( L in 3 , 4 ) at villus ( 1 , 3 ) or submucosa ( 2 , 4 ) . White / yellow scale bars = 100 or 20 μm , respectively . ( D ) The numbers of apoptotic BECs and LECs per 0 . 1 mm2 blood vessel ( CD31 + LYVE1 - ) and lymphatic vessel ( CD31 + LYVE1 + ) area in intestinal mucosa were quantified respectively based on Figure 5C . Data are box - and - whisker plots , Mann Whitney U test , each symbol represents one mouse , N = 3 ~ 6 , * * P < 0 . 01 , n . s . = not significant .

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

(C) Representative TUNEL/CD31/LYVE1/DAPI immunostaining images of distal jejunums for the analysis of apoptotic BECs/LECs in intestinal paraffin sections (15 µm). High magnification images are from the dotted line boxes (1~4). Arrow heads show the apoptotic BECs (B in 1, 2) and LECs (L in 3, 4) at villus (1, 3) or submucosa (2, 4). White/yellow scale bars = 100 or 20 μm, respectively. (D) The numbers of apoptotic BECs and LECs per 0.1 mm2 blood vessel (CD31+LYVE1-) and lymphatic vessel (CD31+LYVE1+) area in intestinal mucosa were quantified respectively based on Figure 5C. Data are box-and-whisker plots, Mann Whitney U test, each symbol represents one mouse, N = 3~6, **P<0.01, n.s. = not significant.


( D ) Violin plots of the Rspo3 expression in trophocytes and LECs in intestine at I / R - 18 . 5h . Mann - Whitney U test , each symbol represents one cell ; N = 12 and 12 in control and EC - Foxc - DKO trophocytes , respectively ; N = 16 and 5 in control and EC - Foxc - DKO LECs .

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

(D) Violin plots of the Rspo3 expression in trophocytes and LECs in intestine at I/R-18.5h. Mann-Whitney U test, each symbol represents one cell; N = 12 and 12 in control and EC-Foxc-DKO trophocytes, respectively; N=16 and 5 in control and EC-Foxc-DKO LECs.


( E ) Validation study by qPCR for the detection of relative mRNA expression of Rspo3 in the sorted intestinal LECs at I / R - 18 . 5h . Data are box - and - whisker plots , Mann - Whitney U test , each symbol represents one mouse , N = 5 ~ 6 , * * P < 0 . 01 . The box - and - whisker plots display the median value ( central band in the box ) , second and third quartiles ( bottom and top ends of the box , respectively ) as well as minimum / maximum values ( whiskers blow / above the box ) of the data sets .

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

(E) Validation study by qPCR for the detection of relative mRNA expression of Rspo3 in the sorted intestinal LECs at I/R-18.5h. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 5~6, **P<0.01. The box-and-whisker plots display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.


( D ) Violin plots of the Cxcl12 expression in fibroblasts and telocytes / trophocytes . Mann - Whitney U test , each symbol represents one cell , N = 67 and 98 in control and EC - Foxc - DKO mice , respectively ; n . s . = not significant .

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

(D) Violin plots of the Cxcl12 expression in fibroblasts and telocytes/trophocytes. Mann-Whitney U test, each symbol represents one cell, N = 67 and 98 in control and EC-Foxc-DKO mice, respectively; n.s. = not significant.


( F ) Relative mRNA expression of Cxcl12 in Dynabeads - isolated ECs ( CD45 - CD31 + ) from distal jejunum . Data are box - and - whisker plots , Mann - Whitney U test , each symbol represents one mouse , N = 6 ~ 10 , * P < 0 . 05 . The box - and - whisker plots display the median value ( central band in the box ) , second and third quartiles ( bottom and top ends of the box , respectively ) as well as minimum / maximum values ( whiskers blow / above the box ) of the data sets .

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

(F) Relative mRNA expression of Cxcl12 in Dynabeads-isolated ECs (CD45-CD31+) from distal jejunum. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 6~10, *P<0.05. The box-and-whisker plots display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.


FOXC1 and FOXC2 co - occupy as well as the ECRs and promoters of CXCL12 in HUVECs ( D ) . Quantitative - ChIP assay was performed using rabbit ( R ) anti - FOXC1 and mouse ( M ) anti - FOXC2 antibodies to analyze the recruitment of FOXC on promoters and / or ECRs in HUVECs respectively . Values were quantified against IgG controls . Data are mean ± SEM , paired t - test , each symbol represents data collected from one experiment , N = 4 , * P < 0 . 05 , * * P < 0 . 01 , n . s . = not significant .

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

FOXC1 and FOXC2 co-occupy as well as the ECRs and promoters of CXCL12 in HUVECs (D). Quantitative-ChIP assay was performed using rabbit (R) anti-FOXC1 and mouse (M) anti-FOXC2 antibodies to analyze the recruitment of FOXC on promoters and/or ECRs in HUVECs respectively. Values were quantified against IgG controls. Data are mean ± SEM, paired t-test, each symbol represents data collected from one experiment, N = 4, * P<0.05, ** P<0.01, n.s.=not significant.


In RSPO3 rescue experiment , each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro - orbital injection 30 min before ischemia . PBS treated mice were used as vehicle control . ( C ) Representative images of crypts immunostained with OLFM4 and β - catenin in PBS / RSPO3 treated EC - Foxc - DKO mice 24h after I / R . The accumulation of β - catenin in the nuclei of ISCs ( dotted circles ) was found in RSPO3 rescued mice . Paraffin sections ( 4 µm ) , ( D ) Quantification of relative fluorescent intensity ( FI ) of β - catenin immunostaining within ISC and ( E ) quantification of the number of OLFM4 + ISCs were performed based on Figure 9C . Data are box - and - whisker plots , Mann - Whitney U test , each symbol represents one mouse , N = 4 ~ 6 , * P < 0 . 05 .

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

In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. (C) Representative images of crypts immunostained with OLFM4 and β-catenin in PBS/RSPO3 treated EC-Foxc-DKO mice 24h after I/R. The accumulation of β-catenin in the nuclei of ISCs (dotted circles) was found in RSPO3 rescued mice. Paraffin sections (4 µm), (D) Quantification of relative fluorescent intensity (FI) of β-catenin immunostaining within ISC and (E) quantification of the number of OLFM4+ ISCs were performed based on Figure 9C. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 4~6, *P<0.05.


In RSPO3 rescue experiment , each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro - orbital injection 30 min before ischemia . PBS treated mice were used as vehicle control . ( F ) Representative images of immunostaining of CCND1 in intestines in PBS / RSPO3 treated EC - Foxc - DKO mice at I / R - 24h . Scale bars = 100 µm . ( G ) Quantification of the number of CCND1 + epithelial cells per crypt at I / R - 24h are based on Figure 9F . Data are box - and - whisker plots , Mann - Whitney U test , each symbol represents one mouse , N = 7 , * * P < 0 . 01 .

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

In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. (F) Representative images of immunostaining of CCND1 in intestines in PBS/RSPO3 treated EC-Foxc-DKO mice at I/R-24h. Scale bars = 100 µm. (G) Quantification of the number of CCND1+ epithelial cells per crypt at I/R-24h are based on Figure 9F. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 7, **P<0.01.


In RSPO3 rescue experiment , each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro - orbital injection 30 min before ischemia . PBS treated mice were used as vehicle control . ( H ) Relative mRNA expression of Rspo3 in sorted LECs and Cxcl12 in sorted BECs from intestines of PBS / RSPO3 treated EC - Foxc - DKO mice at I / R - 18 . 5h . Data are box - and - whisker plots , Mann - Whitney U test , each symbol represents one mouse , N = 4 ~ 6 , * P < 0 . 05 .

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

In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. (H) Relative mRNA expression of Rspo3 in sorted LECs and Cxcl12 in sorted BECs from intestines of PBS/RSPO3 treated EC-Foxc-DKO mice at I/R-18.5h. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 4~6, *P<0.05.


In RSPO3 rescue experiment , each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro - orbital injection 30 min before ischemia . PBS treated mice were used as vehicle control . Representative images of H & E staining show the rescue effects of RSPO3 in intestinal mucosa LEC - Foxc - DKO ( I ) mice as well as their control mice 24h after I / R . Red numbers indicate the Chiu scores . Quantification of Chiu Score for the intestines at I / R - 24h are based H & E staining as shown in Figure 9A and 9I . Data are box - and - whisker plots , Mann - Whitney U test , each symbol represents one mouse , N = 6 ~ 13 , * * P < 0 . 01 .

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

In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. Representative images of H&E staining show the rescue effects of RSPO3 in intestinal mucosa LEC-Foxc-DKO (I) mice as well as their control mice 24h after I/R. Red numbers indicate the Chiu scores. Quantification of Chiu Score for the intestines at I/R-24h are based H&E staining as shown in Figure 9A and 9I. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 6~13, **P<0.01.


In RSPO3 rescue experiment , each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro - orbital injection 30 min before ischemia . PBS treated mice were used as vehicle control . ( K ) Representative images of CD31 / LYVE1 immunostaining in the intestines of PBS / RSPO3 treated LEC - Foxc - DKO mice at I / R - 24h . ( L , M ) Quantification of the vessel density ( L ) ( = vessel area / total intestinal tissue area x 100 % ) for the blood ( B ) and / or lymphatic ( L ) vessels ( markers listed below the graph were used to identify B and L ) as well as the measurement of lacteal length ( M ) was performed based on Figure 9K . The Data are box - and - whisker plots , Mann - Whitney U test , each symbol represents one mouse , N = 7 , * * P < 0 . 01 , n . s . = not significant .

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

In RSPO3 rescue experiment, each mouse was treated with 5 μg RSPO3 in 100μL PBS by retro-orbital injection 30 min before ischemia. PBS treated mice were used as vehicle control. (K) Representative images of CD31/LYVE1 immunostaining in the intestines of PBS/RSPO3 treated LEC-Foxc-DKO mice at I/R-24h. (L, M) Quantification of the vessel density (L) (= vessel area/total intestinal tissue area x 100%) for the blood (B) and/or lymphatic (L) vessels (markers listed below the graph were used to identify B and L) as well as the measurement of lacteal length (M) was performed based on Figure 9K. The Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 7, **P<0.01, n.s.=not significant.


In CXCL12 rescue experiments , mice were treated with 50μg / kg CXCL12 in PBS by retro - orbital injection 30 min before ischemia . Mice treated with PBS were used as control . ( H ) Representative confocal images of CD31 immunostaining of distal jejuna in PBS - and CXCL12 - treated EC - Foxc - DKO mice after I / R at 24h . Paraffin sections ( 15 µm ) , scale bars = 100 μm . L represents the lacteal length measured in Figure 10J . ( I , J ) ( I ) Quantification of CD31 + vessel density ( % = total CD31 + vessel area / total intestinal tissue area x 100 % ) and ( J ) quantification of lacteal length were performed based on Figure 10H . Data are box - and - whisker plots , Mann - Whitney U test , each symbol represents one mouse , N = 4 ~ 6 , * * P < 0 . 01 .

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

In CXCL12 rescue experiments, mice were treated with 50μg/kg CXCL12 in PBS by retro-orbital injection 30 min before ischemia. Mice treated with PBS were used as control. (H) Representative confocal images of CD31 immunostaining of distal jejuna in PBS- and CXCL12- treated EC-Foxc-DKO mice after I/R at 24h. Paraffin sections (15 µm), scale bars = 100 μm. L represents the lacteal length measured in Figure 10J. (I, J) (I) Quantification of CD31+ vessel density (% = total CD31+ vessel area/total intestinal tissue area x 100%) and (J) quantification of lacteal length were performed based on Figure 10H. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 4~6, **P<0.01.


In CXCL12 rescue experiments , mice were treated with 50μg / kg CXCL12 in PBS by retro - orbital injection 30 min before ischemia . Mice treated with PBS were used as control . ( K ) Relative mRNA expression of Rspo3 in sorted intestinal LECs from PBS / CXCL12 treated EC - Foxc - DKO mice at I / R - 18 . 5h . Data are box - and - whisker plots , Mann - Whitney U test , each symbol represents one mouse , N = 5 ~ 6 , n . s . = not significant . The box - and - whisker plots in display the median value ( central band in the box ) , second and third quartiles ( bottom and top ends of the box , respectively ) as well as minimum / maximum values ( whiskers blow / above the box ) of the data sets .

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

In CXCL12 rescue experiments, mice were treated with 50μg/kg CXCL12 in PBS by retro-orbital injection 30 min before ischemia. Mice treated with PBS were used as control. (K) Relative mRNA expression of Rspo3 in sorted intestinal LECs from PBS/CXCL12 treated EC-Foxc-DKO mice at I/R-18.5h. Data are box-and-whisker plots, Mann-Whitney U test, each symbol represents one mouse, N = 5~6, n.s.=not significant. The box-and-whisker plots in display the median value (central band in the box), second and third quartiles (bottom and top ends of the box, respectively) as well as minimum/maximum values (whiskers blow/above the box) of the data sets.


Confocal images of HeLa cells stained with NF45 / NF90 antibodies and antibodies against nucleolin and fibrillarin . Scale bar , 10 μm .

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

Confocal images of HeLa cells stained with NF45/NF90 antibodies and antibodies against nucleolin and fibrillarin. Scale bar, 10 μm.


HEK293 cells were infected with lentiviruses containing negative control shRNA ( shNC ) or NF45 / NF90 specific shRNA ( shNF45 # 1 , shNF45 # 2 , shNF90 # 1 and shNF90 # 2 ) for 24 h , and then transfected with rDNA - Luc . Luciferase activity was measured after a further 24 h ( n = 3 ) . Western blots show the protein levels of NF45 and NF90 . Empty vector ( EV ) , FLAG - NF45 plasmid ( NF45 ) or FLAG - NF90 plasmid ( NF90 ) were transfected into HEK293 cells for 24 h , and then rDNA - Luc was co - transfected into the cells . Luciferase activity was measured after a further 24 h ( n = 3 ) . Western blots show the protein levels of FLAG - NF45 and FLAG - NF90 .

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

HEK293 cells were infected with lentiviruses containing negative control shRNA (shNC) or NF45/NF90 specific shRNA (shNF45#1, shNF45#2, shNF90#1 and shNF90#2) for 24 h, and then transfected with rDNA-Luc. Luciferase activity was measured after a further 24 h (n = 3). Western blots show the protein levels of NF45 and NF90.   Empty vector (EV), FLAG-NF45 plasmid (NF45) or FLAG-NF90 plasmid (NF90) were transfected into HEK293 cells for 24 h, and then rDNA-Luc was co-transfected into the cells. Luciferase activity was measured after a further 24 h (n=3). Western blots show the protein levels of FLAG-NF45 and FLAG-NF90.


HEK293 cells were infected with lentiviruses containing negative control shRNA ( shNC ) or NF45 / NF90 specific shRNA ( shNF45 # 1 , shNF45 # 2 , shNF90 # 1 and shNF90 # 2 ) for 24 h , and then transfected with rDNA - Luc . Luciferase activity was measured after a further 24 h ( n = 3 ) . Western blots show the protein levels of NF45 and NF90 .

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

HEK293 cells were infected with lentiviruses containing negative control shRNA (shNC) or NF45/NF90 specific shRNA (shNF45#1, shNF45#2, shNF90#1 and shNF90#2) for 24 h, and then transfected with rDNA-Luc. Luciferase activity was measured after a further 24 h (n = 3). Western blots show the protein levels of NF45 and NF90.


E - F The qPCR analysis of 45S pre - rRNA from HEK293 cells treated with indicated shRNAs ( E ) and plasmids ( F ) for 24 h . Western blots show the expression levels of NF45 , NF90 , FLAG - NF45 and FLAG - NF90 . ( n = 3 )

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

E-F The qPCR analysis of 45S pre-rRNA from HEK293 cells treated with indicated shRNAs (E) and plasmids (F) for 24 h. Western blots show the expression levels of NF45, NF90, FLAG-NF45 and FLAG-NF90. (n = 3)


In Jurkat cells , a ChIP assay was performed with control IgG , NF45 and NF90 antibodies , and then the precipitated DNA was analyzed using qPCR with the aforementioned primers . The relative rDNA fold - enrichment was normalized to control IgG treatment . ( n = 3 )

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

In Jurkat cells, a ChIP assay was performed with control IgG, NF45 and NF90 antibodies, and then the precipitated DNA was analyzed using qPCR with the aforementioned primers. The relative rDNA fold-enrichment was normalized to control IgG treatment. (n=3)


EMSA assay . Lane 1 ) Biotin - labled rDNA probe . Lane 2 ) Biotin - labled rDNA probe + nuclear protein . Lane 3 ) Biotin - labled rDNA probe + nuclear protein + 100 - fold molar excess of biotin - unlabeled specific competitor . Lane 4 ) Biotin - labled rDNA probe + nuclear protein + 100 - fold molar excess of biotin - unlabeled mutant competitor . Lane 5 ) Biotin - labled rDNA probe + nuclear protein + NF90 antibody . Lane 6 ) Biotin - labled rDNA probe + nuclear protein + NF45 antibody . The details were described in Materials and Methods section .

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

EMSA assay. Lane 1) Biotin-labled rDNA probe. Lane 2) Biotin-labled rDNA probe + nuclear protein. Lane 3) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled specific competitor. Lane 4) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled mutant competitor. Lane 5) Biotin-labled rDNA probe + nuclear protein + NF90 antibody. Lane 6) Biotin-labled rDNA probe + nuclear protein + NF45 antibody. The details were described in Materials and Methods section.


The relative luciferase activity of HEK293T cells transfected with empty vector ( EV ) or FLAG - NF90 ( NF90 ) in combination with rDNA - Luc wild type ( WT ) or mutated rDNA - Luc reporter plasmid ( Mutant ) for 24 h . ( n = 3 ) Western blot shows FLAG - NF90 protein level .

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

The relative luciferase activity of HEK293T cells transfected with empty vector (EV) or FLAG-NF90 (NF90) in combination with rDNA -Luc wild type (WT) or mutated rDNA-Luc reporter plasmid (Mutant) for 24 h. (n=3) Western blot shows FLAG-NF90 protein level.


The relative luciferase activity of HEK293T cells transfected with empty vector ( EV ) or FLAG - NF90 ( NF90 ) in combination with

The relative luciferase activity of HEK293T cells transfected with empty vector (EV) or FLAG-NF90 (NF90) in combination with rDNA


Co - IP analysis of nucleolar proteins with NF90 in HEK293 cells transfected with EV , and the following plasmids : GFP - S5 , GFP - L9 , GFP - B23 , GFP - UBF , GFP - C23 . * represents the target protein .

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

Co-IP analysis of nucleolar proteins with NF90 in HEK293 cells transfected with EV, and the following plasmids: GFP-S5, GFP-L9, GFP-B23, GFP-UBF, GFP-C23. * represents the target protein.


Co - IP analysis of endogenous UBF with NF45 and NF90 in HEK293 cells .

Co-IP analysis of endogenous UBF with NF45 and NF90 in HEK293 cells.


HEK293 cells were infected by lentivirus containing NF45 - specfic shRNA or NF90 - specfic shRNA , and then stimulated in serum for 0 , 3 and 6 h . Cell lysates were prepared to analyze the expression levels of the indicated proteins by western blot .

HEK293 cells were infected by lentivirus containing NF45-specfic shRNA or NF90-specfic shRNA, and then stimulated in serum for 0, 3 and 6 h. Cell lysates were prepared to analyze the expression levels of the indicated proteins by western blot.


ChIP analysis was used to determinate the binding ability of NF45 and NF90 to rDNA promoters in HeLa cells stimulated with serum for 0 , 3 and 6 h . ( n = 3 )

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

ChIP analysis was used to determinate the binding ability of NF45 and NF90 to rDNA promoters in HeLa cells stimulated with serum for 0, 3 and 6 h. (n = 3)


ChIP analysis of NF45 and NF90 binding to the H42 . 9 loci in HeLa cells with the treatment of CX5461 or DMSO for 2 h . ( n = 3 )

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

ChIP analysis of NF45 and NF90 binding to the H42.9 loci in HeLa cells with the treatment of CX5461 or DMSO for 2 h. (n = 3)


Confocal images of HeLa cells treated with CX5461 for 2 h showing NF45 and NF90 colocalized with fibrillarin . Scale bar , 10 μm .

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Confocal images of HeLa cells treated with CX5461 for 2 h showing NF45 and NF90 colocalized with fibrillarin. Scale bar, 10 μm.


ChIP analysis of NF45 and NF90 binding to the H42 . 9 loci in CD3 + T cells stimulated with P / I for 6 h . ( n = 3 )

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ChIP analysis of NF45 and NF90 binding to the H42.9 loci in CD3+ T cells stimulated with P/I for 6 h. (n=3)


qPCR analysis of pre - rRNA in CD3 + T cells treated with P / I for 6 h . ( n = 3 )

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qPCR analysis of pre-rRNA in CD3+ T cells treated with P/I for 6 h. (n=3)


CD3 + T cells were first silenced with shNC , shNF45 or shNF90 , and then treated with P / I to extract total RNA for qPCR analysis of pre - rRNA . ( n = 3 )

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CD3+ T cells were first silenced with shNC, shNF45 or shNF90, and then treated with P/I to extract total RNA for qPCR analysis of pre-rRNA. (n=3)


The analysis of rDNA - luc activity in Jurkat cells with or without NF90 silence upon P / I stimulation or not . ( n = 3 )

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The analysis of rDNA-luc activity in Jurkat cells with or without NF90 silence upon P/I stimulation or not. (n=3)


Confocal images of Jurkat cells to show NF45 ( left panel ) or NF90 ( right panel ) ( green fluorescence ) colocalized with NFATc2 ( red fluorescence ) in nucleoli . Scale bar , 10 μm .

Confocal images of Jurkat cells to show NF45 (left panel) or NF90 (right panel) (green fluorescence) colocalized with NFATc2 (red fluorescence) in nucleoli. Scale bar, 10 μm.


Co - IP analysis of NFATc2 and NFATc1 with NF90 in Jurkat cells treated with P / I .

Co-IP analysis of NFATc2 and NFATc1 with NF90 in Jurkat cells treated with P/I.


ChIP analysis of NFATc2 binding to the rDNA loci in CD3 + T cells stimulated with P / I for 6 h . ( n = 3 )

ChIP analysis of NFATc2 binding to the rDNA loci in CD3+ T cells stimulated with P/I for 6 h. (n=3)


Jurkat cells co - transfected with rDNA - luc ( wild type or mutant plasmid ) with NF90 and / or NFATc2 for 24 h , then rDNA - luc activity was detected after P / I sitmulation or not ( DMSO ) . ( n = 3 )

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Jurkat cells co-transfected with rDNA-luc (wild type or mutant plasmid) with NF90 and/or NFATc2 for 24 h, then rDNA-luc activity was detected after P/I sitmulation or not (DMSO). (n=3)


ChIP analysis of NFATc2 binding to the H42 . 9 loci in NF90 - slienced CD3 + T cells stimulated with P / I for 6 h . ( n = 3 )

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ChIP analysis of NFATc2 binding to the H42.9 loci in NF90-slienced CD3+ T cells stimulated with P/I for 6 h. (n=3)


CD69 expression , an early marker of lymphocyte activation , in CD3 + T cells infected with shNC , shNF45 or shNF90 lentivirus upon stimulation with P / I for 12 h .

CD69 expression, an early marker of lymphocyte activation, in CD3+ T cells infected with shNC, shNF45 or shNF90 lentivirus upon stimulation with P/I for 12 h.


B - C PBMC cells with shNC , shNF45 or shNF90 lentivirus infection were stimulated with P / I for 12h , then the freshly replaced medium without P / I after another 24 h culture was harvested for IL - 2 and IFN - γ detections by ELISA . ( n = 3 )

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B-C PBMC cells with shNC, shNF45 or shNF90 lentivirus infection were stimulated with P/I for 12h, then the freshly replaced medium without P/I after another 24 h culture was harvested for IL-2 and IFN-γ detections by ELISA.(n=3)


The proliferation of shNC , shNF45 or shNF90 - infected PBMCs was monitored using a CCK - 8 assay . ( n = 3 )

The proliferation of shNC, shNF45 or shNF90-infected PBMCs was monitored using a CCK-8 assay. (n = 3)


Confocal images to show the subcellular localization of NFATc2 in CD3 + T cells treated with P / I and FK506 or CX5461 . Scale bar , 10 μm .

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Confocal images to show the subcellular localization of NFATc2 in CD3+ T cells treated with P/I and FK506 or CX5461. Scale bar, 10 μm.


Flow cytometry analysis of CD69 expression in P / I - stimulated CD3 + T cells treated with or without FK506 or CX5461 .

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Flow cytometry analysis of CD69 expression in P/I-stimulated CD3+ T cells treated with or without FK506 or CX5461.


G - H ELISA assays of IL - 2 and IFN - γ in CD3 + T cells with the indicated treatments . ( n = 3 )

G-H ELISA assays of IL-2 and IFN-γ in CD3+ T cells with the indicated treatments. (n = 3)


Flow cytometry analysis of CFSE - labeled CD3 + T cells in the presence of DMSO , FK506 and CX5461 for 0 , 3 and 7 d .

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Flow cytometry analysis of CFSE-labeled CD3+ T cells in the presence of DMSO, FK506 and CX5461 for 0, 3 and 7 d.


qPCR analysis of pre - rRNA expression from skin - and heart - transplanted mouse spleens . ( n = 5 )

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qPCR analysis of pre-rRNA expression from skin- and heart-transplanted mouse spleens. (n = 5)


pre - rRNA expression levels of PBMC from renal transplantation patients . ( Stable group , n = 7 . ABMR group , n = 8 . TCMR group , n = 8 ) .

pre-rRNA expression levels of PBMC from renal transplantation patients. (Stable group, n = 7. ABMR group, n = 8. TCMR group, n = 8).


Skin graft survival curve in mice treated with DMSO , 2 . 0 mg / kg FK506 or 0 . 5 - 2 . 0 mg / kg CX5461 . ( n = 5 )

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Skin graft survival curve in mice treated with DMSO, 2.0 mg/kg FK506 or 0.5 -2.0 mg/kg CX5461. (n = 5)


Representative images of HE staining of skin graft at seven days post transplantation . Scale bar , 50 μm .

Representative images of HE staining of skin graft at seven days post transplantation. Scale bar, 50 μm.


CD3 + T cells ( upper ) or CD4 + and CD8 + T cells ( lower ) in the spleens of skin - graft recipient mice were analyzed by FACS .

CD3+ T cells (upper) or CD4+ and CD8+ T cells (lower) in the spleens of skin-graft recipient mice were analyzed by FACS.


G - H ELISA analysis of IL - 2 and IFN - γ levels in skin - graft recipient mouse serum at 4th day post transplantation . ( n = 5 )

G-H ELISA analysis of IL-2 and IFN-γ levels in skin-graft recipient mouse serum at 4th day post transplantation. (n = 5)


The survival time ( days ) of cardiac graft in mice treated with DMSO , 2 . 0 mg / kg FK506 or 2 . 0 mg / kg CX5461 . ( n = 5 )

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The survival time (days) of cardiac graft in mice treated with DMSO, 2.0 mg/kg FK506 or 2.0 mg/kg CX5461. (n=5)


Representative images of HE and CD3 - IHC staining of cardiac graft at seven days post transplantation . NC , Scale bar , 100 μm .

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Representative images of HE and CD3-IHC staining of cardiac graft at seven days post transplantation. NC, Scale bar, 100 μm.


CD3 + T cells ( upper ) or CD4 + and CD8 + T cells ( lower ) in the spleens of heart - graft recipient mice were analyzed by FACS .

CD3+ T cells (upper) or CD4+ and CD8+ T cells (lower) in the spleens of heart-graft recipient mice were analyzed by FACS.


L - M ELISA analysis of IL - 2 and IFN - γ levels in heart - graft recipient mouse serum at 7th day post transplantation . ( n = 5 )

L-M ELISA analysis of IL-2 and IFN-γ levels in heart-graft recipient mouse serum at 7th day post transplantation. (n = 5)


qPCR analysis of pre - rRNA expression from transplanted mouse spleen . ( n = 5 )

qPCR analysis of pre-rRNA expression from transplanted mouse spleen. (n = 5)


FACS analysis of CD4 + , CD25 + and Foxp3 + Treg cells isolated from superficial cervical lymph nodes of heart - transplanted mice .

FACS analysis of CD4+, CD25+ and Foxp3+ Treg cells isolated from superficial cervical lymph nodes of heart-transplanted mice.


Each mitochondrial protein is colored according to ( A ) its fold change calculated by standard differential expression using the limma package ( Ritchie et al , 2015 ; Phipson et al , 2016 ) ; ( B ) its CNV value ( 5 proteins with the highest CNV value and 5 proteins with the lowest CNV values are highlighted and annotated in boxes ) ; and ( C ) the absolute difference between the two

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Each mitochondrial protein is colored according to (A) its fold change calculated by standard differential expression using the limma package (Ritchie et al, 2015; Phipson et al, 2016); (B) its CNV value (5 proteins with the highest CNV value and 5 proteins with the lowest CNV values are highlighted and annotated in boxes); and (C) the absolute difference between the two values


Analysis of the abundance distribution for ( A ) cytoplasmic , ( B ) mitochondrial , and ( C ) extracellular proteins across 5 age groups in C . elegans ( Walther et al , 2015 ) . Each age group was compared against a pool reference sample using SILAC ( Ong et al , 2002 ) . Distribution of protein fold changes for the different time points are shown as density plots and as as boxplots ( inset ) ; below the boxplots , asterisks mark the time points that show significantly different distribution of fold changes compared to the first time point ( Mann - Whitney test p < 0 . 01 )

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Analysis of the abundance distribution for (A) cytoplasmic, (B) mitochondrial, and (C) extracellular proteins across 5 age groups in C.elegans (Walther et al, 2015). Each age group was compared against a pool reference sample using SILAC (Ong et al, 2002). Distribution of protein fold changes for the different time points are shown as density plots and as as boxplots (inset); below the boxplots, asterisks mark the time points that show significantly different distribution of fold changes compared to the first time point (Mann-Whitney test p<0.01)


Abundance variation of complex members for ( D ) three mitochondrial complexes ( respiratory complex I , mitochondrial ribosome and mitochondrial import / export proteins ) . For each complex , the abundance variation of each member ( colored in grey ) over the 5 age groups is reported with the Δ SILAC ratio ( difference between the experimental SILAC ratio of a time point and the SILAC ratio at day 1 , left ) and the CNV value ( right ) . For each age group , the box plot ( colored as the corresponding time - point in the panels A - B - C ) represents the variance between the abundances of the complex members . Significant changes ( Mann - Whitney test p < 0 . 05 ) are marked with a red

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Abundance variation of complex members for (D) three mitochondrial complexes (respiratory complex I, mitochondrial ribosome and mitochondrial import/export proteins). For each complex, the abundance variation of each member (colored in grey) over the 5 age groups is reported with the Δ SILAC ratio (difference between the experimental SILAC ratio of a time point and the SILAC ratio at day 1, left) and the CNV value (right). For each age group, the box plot (colored as the corresponding time-point in the panels A-B-C) represents the variance between the abundances of the complex members. Significant changes (Mann-Whitney test p<0.05) are marked with a red star


( E ) Variation of the mitochondrial proteome over the lifespan of C . elegans ( day 1 and day 22 ) . Respiratory chain complex I and mitochondrial ribosomal proteins are highlighted in green and purple , respectively ( selected proteins , with extreme CNV values , are indicated )

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(E) Variation of the mitochondrial proteome over the lifespan of C.elegans (day 1 and day 22). Respiratory chain complex I and mitochondrial ribosomal proteins are highlighted in green and purple, respectively (selected proteins, with extreme CNV values, are indicated)


( F ) Variation of the extracellular proteome abundance over the lifespan of C . elegans ( day 1 and day 22 ) . Four proteins have been selected ( highlighted with a colored circle : purple for NAS - 34 , green for Y45F10C . 4 , blue for Vitellogenin - 1 , black for Y45F10C . 2 ) for their slower or accelerated increase of abundance compared to the rest of extracellular proteins . The variation of the selected proteins ( highlighted with the same colors ) throughout the worm lifespan is represented in the middle box , with the average of the extracellular proteome colored in yellow . Related to Figure EV5 and Dataset

(F) Variation of the extracellular proteome abundance over the lifespan of C.elegans (day 1 and day 22). Four proteins have been selected (highlighted with a colored circle: purple for NAS-34, green for Y45F10C.4, blue for Vitellogenin-1, black for Y45F10C.2) for their slower or accelerated increase of abundance compared to the rest of extracellular proteins. The variation of the selected proteins (highlighted with the same colors) throughout the worm lifespan is represented in the middle box, with the average of the extracellular proteome colored in yellow. Related to Figure EV5 and Dataset EV6


( A ) Death rate versus pre - starvation growth rate on a semilog - plot . CARLOS conditions are defined in legend . CARLOS conditions that perturb nutrient quality ( C , A , L and S ) lead to an exponential increase of death rate . Conditions that stress the proteome ( R and O ) show an orthogonal exponential increase with slower growth . Each data point is a single experiment . Chemostat ( C ) and parts of ( L ) are from Ref . ( Biselli et al . , 2020 ) . See Dataset EV1 for data .

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(A) Death rate versus pre-starvation growth rate on a semilog-plot. CARLOS conditions are defined in legend. CARLOS conditions that perturb nutrient quality (C, A, L and S) lead to an exponential increase of death rate. Conditions that stress the proteome (R and O) show an orthogonal exponential increase with slower growth. Each data point is a single experiment. Chemostat (C) and parts of (L) are from Ref. (Biselli et al., 2020). See Dataset EV1 for data.


( C ) Z - values are merged into a single ' Survival score ' for each protein that measures correlation of abundance with survival across all experiments . A detailed description of the construction of the score is given in Box 1 . Several GO - processes are significantly enriched ( FDR levels of the Benjamini - Hochberg step - up procedure ) towards higher scores . ∆ Survival score is calculated as the difference in median Survival score between a process and the background of all proteins .

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(C) Z-values are merged into a single 'Survival score' for each protein that measures correlation of abundance with survival across all experiments. A detailed description of the construction of the score is given in Box 1. Several GO-processes are significantly enriched (FDR levels of the Benjamini-Hochberg step-up procedure) towards higher scores. ∆Survival score is calculated as the difference in median Survival score between a process and the background of all proteins.


( B ) Knock - out of several genes with different functionalities in the cell envelope increase death rates . Growth in glycerol minimal medium , starvation induced by glycerol depletion .

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(B) Knock-out of several genes with different functionalities in the cell envelope increase death rates. Growth in glycerol minimal medium, starvation induced by glycerol depletion.


( C ) 8 µg / ml Polymyxin B and 4 µg / ml Colistin , added daily to cultures previously grown on glucose minimal medium ( reference condition ) with the first dose at 24h . Antibiotic concentration was 10x minimum inhibition concentration during growth ( MIC ) . Each data point is the average of two biological repeats . Error bars are not shown because the variation is smaller than symbol size . Viabilities indicated at < 101 mean no colonies were detected from 100 µl culture .

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(C) 8 µg/ml Polymyxin B and 4 µg/ml Colistin, added daily to cultures previously grown on glucose minimal medium (reference condition) with the first dose at 24h. Antibiotic concentration was 10x minimum inhibition concentration during growth (MIC). Each data point is the average of two biological repeats. Error bars are not shown because the variation is smaller than symbol size. Viabilities indicated at <101 mean no colonies were detected from 100 µl culture.


( D ) Death rates for different Mg2 + concentrations . Reference condition ( black circle in Fig . 1A ) has 0 . 41 mM Mg2 + . DNP + cultures were supplemented with 1 mM 2 , 4 - dinitrophenol during starvation in reference medium . Each data point is from a single experiment . All cultures were grown in glucose minimal medium . P - values ( Student ' s t - test , two - tailed ) are indicated in comparison to the reference condition .

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(D) Death rates for different Mg2+ concentrations. Reference condition (black circle in Fig. 1A) has 0.41 mM Mg2+. DNP+ cultures were supplemented with 1 mM 2,4-dinitrophenol during starvation in reference medium. Each data point is from a single experiment. All cultures were grown in glucose minimal medium. P-values (Student's t-test, two-tailed) are indicated in comparison to the reference condition.


Death rate plotted versus growth rate on a semi - log scale for experiments in ' osmo - balanced ' medium . Colors indicate carbon limitation ( C , ' blue ' , acetate and glycerol ) , anabolic limitation ( A , ' green ' , 3µM IPTG ) , ribosome limitation ( R , ' yellow ' , 4µM Chloramphenicol ) , rich medium ( L , ' red ' , LB ) , LacZ overexpression ( O , ' grey ' , 10ng / ml cTc ) , stationary phase ( S , ' white ' ) and the glucose reference condition ( ' black ' ) , as defined in Fig . 1A . The data shows weak anti - correlation with growth rate ; solid black line is an exponential fit to the data ( R2 = 0 . 34 ) . Dashed lines are the exponential fits to the CARLOS data in Fig . 1A .

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Death rate plotted versus growth rate on a semi-log scale for experiments in 'osmo-balanced' medium. Colors indicate carbon limitation (C, 'blue', acetate and glycerol), anabolic limitation (A, 'green', 3µM IPTG), ribosome limitation (R, 'yellow', 4µM Chloramphenicol), rich medium (L, 'red', LB), LacZ overexpression (O, 'grey', 10ng/ml cTc), stationary phase (S, 'white') and the glucose reference condition ('black'), as defined in Fig. 1A. The data shows weak anti-correlation with growth rate; solid black line is an exponential fit to the data (R2 = 0.34). Dashed lines are the exponential fits to the CARLOS data in Fig. 1A.


B atg19Δ cells containing HTB ‐ Atg19 were grown to mid ‐ log phase . Atg19 was affinity purified and subjected to mass spectrometric phosphorylation mapping . Phosphorylation sites : enlarged ; lysine substitutions : gray ; Atg11 binding region : green .

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B atg19Δ cells containing HTB‐Atg19 were grown to mid‐log phase. Atg19 was affinity purified and subjected to mass spectrometric phosphorylation mapping. Phosphorylation sites: enlarged; lysine substitutions: gray; Atg11 binding region: green.


C atg19Δ cells containing GFP ‐ Atg19 wild ‐ type or mutants as indicated were grown to mid ‐ log phase . Processing of endogenous Ape1 was analyzed by Western blotting and quantified by calculating the ratio of cleaved versus uncleaved Ape1 normalized to the wild ‐ type . Atg19 expression was assessed by anti ‐ GFP Western blotting .

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C atg19Δ cells containing GFP‐Atg19 wild‐type or mutants as indicated were grown to mid‐log phase. Processing of endogenous Ape1 was analyzed by Western blotting and quantified by calculating the ratio of cleaved versus uncleaved Ape1 normalized to the wild‐type. Atg19 expression was assessed by anti‐GFP Western blotting.


D atg19Δ GFP ‐ Atg11 Atg1 ‐ TAP cells containing myc ‐ Atg19 as indicated were grown to log phase . Atg11 was immunoprecipitated , and bound Atg19 was analyzed by Western blotting .

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D atg19Δ GFP‐Atg11 Atg1‐TAP cells containing myc‐Atg19 as indicated were grown to log phase. Atg11 was immunoprecipitated, and bound Atg19 was analyzed by Western blotting.


E GST ‐ fused propeptide of Ape1 , Atg19 as indicated , and a His ‐ tagged fragment of Atg11 ( amino acids 685 - 1178 ) were purified from Escherichia coli , mixed , and propeptide ‐ bound proteins were isolated and analyzed by Coomassie staining . Quantification of three individual experiments is shown . Error bars represent standard deviation .

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E GST‐fused propeptide of Ape1, Atg19 as indicated, and a His‐tagged fragment of Atg11 (amino acids 685-1178) were purified from Escherichia coli, mixed, and propeptide‐bound proteins were isolated and analyzed by Coomassie staining. Quantification of three individual experiments is shown. Error bars represent standard deviation.


A GFP ‐ Atg11 Ape1 ‐ mRuby atg19Δ cells containing myc ‐ Atg19 as indicated and Cup1 ‐ Ape1 were grown to log phase . Overexpression of Ape1 was induced by addition of 250 μM copper sulfate for 3 h , and autophagy was induced by treating cells for 1 h with rapamycin . Scale bar , 5 μm .

A GFP‐Atg11 Ape1‐mRuby atg19Δ cells containing myc‐Atg19 as indicated and Cup1‐Ape1 were grown to log phase. Overexpression of Ape1 was induced by addition of 250 μM copper sulfate for 3 h, and autophagy was induced by treating cells for 1 h with rapamycin. Scale bar, 5 μm.


B Ape1 ‐ mRuby atg19Δ cells containing GFP ‐ Atg19 wild ‐ type or GFP ‐ Atg19 ‐ 3A were analyzed in log phase . Scale bar , 5 μm .

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B Ape1‐mRuby atg19Δ cells containing GFP‐Atg19 wild‐type or GFP‐Atg19‐3A were analyzed in log phase. Scale bar, 5 μm.


C GFP ‐ Atg8 Ape1 ‐ mRuby atg19Δ cells containing myc ‐ Atg19 as indicated and Cup1 ‐ Ape1 were analyzed as in ( A ) . Scale bar , 5 μm .

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C GFP‐Atg8 Ape1‐mRuby atg19Δ cells containing myc‐Atg19 as indicated and Cup1‐Ape1 were analyzed as in (A). Scale bar, 5 μm.


B atg34Δ yeast cells containing HTB ‐ Atg34 were grown to mid ‐ log phase and treated with rapamycin . Atg34 was affinity purified and subjected to mass spectrometric phosphorylation mapping . Phosphorylation sites : enlarged ; Atg11 binding region : green ; lysine substitution : gray .

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B atg34Δ yeast cells containing HTB‐Atg34 were grown to mid‐log phase and treated with rapamycin. Atg34 was affinity purified and subjected to mass spectrometric phosphorylation mapping. Phosphorylation sites: enlarged; Atg11 binding region: green; lysine substitution: gray.


C Ams1 ‐ GFP atg19Δatg34Δ cells containing protein A ( Stag ) ‐ tagged Atg34 as indicated were starved for 4 h . Processing of endogenous Ams1 ‐ GFP was analyzed by Western blotting and quantified by calculating the ratio of free GFP versus uncleaved Ams1 ‐ GFP normalized to the wild ‐ type . Expression of Atg34 proteins was assessed by anti ‐ protein A Western blotting .

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C Ams1‐GFP atg19Δatg34Δ cells containing protein A (Stag)‐tagged Atg34 as indicated were starved for 4 h. Processing of endogenous Ams1‐GFP was analyzed by Western blotting and quantified by calculating the ratio of free GFP versus uncleaved Ams1‐GFP normalized to the wild‐type. Expression of Atg34 proteins was assessed by anti‐protein A Western blotting.


E GST , GST ‐ Atg19 , and GST ‐ Atg34 were purified from Escherichia coli and in vitro phosphorylated with immunoprecipitated Atg1 ‐ TAP bound to IgG magnetic beads .

E GST, GST‐Atg19, and GST‐Atg34 were purified from Escherichia coli and in vitro phosphorylated with immunoprecipitated Atg1‐TAP bound to IgG magnetic beads.


F In vitro phosphorylation of recombinant Atg19 , Atg19 ‐ 3A , and Atg19 ‐ 3D using wild ‐ type and kinase ‐ dead Atg1 ‐ D211A as described in ( E ) .

F In vitro phosphorylation of recombinant Atg19, Atg19‐3A, and Atg19‐3D using wild‐type and kinase‐dead Atg1‐D211A as described in (E).


A Wild ‐ type , hrr25 ‐ ts , and hrr25 ‐ ts cells containing HRR25 on a centromeric plasmid were grown to mid ‐ log phase at 18 ° C followed by 2 and 4 h of log ‐ phase growth at 37 ° C . Ape1 processing was analyzed by Western blotting .

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A Wild‐type, hrr25‐ts, and hrr25‐ts cells containing HRR25 on a centromeric plasmid were grown to mid‐log phase at 18°C followed by 2 and 4 h of log‐phase growth at 37°C. Ape1 processing was analyzed by Western blotting.


B atg19Δ and atg19Δ hrr25 ‐ ts cells containing GFP ‐ Atg19 as indicated were analyzed for Ape1 processing in log phase after 2 h at 37 ° C .

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B atg19Δ and atg19Δ hrr25‐ts cells containing GFP‐Atg19 as indicated were analyzed for Ape1 processing in log phase after 2 h at 37°C.


C GFP ‐ Atg11 , GFP ‐ Atg11 hrr25 ‐ ts , and GFP ‐ Atg11 atg1 ‐ D211A cells were analyzed for GFP ‐ Atg11 dot formation . Quantification of at least 70 cells in three individual experiments is shown . Error bars represent standard deviation . Scale bar , 3 μm .

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C GFP‐Atg11, GFP‐Atg11 hrr25‐ts, and GFP‐Atg11 atg1‐D211A cells were analyzed for GFP‐Atg11 dot formation. Quantification of at least 70 cells in three individual experiments is shown. Error bars represent standard deviation. Scale bar, 3 μm.


D GFP ‐ Atg11 , GFP ‐ Atg11 hrr25 ‐ ts , and GFP ‐ Atg11 atg19Δ cells containing BFP ‐ Ape1 and Cup1 ‐ Ape1 were grown to log phase . Overexpression of Ape1 was induced by addition of 250 μM CuSO4 for 3 h . coloc . : Cells with GFP ‐ Atg11 / BFP ‐ Ape1 co ‐ localization . n = 50 ( wt ) , 84 ( hrr25 ‐ ts ) , 127 ( atg19Δ ) . Scale bar , 3 μm .

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D GFP‐Atg11, GFP‐Atg11 hrr25‐ts, and GFP‐Atg11 atg19Δ cells containing BFP‐Ape1 and Cup1‐Ape1 were grown to log phase. Overexpression of Ape1 was induced by addition of 250 μM CuSO4 for 3 h. coloc.: Cells with GFP‐Atg11/BFP‐Ape1 co‐localization. n = 50 (wt), 84 (hrr25‐ts), 127 (atg19Δ). Scale bar, 3 μm.


E Hrr25 was immunoprecipitated from Hrr25 ‐ TAP cells and as a control from a non ‐ tagged wild ‐ type strain . In vitro phosphorylation of recombinant Atg19 , Atg19 ‐ 3A , Atg34 , and GST using the kinase ‐ bound beads was performed as described in Fig E . Both Atg19 panels are from the same gel with the same exposure .

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E Hrr25 was immunoprecipitated from Hrr25‐TAP cells and as a control from a non‐tagged wild‐type strain. In vitro phosphorylation of recombinant Atg19, Atg19‐3A, Atg34, and GST using the kinase‐bound beads was performed as described in Fig E. Both Atg19 panels are from the same gel with the same exposure.


A ) Treadmill analysis ( n = 8 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by unpaired , two - tailed Student ' s t test * * * p = 0 . 005 ; * * * p = 0 . 0007 ( T3 ) and * * * * p < 0 . 0001 ( T4 ) . Only p values for rapamycin - treated vs . untreated Cox15sm / sm mice are shown

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A) Treadmill analysis (n=8/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by unpaired, two-tailed Student's t test ***p=0.005; ***p=0.0007 (T3) and ****p<0.0001 (T4). Only p values for rapamycin-treated vs. untreated Cox15sm/sm mice are shown


B ) Histological and histochemical characterization of skeletal muscle in rapamycin - treated and untreated Cox15sm / sm and WT mice . H & E : hematoxylin and eosin ; PAS : periodic acid Schiff reaction ; COX : cytochrome c oxidase ; SDH : succinate dehydrogenase . White bars correspond to 50µm

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B) Histological and histochemical characterization of skeletal muscle in rapamycin-treated and untreated Cox15sm/sm and WT mice. H&E: hematoxylin and eosin; PAS: periodic acid Schiff reaction; COX: cytochrome c oxidase; SDH: succinate dehydrogenase. White bars correspond to 50µm


C ) Analysis of the cross - sectional area of muscle fibers in the different genotypes ( n = 3 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test : * * * p = 0 . 001 ; * * * p = 0 . 006 , * * * * p < 0 . 0001

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C) Analysis of the cross-sectional area of muscle fibers in the different genotypes (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test: ***p=0.001; ***p=0.006, ****p<0.0001


D ) Analysis of the number of centralized nuclei in muscle fibers ( n = 3 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * p = 0 . 0018 , * * p = 0 . 0052

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D) Analysis of the number of centralized nuclei in muscle fibers (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0018, **p=0.0052


E ) Analysis of PAS - reaction intensity in muscle fibers ( n = 4 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * p = 0 . 0293 ( WT vs . Cox15sm / sm ) and * p = 0 . 0150 ( Cox15sm / sm vs . Cox15sm / sm Rapa )

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E) Analysis of PAS-reaction intensity in muscle fibers (n=4/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0293 (WT vs. Cox15sm/sm) and *p=0.0150 (Cox15sm/sm vs. Cox15sm/sm Rapa)


F ) Analysis of COX - reaction intensity in muscle fibers ( n = 3 ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * * * p < 0 . 0001 ; * * p = 0 . 0031

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F) Analysis of COX-reaction intensity in muscle fibers (n=3). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ****p<0.0001; **p=0.0031


G ) Analysis of SDH - reaction intensity in muscle fibers ( n = 3 ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * p = 0 . 0199 , * * p = 0 . 0091

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G) Analysis of SDH-reaction intensity in muscle fibers (n=3). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0199, **p=0.0091


A ) Spectrophotometric activities of the respiratory chain ( n = 4 - 5 / group ) . CS : citrate synthase ; CIV : complex IV . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by two way ANOVA with Tukey ' s correction * * * * p < 0 . 0001 ( CS : WT vs . Cox15sm / sm ) , * * * p = 0 . 0010 ( CS : Cox15sm / sm vs . Cox15sm / sm rapamycin ) , * * * * p < 0 . 0001 ( CIV : WT vs . Cox15sm / sm ) , * * p = 0 . 0060 ( CIV / CS : WT vs . Cox15sm / sm )

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A) Spectrophotometric activities of the respiratory chain (n=4-5/group). CS: citrate synthase; CIV: complex IV. Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by two way ANOVA with Tukey's correction ****p<0.0001 (CS: WT vs. Cox15sm/sm), ***p=0.0010 (CS: Cox15sm/sm vs. Cox15sm/sm rapamycin), ****p<0.0001 (CIV: WT vs. Cox15sm/sm), **p=0.0060 (CIV/CS: WT vs. Cox15sm/sm)


B ) Western blot immunovisualization of subunits of the respiratory complexes . Note the increased protein levels in Cox15sm / sm vs . WT samples , which are reduced to normal levels upon rapamycin treatment . Additional samples were run on a separate gel ( not shown )

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B) Western blot immunovisualization of subunits of the respiratory complexes. Note the increased protein levels in Cox15sm/sm vs. WT samples, which are reduced to normal levels upon rapamycin treatment. Additional samples were run on a separate gel (not shown)


C ) BNGE in gel activity for cIV . Sc : supercomplexes . Note that the COX reaction is slightly increased in rapamycin - treated vs . untreated Cox15sm / sm samples

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C) BNGE in gel activity for cIV. Sc: supercomplexes. Note that the COX reaction is slightly increased in rapamycin-treated vs. untreated Cox15sm/sm samples


D ) Immunoblot of 1D - BNGE using an anti - cIV antibody ( COX1 ) . Note that COX amount is slightly increased in rapamycin treated vs . untreated Cox15sm / sm muscles . SDHB was used as a loading control

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D) Immunoblot of 1D-BNGE using an anti-cIV antibody (COX1). Note that COX amount is slightly increased in rapamycin treated vs. untreated Cox15sm/sm muscles. SDHB was used as a loading control


E ) State III ( succinate - driven ) oxygen consumption ( n = 3 / group ) . Error bars represent SEM . The asterisks represent the significance levels calculated by unpaired , two tail Student ' s t test * * p = 0 . 0016 ( WT vs . Cox15sm / sm ) ; * p = 0 . 047 ( Cox15sm / sm vs . Cox15sm / sm rapamycin )

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E) State III (succinate-driven) oxygen consumption (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by unpaired, two tail Student's t test **p=0.0016 (WT vs. Cox15sm/sm); *p=0.047 (Cox15sm/sm vs. Cox15sm/sm rapamycin)


F ) Analysis of mtDNA amount ( n = 9 / group ) . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey ' s correction : * * p = 0 . 0020 ; * p = 0 . 0296

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F) Analysis of mtDNA amount (n=9/group). Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey's correction: **p=0.0020; *p=0.0296


Note the accumulation of profoundly altered mitochondria in Cox15sm / sm muscles ( white arrows ) , possibly as a result of partially digested organelles within endolysosomes . Black arrows indicate examples of normal mitochondria . In rapamycin - treated Cox15sm / sm animals only a few of these structures are present , which can be detected also in rapamycin - treated WT samples . The bar corresponds to 1136 nm ( 4400X )

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Note the accumulation of profoundly altered mitochondria in Cox15sm/sm muscles (white arrows), possibly as a result of partially digested organelles within endolysosomes. Black arrows indicate examples of normal mitochondria. In rapamycin-treated Cox15sm/sm animals only a few of these structures are present, which can be detected also in rapamycin-treated WT samples. The bar corresponds to 1136 nm (4400X)


A ) Representative western blot of autophagy markers at steady state in skeletal muscle . GAPDH was used as a loading control . B ) Densitometric analysis of immunoblots similar to that shown in A ( n = 4 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * p = 0 . 0068 ( LC3 - II WT vs . Cox15sm / sm ) , * * p = 0 . 0063 ( P62 WT vs . Cox15sm / sm ) * p = 0 . 025 ( P62 Cox15sm / sm vs . Cox15sm / sm + rapamycin ) , * * p = 0 . 0098 ( S6 WT vs . Cox15sm / sm ) , * p = 0 . 034 ( P - S6 WT vs WT + rapamycin ) , * * * p = 0 . 0002 ( Cox15sm / sm vs . Cox15sm / sm + rapamycin ) , * p = 0 . 014 ( P - S6 WT vs . WT + rapamycin ) * * * p = 0 . 0007 ( Cox15sm / sm vs . Cox15sm / sm + rapamycin )

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A) Representative western blot of autophagy markers at steady state in skeletal muscle. GAPDH was used as a loading control. B) Densitometric analysis of immunoblots similar to that shown in A (n=4/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0068 (LC3-II WT vs. Cox15sm/sm), **p=0.0063 (P62 WT vs. Cox15sm/sm) *p=0.025 (P62 Cox15sm/sm vs. Cox15sm/sm+rapamycin), **p=0.0098 (S6 WT vs. Cox15sm/sm), *p=0.034 (P-S6 WT vs WT+rapamycin), ***p=0.0002 (Cox15sm/sm vs. Cox15sm/sm+rapamycin), *p=0.014 (P-S6 WT vs. WT+rapamycin) ***p=0.0007 (Cox15sm/sm vs. Cox15sm/sm+rapamycin)


C ) Analysis of the autophagic flux ( n = 3 / group ) . Upper panel : representative western blot of Cox15sm / sm and WT muscles treated with colchicine , rapamycin , or rapamycin plus colchicine . Note that LC3 - II was increased in Cox15sm / sm vs . WT samples . Colchicine did not increase LC3 - II levels suggesting a block in the autophagic flux ; rapamycin plus colchicine treated Cox15sm / sm mice showed higher levels of LC3 - II , suggesting that rapamycin increased the autophagic flux in Cox15sm / sm muscles . Black : WT ; dark grey : colchicine - treated WT ; middle grey : rapamycin - treated WT ; light grey : colchicine plus rapamycin treated mice ; red : untreated Cox15sm / sm ; orange : colchicine - treated ; blue : rapamycin - treated Cox15sm / sm ; purple : colchicine plus rapamycin treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * p = 0 . 0049 ( WT vs . WT + colchicine ) , * * p = 0 . 0046 ( WT + rapamycin vs . WT + rapamycin + colchicine ) , * p = 0 . 0225 ( WT vs . Cox15sm / sm ) , * * p = 0 . 0057 ( Cox15sm / sm colchicine vs . Cox15sm / sm + rapamycin + colchicine ) , * * * p = 0 . 0003 ( Cox15sm / sm rapamycin vs . Cox15sm / sm + rapamycin + colchicine )

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C) Analysis of the autophagic flux (n=3/group). Upper panel: representative western blot of Cox15sm/sm and WT muscles treated with colchicine, rapamycin, or rapamycin plus colchicine. Note that LC3-II was increased in Cox15sm/sm vs. WT samples. Colchicine did not increase LC3-II levels suggesting a block in the autophagic flux; rapamycin plus colchicine treated Cox15sm/sm mice showed higher levels of LC3-II, suggesting that rapamycin increased the autophagic flux in Cox15sm/sm muscles. Black: WT; dark grey: colchicine-treated WT; middle grey: rapamycin-treated WT; light grey: colchicine plus rapamycin treated mice; red: untreated Cox15sm/sm; orange: colchicine-treated; blue: rapamycin-treated Cox15sm/sm; purple: colchicine plus rapamycin treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0049 (WT vs. WT+colchicine), **p=0.0046 (WT+rapamycin vs. WT+rapamycin+colchicine), *p=0.0225 (WT vs. Cox15sm/sm), **p=0.0057 (Cox15sm/sm colchicine vs. Cox15sm/sm+rapamycin+colchicine), ***p=0.0003 (Cox15sm/sm rapamycin vs. Cox15sm/sm+rapamycin+colchicine)


A ) Analysis of the autophagic flux ( n = 4 / group ) . Upper panel : representative western blot of Cox15sm / sm and WT muscles treated with colchicine , rilmenidine , or rilmenidine plus colchicine . Colchicine alone increased LC3 - II in WT but not Cox15sm / sm mice ; rilmenidine had no effect on WT LC3 - II levels , but reduced them in Cox15sm / sm mice . Rilmenidine plus colchicine increased LC3 - II levels in both WT and Cox15sm / sm mice . Black : WT ; dark grey : colchicine - treated WT ; middle grey : rapamycin - treated WT ; light grey : colchicine plus rapamycin treated mice ; red : untreated Cox15sm / sm ; orange : colchicine - treated ; blue : rapamycin - treated Cox15sm / sm ; purple : colchicine plus rapamycin treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test : * * p = 0 . 0093 ( WT vs . WT + colchicine ) , * * * * p < 0 . 0001 ( WT + rilmenidine vs . WT + rilmenidine + colchicine ) , * * p = 0 . 0001 ( WT vs . Cox15sm / sm ) , * * p = 0 . 0083 ( Cox15sm / sm vs . Cox15sm / sm + rilmenidine ) , * p = 0 . 0112 ( Cox15sm / sm vs . Cox15sm / sm + rilmenidine + colchicine ) , * * * * p < 0 .

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A) Analysis of the autophagic flux (n=4/group). Upper panel: representative western blot of Cox15sm/sm and WT muscles treated with colchicine, rilmenidine, or rilmenidine plus colchicine. Colchicine alone increased LC3-II in WT but not Cox15sm/sm mice; rilmenidine had no effect on WT LC3-II levels, but reduced them in Cox15sm/sm mice. Rilmenidine plus colchicine increased LC3-II levels in both WT and Cox15sm/sm mice. Black: WT; dark grey: colchicine-treated WT; middle grey: rapamycin-treated WT; light grey: colchicine plus rapamycin treated mice; red: untreated Cox15sm/sm; orange: colchicine-treated; blue: rapamycin-treated Cox15sm/sm; purple: colchicine plus rapamycin treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test: **p=0.0093 (WT vs. WT+colchicine), ****p<0.0001 (WT+rilmenidine vs. WT+rilmenidine+colchicine), **p=0.0001 (WT vs. Cox15sm/sm), **p=0.0083 (Cox15sm/sm vs. Cox15sm/sm+rilmenidine), *p=0.0112 (Cox15sm/sm vs. Cox15sm/sm+rilmenidine+colchicine), ****p<0.000


B ) Treadmill analysis ( n = 4 / group ) . Black : untreated WT ; grey : rilmenidine - treated WT ; red : untreated Cox15sm / sm ; blue : rilmenidine - treated Cox15sm / sm . Error bars represent SEM

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B) Treadmill analysis (n=4/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM


C ) Histological and histochemical characterization of skeletal muscle in rapamycin - treated and untreated Cox15sm / sm and WT mice . H & E : hematoxylin and eosin ; GT : Gomori trichrome ; PAS : periodic acid Schiff reaction

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C) Histological and histochemical characterization of skeletal muscle in rapamycin-treated and untreated Cox15sm/sm and WT mice. H&E: hematoxylin and eosin; GT: Gomori trichrome; PAS: periodic acid Schiff reaction


D ) Analysis of the cross - sectional area of muscle fibers in the different genotypes ( n = 3 / group ) . Black : untreated WT ; grey : rilmenidine - treated WT ; red : untreated Cox15sm / sm ; blue : rilmenidine - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * * p = 0 . 0002 , * * * * p < 0 . 0001

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D) Analysis of the cross-sectional area of muscle fibers in the different genotypes (n=3/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ***p=0.0002, ****p<0.0001


E ) Analysis of the number of centralized nuclei in muscle fibres ( n = 3 / group ) . Black : untreated WT ; grey : rilmenidine - treated WT ; red : untreated Cox15sm / sm ; blue : rilmenidine - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * p = 0 . 0091

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E) Analysis of the number of centralized nuclei in muscle fibres (n=3/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0091


A ) anti - TFEB immunofluorescence on rapamycin - and rilmenidine - treated WT and Cox15sm / sm muscles vs . untreated ( UT ) samples . CD11C ( blue signal ) indicates inflammatory cells . Right panel : quantification of n = 3 animals / group . The scale bars correspond to 30µm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test B ) Quantification of the TFEB - positive nuclei ( n = 3 / group ) . Quantification was performed using Imaris spots surface excluding the CD11C positive nuclei . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * p = 0 . 0199 ( WT vs . WT + rapamycin ) , * p = 0 . 0150 ( Cox15sm / sm vs . Cox15sm / sm + rapamycin ) , * * * p = 0 . 0001

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A) anti-TFEB immunofluorescence on rapamycin- and rilmenidine-treated WT and Cox15sm/sm muscles vs. untreated (UT) samples. CD11C (blue signal) indicates inflammatory cells. Right panel: quantification of n=3 animals/group. The scale bars correspond to 30µm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test B) Quantification of the TFEB-positive nuclei (n=3/group). Quantification was performed using Imaris spots surface excluding the CD11C positive nuclei. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0199 (WT vs. WT+rapamycin), *p=0.0150 (Cox15sm/sm vs. Cox15sm/sm+rapamycin), ***p=0.0001


A ) Real - time PCR analysis of Lamp1 transcript ( n = 3 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by Student ' s t test * * p = 0 . 008 , * p = 0 . 038

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A) Real-time PCR analysis of Lamp1 transcript (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by Student's t test **p=0.008, *p=0.038


B ) Anti - LAMP1 staining in rapamycin - and rilmenidine - treated WT and Cox15sm / sm muscles vs . untreated ( UT ) samples . Rapamycin , but not rilmendine , increases the number of LAMP1 - positive vesicles in Cox15sm / sm samples ( inset ) . The scale bars correspond to 30µm

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B) Anti-LAMP1 staining in rapamycin- and rilmenidine-treated WT and Cox15sm/sm muscles vs. untreated (UT) samples. Rapamycin, but not rilmendine, increases the number of LAMP1-positive vesicles in Cox15sm/sm samples (inset). The scale bars correspond to 30µm


C ) Quantification of LAMP1 - positive vesicles ( n = 3 / group ) . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * * * p < 0 . 0001

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C) Quantification of LAMP1-positive vesicles (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ****p<0.0001


D ) Cathepsin L specific activity ( n = 3 / group ) . Error bars represent SEM . The asterisks represent the significance levels calculated by Student ' s t test * p = 0 . 0133

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D) Cathepsin L specific activity (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by Student's t test *p=0.0133


C , Immunostaining of endogenous Trnp1 and DAPI in P19 cells and E14 cortical cells at 1 day in vitro ( div ) .

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C, Immunostaining of endogenous Trnp1 and DAPI in P19 cells and E14 cortical cells at 1 day in vitro (div).


D , Co - IP with anti - GFP and WB with anti - Trnp1 antibodies in lysates from P19 cells transfected with plasmids expressing Trnp1 - GFP fusion protein and / or Trnp1 - IRES - RFP for 24h . Mw ( molecular weights ) : 55 kDa ( black line ) ; 35 kDa ( blue line ) ; 25 kDa ( red line ) . Input : 0 , 1 % ; co - IP : all immunoprecipitated proteins .

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D, Co-IP with anti-GFP and WB with anti-Trnp1 antibodies in lysates from P19 cells transfected with plasmids expressing Trnp1-GFP fusion protein and/or Trnp1-IRES-RFP for 24h. Mw (molecular weights): 55 kDa (black line); 35 kDa (blue line); 25 kDa (red line). Input: 0,1%; co-IP: all immunoprecipitated proteins.


E , Representative phase contrast images of in vitro phase separation of the recombinant proteins YFP ( upper row ) or Trnp1 ( lower row ) at the indicated concentration in 50 mM Sorenson ' s buffer ( pH 7 , 6 ) containing 150 mM salt and 2mM DTT with the crowding agent dextran or RNA when depicted .

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E, Representative phase contrast images of in vitro phase separation of the recombinant proteins YFP (upper row) or Trnp1 (lower row) at the indicated concentration in 50 mM Sorenson's buffer (pH 7,6) containing 150 mM salt and 2mM DTT with the crowding agent dextran or RNA when depicted.


B , GFP and Trnp1 immunostaining with DAPI labelling in E14 cortical cells transfected for 24 h with plasmids ( indicated by arrows ) expressing the different truncated / mutant forms of Trnp1 and GFP showing Trnp1 localization .

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B, GFP and Trnp1 immunostaining with DAPI labelling in E14 cortical cells transfected for 24 h with plasmids (indicated by arrows) expressing the different truncated/mutant forms of Trnp1 and GFP showing Trnp1 localization.


C , WB of cytoplasmic and nuclear enriched fractions of P19 cells transfected with the different truncated / mutant forms of Trnp1 for 24h . Hist1 and actin serve as loading controls for nuclear and cytoplasmic fractions , respectively . The values under the anti - Trnp1 blots indicate the percentage of the signal relative to the control set to 100 % after normalization by α - actin ( Image J quantification ) . The values under the anti - actin and anti - Hist1 blots indicate the percentage of the signal considering that nuclear and cytoplasmic fractions together is 100 % .

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C, WB of cytoplasmic and nuclear enriched fractions of P19 cells transfected with the different truncated/mutant forms of Trnp1 for 24h. Hist1 and actin serve as loading controls for nuclear and cytoplasmic fractions, respectively. The values under the anti-Trnp1 blots indicate the percentage of the signal relative to the control set to 100% after normalization by α-actin (Image J quantification). The values under the anti-actin and anti-Hist1 blots indicate the percentage of the signal considering that nuclear and cytoplasmic fractions together is 100%.


D , WB showing mutCC Trnp1 and ∆ 1 - 16 Trnp1 protein turnover in P19 cells 24 h after transfection in the presence of cycloheximide ( CHX ) for the time indicated below in hours ( h ) . GFP ( half live : 24h ) was used as a loading control .

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D, WB showing mutCC Trnp1 and ∆1-16 Trnp1 protein turnover in P19 cells 24 h after transfection in the presence of cycloheximide (CHX) for the time indicated below in hours (h). GFP (half live:24h) was used as a loading control.


E , Co - IP with anti - FLAG magnetic beads and WB with anti - Trnp1 antibodies showing that FLAGTrnp1 interacts with the GFP - fusion and the ∆ 1 - 16Trnp1 proteins but not with other truncated Trnp1 proteins indicating IDRs are crucial for Trnp1 homo - oligomerization . Samples were collected 24h after transfection of P19 cells with plasmids expressing the indicated proteins depicted in the panel .

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E, Co-IP with anti-FLAG magnetic beads and WB with anti-Trnp1 antibodies showing that FLAGTrnp1 interacts with the GFP-fusion and the ∆1-16Trnp1 proteins but not with other truncated Trnp1 proteins indicating IDRs are crucial for Trnp1 homo-oligomerization. Samples were collected 24h after transfection of P19 cells with plasmids expressing the indicated proteins depicted in the panel.


F , Representative phase contrast images of in vitro phase separation of the recombinant ∆ 1 - 16Trnp1 , ∆ 1 − 140Trnp1 and ∆ 95 - 223Trnp1 proteins at the indicated concentrations in 50 mM Sorenson ' s buffer ( pH 7 , 6 ) containing 150 mM salt and 2mM DTT plus Dextran or RNA when indicated . G , Violin Dot Plots illustrating quantification of the area of single droplets ( N = 700 ) formed by the recombinant Trnp1 , ∆ 1 - 16Trnp1 , ∆ 1 − 140Trnp1 , ∆ 95 - 223Trnp1 proteins at 25 μM concentration in a representative experiment of three repetitions under the conditions shown in F .

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F, Representative phase contrast images of in vitro phase separation of the recombinant ∆1-16Trnp1, ∆1−140Trnp1 and ∆95-223Trnp1 proteins at the indicated concentrations in 50 mM Sorenson's buffer (pH 7,6) containing 150 mM salt and 2mM DTT plus Dextran or RNA when indicated. G, Violin Dot Plots illustrating quantification of the area of single droplets (N=700) formed by the recombinant Trnp1, ∆1-16Trnp1, ∆1−140Trnp1, ∆95-223Trnp1 proteins at 25 μM concentration in a representative experiment of three repetitions under the conditions shown in F.


H , Histogram depicting the percent of proliferating ( Ki67 + ) cells transfected for 24 h with plasmids expressing control ( GFP ) or Trnp1 - IRES - GFP and its different mutant / truncated forms - IRES - GFP in dissociated E14 cortical cells . Biological replicates : GFP = 21 ; Trnp1 = 15 ; FLAGTrnp1 = 10 ; mutCC = 5 ; ∆ 1 - 16 = 7 ; ∆ 1 - 1 - 56 , ∆ 1 - 97 , ∆ 1 - 140 = 3 , ∆ 95 - 223 = 5 and ∆ 140 - 223 = 4 .

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H, Histogram depicting the percent of proliferating (Ki67+) cells transfected for 24 h with plasmids expressing control (GFP) or Trnp1-IRES-GFP and its different mutant/truncated forms-IRES-GFP in dissociated E14 cortical cells. Biological replicates: GFP=21; Trnp1=15; FLAGTrnp1=10; mutCC=5; ∆1-16=7; ∆1-1-56, ∆1-97, ∆1-140=3, ∆95-223=5 and ∆140-223=4.


A - C , Micrographs showing examples of sections of E15 cerebral cortex 2 days after IUE of the plasmids indicated in ( A - C ) with magnifications showing the indicated immunostainings . Scale bars : 100 µm and 20 µm for magnifications . Filled arrows show electroporated cells expressing Pax6 and Ki67 proteins . Open arrows show electroporated cells negative for Pax6 and Ki67 staining . VZ : ventricular zone ; SVZ : subventricular zone ; IZ : intermediate zone ; CP : cortical plate ; V : ventricle . D , Bar and dot plot showing the distribution as percent of GFP + cells / bin after IUE and staining as in A - C . A cortical column reaching from the ventricular surface ( bottom in A ) to the pial surface ( top in A ) was divided into equal sized bins with bin1 at the ventricular surface and bin 5 at the pial surface .

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A-C, Micrographs showing examples of sections of E15 cerebral cortex 2 days after IUE of the plasmids indicated in (A-C) with magnifications showing the indicated immunostainings. Scale bars: 100 µm and 20 µm for magnifications. Filled arrows show electroporated cells expressing Pax6 and Ki67 proteins. Open arrows show electroporated cells negative for Pax6 and Ki67 staining. VZ: ventricular zone; SVZ: subventricular zone; IZ: intermediate zone; CP: cortical plate; V: ventricle. D, Bar and dot plot showing the distribution as percent of GFP+ cells/bin after IUE and staining as in A-C. A cortical column reaching from the ventricular surface (bottom in A) to the pial surface (top in A) was divided into equal sized bins with bin1 at the ventricular surface and bin 5 at the pial surface.


E - G , Bar and dot plots of the proportion of proliferating ( Ki67 + ) cells ( E ) , the number of NSCs ( Pax6 + ) ( F ) and the proportion of proliferating ( BrdU + ) TAPs ( Tbr2 + ) ( G ) as in A - C showing a significant increase in proliferating NSCs upon Trnp1 IUE compared to control or ∆ 1 - 16Trnp1 proteins ( E - F ) and a significant decrease in proliferating TAPs upon Trnp1 IUE and increase upon ∆ 1 - 16Trnp1 IUE compared to control ( G ) at E15 , 2 days after IUE .

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E-G, Bar and dot plots of the proportion of proliferating (Ki67+) cells (E), the number of NSCs (Pax6+) (F) and the proportion of proliferating (BrdU+) TAPs (Tbr2+)(G) as in A-C showing a significant increase in proliferating NSCs upon Trnp1 IUE compared to control or ∆1-16Trnp1 proteins (E- F) and a significant decrease in proliferating TAPs upon Trnp1 IUE and increase upon ∆1-16Trnp1 IUE compared to control (G) at E15, 2 days after IUE.


A , WB showing co - IP performed with the antibodies indicated on top of the lanes in nuclear lysates treated with the nucleases indicated to the right of the panel from P19 cells transfected with plasmids expressing FLAGTrnp1 for 24h immunoblotted with anti - Trnp1 antibodies . As negative control no antibody was added to the co - IP .

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A, WB showing co-IP performed with the antibodies indicated on top of the lanes in nuclear lysates treated with the nucleases indicated to the right of the panel from P19 cells transfected with plasmids expressing FLAGTrnp1 for 24h immunoblotted with anti-Trnp1 antibodies. As negative control no antibody was added to the co-IP.


B - C , Volcano plot showing the mean difference of the protein iBAQ between FLAGTrnp1 ( B ) and FLAG ∆ 1 - 16Trnp1 ( C ) interacting proteins versus control plotted against the p - value . Proteins used to experimentally confirm the MS results are indicated with squares and the protein name in ( B ) .

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B-C, Volcano plot showing the mean difference of the protein iBAQ between FLAGTrnp1 (B) and FLAG∆1-16Trnp1 (C) interacting proteins versus control plotted against the p-value. Proteins used to experimentally confirm the MS results are indicated with squares and the protein name in (B).


D , STRING analysis of Trnp1 interactors The disconnected nodules are not shown . Proteins cluster have been color coded ( green for chromatin association , red for RNA splicing , blue for proteins involved in nucleolus function and light yellow for the rest ) .

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D, STRING analysis of Trnp1 interactors The disconnected nodules are not shown. Proteins cluster have been color coded (green for chromatin association, red for RNA splicing, blue for proteins involved in nucleolus function and light yellow for the rest).


A , Superresolution confocal image of endogenous Nucleophosmin ( B23 ) , Nucleolin ( Ncl ) and Trnp1 proteins in E14 cortical cells at 1div . The yellow line indicates the position of the intensity line scan plot ( below confocal panels ) measuring the grey value of each staining ( Trnp1 = grey ; Ncl = purple and B23 = green ) .

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A, Superresolution confocal image of endogenous Nucleophosmin (B23), Nucleolin (Ncl) and Trnp1 proteins in E14 cortical cells at 1div. The yellow line indicates the position of the intensity line scan plot (below confocal panels) measuring the grey value of each staining (Trnp1=grey; Ncl=purple and B23=green).


B , Co - IP experiments with lysates from P19 cells expressing FLAGTrnp1 for 24h . IPs and WB with the antibodies indicated show interaction between Trnp1 and Ncl but not with Fbl1 consistent with the MS data .

B, Co-IP experiments with lysates from P19 cells expressing FLAGTrnp1 for 24h. IPs and WB with the antibodies indicated show interaction between Trnp1 and Ncl but not with Fbl1 consistent with the MS data.


Ultrastructural analysis depicting in the nucleolus ( arrows for nucleolar interstice ) ( C ) , condensed chromatin blocks ( arrows ) ( D ) and nuclear speckles or interchromatin granule clusters ( arrows for dense cords ) Nu : nucleolus ; G : granular component ; D : dense fibrillar component ; FC : fibrillar center ; C : condensed chromatin .

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Ultrastructural analysis depicting in the nucleolus (arrows for nucleolar interstice) (C), condensed chromatin blocks (arrows) (D) and nuclear speckles or interchromatin granule clusters (arrows for dense cords) Nu: nucleolus; G: granular component; D: dense fibrillar component; FC: fibrillar center; C: condensed chromatin.