D , Representative images of the invading front of the tumors . The scale bar corresponds to 4 μM . E , Percentage of tumors showing muscle invasion .

O O O O O 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, Representative images of the invading front of the tumors. The scale bar corresponds to 4 μM. E, Percentage of tumors showing muscle invasion.


F , G , In vivo tumor growth of MTO cells subcutaneously injected in NOD / SCID mice ( F ) ( 5x105 cells / flank ; eight tumors per condition ) and treated with WntC59 ( 10 mg / kg / day in the drinking water ) or vehicle . Tumor weight of NOD / SCID mice at day 26 is presented in G . In F and G , the mean ± SD of the eight tumors is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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, G, In vivo tumor growth of MTO cells subcutaneously injected in NOD/SCID mice (F) (5x105 cells/flank; eight tumors per condition) and treated with WntC59 (10 mg/kg/day in the drinking water) or vehicle. Tumor weight of NOD/SCID mice at day 26 is presented in G. In F and G, the mean ± SD of the eight tumors is shown.


I , J , WB ( I ) and RNA analysis ( J ) of MTO cells infected with shRor2 or shCtl . Quantification of the WB is also presented ( I , right ) . Results are the mean ± SD of three biological replicates ( performed in triplicated in J ) .

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

I, J, WB (I) and RNA analysis (J) of MTO cells infected with shRor2 or shCtl. Quantification of the WB is also presented (I, right). Results are the mean ± SD of three biological replicates (performed in triplicated in J).


N , O , C57BL6 mice were injected in the spleen with MTO cells and treated since day 1 with WntC59 or vehicle . In N , kinetics of in vivo luminescence ( mean ± SD of the values in five mice ) ; in O , the percentage of mice showing in vivo luminescence .

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

N, O, C57BL6 mice were injected in the spleen with MTO cells and treated since day 1 with WntC59 or vehicle. In N, kinetics of in vivo luminescence (mean ± SD of the values in five mice); in O, the percentage of mice showing in vivo luminescence.


A - C , Viability was assayed by MTT in the indicated cells treated with LGK when specified and supplemented with cisplatin for 24 h . In B , cisplatin resistance was assessed in shRor2 cells transfected with a Snail1 expression plasmid or the corresponding empty plasmid . The figure shows the mean ± SD of the results of three experiments .

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

A-C, Viability was assayed by MTT in the indicated cells treated with LGK when specified and supplemented with cisplatin for 24 h. In B, cisplatin resistance was assessed in shRor2 cells transfected with a Snail1 expression plasmid or the corresponding empty plasmid. The figure shows the mean ± SD of the results of three experiments.


D , Cells were treated with cisplatin for the indicated times . Total extracts were lysed and analyzed by WB .

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

D, Cells were treated with cisplatin for the indicated times. Total extracts were lysed and analyzed by WB.


I , MTO cell viability was determined as above ( mean ± SD of the results of three - four experiments ) .

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

I, MTO cell viability was determined as above (mean ± SD of the results of three-four experiments).


Quantitative RT - PCR shows that Lpin1 expression is depleted in the skeletal muscles Gastrocnemius ( GC ) and Tibialis Anterior ( TA ) , but not in White Adipose Tissue ( WAT ) from 3 - month - old HSACre / + / Lpin1fEx2 - 3 / fEx2 - 3 mice ( HSA - Cre ) compared to control mice . Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice .

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

Quantitative RT-PCR shows that Lpin1 expression is depleted in the skeletal muscles Gastrocnemius (GC) and Tibialis Anterior (TA), but not in White Adipose Tissue (WAT) from 3-month-old HSACre/+/Lpin1fEx2-3/fEx2-3 mice (HSA-Cre) compared to control mice. Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice.


PAP1 and PAP2 activities were respectively substantially decreased and increased in TA , of HSA - Cre mice as compared to control mice , respectively ( n = 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

PAP1 and PAP2 activities were respectively substantially decreased and increased in TA, of HSA-Cre mice as compared to control mice, respectively (n = 6; *P<0.05).


Measurements of muscle / body weight ratio of GC and TA of 3 - month - old HSA - Cre and control mice .

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

Measurements of muscle/body weight ratio of GC and TA of 3-month-old HSA-Cre and control mice.


Hematoxylin and Eosin ( HE ) staining of representative transverse sections of GC muscles from 3 - month - old HSA - Cre and control mice . The black arrows indicate fibers with centrally located nuclei and yellow arrows indicate mononuclear cell infiltration . Scale bars : 40

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

Hematoxylin and Eosin (HE) staining of representative transverse sections of GC muscles from 3-month-old HSA-Cre and control mice. The black arrows indicate fibers with centrally located nuclei and yellow arrows indicate mononuclear cell infiltration. Scale bars: 40 μm


Relative force measurements after normalization of absolute tetanic force to GC muscle weight from 3 - month - old HSA - Cre and control male mice .

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

Relative force measurements after normalization of absolute tetanic force to GC muscle weight from 3-month-old HSA-Cre and control male mice.


Phosphatidic Acid ( PA ) levels in HSA - Cre and control GC muscles .

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

Phosphatidic Acid (PA) levels in HSA-Cre and control GC muscles.


Phospholipidome in HSA - Cre and control GC muscles . Proportions of indicated phospholipid classes in total phospholipids are represented . PC ; Phosphatidylcholine , PE ; Phosphatidylethanolamine , PI ; Phosphatidylinositol , PS ; Phoshpatidylserine , PA ; Phosphatidic Acid , PG ; Phosphatidylglycerol .

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

Phospholipidome in HSA-Cre and control GC muscles. Proportions of indicated phospholipid classes in total phospholipids are represented. PC; Phosphatidylcholine, PE; Phosphatidylethanolamine, PI; Phosphatidylinositol, PS; Phoshpatidylserine, PA; Phosphatidic Acid, PG; Phosphatidylglycerol.


Amounts of indicated phospholipid classes normalized by protein content in HSA - Cre and control GC muscles . ( D ) Total phospholipid ( PL ) amount normalized by protein content in HSA - Cre and control GC muscles .

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

Amounts of indicated phospholipid classes normalized by protein content in HSA-Cre and control GC muscles. (D) Total phospholipid (PL) amount normalized by protein content in HSA-Cre and control GC muscles.


Distribution of the total carbon number in acyl chains of PC in HSA - Cre and control GC muscles .

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

Distribution of the total carbon number in acyl chains of PC in HSA-Cre and control GC muscles.


Distribution of the number of double bonds in acyl chains of PC in HSA - Cre and control GC muscles .

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

Distribution of the number of double bonds in acyl chains of PC in HSA-Cre and control GC muscles.


Distribution of the total carbon number in acyl chains of PE in HSA - Cre and control GC muscles .

Distribution of the total carbon number in acyl chains of PE in HSA-Cre and control GC muscles.


Distribution of the number of double bonds in acyl chains of PE in HSA - Cre and control GC muscles .

Distribution of the number of double bonds in acyl chains of PE in HSA-Cre and control GC muscles.


HE , Oil Red O ( ORO ) , Cytochrome c Oxidase ( COX ) and Succinate Deshydrogenase ( SDH ) staining of representative frozen sections of TA muscle from 3 - month - old HSA - Cre and control mice .

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

HE, Oil Red O (ORO), Cytochrome c Oxidase (COX) and Succinate Deshydrogenase (SDH) staining of representative frozen sections of TA muscle from 3-month-old HSA-Cre and control mice.


Quantification of triacylglycerol ( TAG ) levels in GC muscles from 3 - month - old HSA - Cre and and control mice .

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

Quantification of triacylglycerol (TAG) levels in GC muscles from 3-month-old HSA-Cre and and control mice.


Content of TAG assessed by MS in HSA - Cre and control GC muscles .

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

Content of TAG assessed by MS in HSA-Cre and control GC muscles.


Cholesterol , ( E ) Cholesteryl ester ( CE ) , ( F ) DAG was assessed by MS in HSA - Cre and control GC muscles .

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

Cholesterol, (E) Cholesteryl ester (CE), (F) DAG was assessed by MS in HSA-Cre and control GC muscles.


Quantitative RT - PCR detection of the level of expression of Fatty acid synthase ( Fasn ) , ATP citrate lyase ( Acly ) , Thyroid hormone - inducible hepatic protein 1 ( Thrsp1 ) , Angiopoietin - like protein 4 / fast - induced adipose factor ( ANGPTL4 / Fiaf ) , Insulin - induced gene 1 ( Insig1 ) and Peroxisome proliferator - activated receptor gamma ( PPARγ ) in TA from 3 - month - old HSA - Cre and control mice . Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice .

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

Quantitative RT-PCR detection of the level of expression of Fatty acid synthase (Fasn), ATP citrate lyase (Acly), Thyroid hormone-inducible hepatic protein 1 (Thrsp1), Angiopoietin-like protein 4/ fast-induced adipose factor (ANGPTL4/Fiaf), Insulin-induced gene 1 (Insig1) and Peroxisome proliferator-activated receptor gamma (PPARγ) in TA from 3-month-old HSA-Cre and control mice. Expression levels are corrected for expression of the control gene Pinin and presented in the graph as fold change relative to control mice.


Immunoblot analysis of Fasn in TA muscle of 3 - month - old HSA - Cre and control mice .

Immunoblot analysis of Fasn in TA muscle of 3-month-old HSA-Cre and control mice.


Quantitative RT - PCR measurement of Carnitine palmitoyltransferase 1B ( Cpt1b ) and Medium - chain acyl - CoA dehydrogenase ( Mcad ) in TA from 3 - month - old HSA - Cre and control mice .

Quantitative RT-PCR measurement of Carnitine palmitoyltransferase 1B (Cpt1b) and Medium-chain acyl-CoA dehydrogenase (Mcad) in TA from 3-month-old HSA-Cre and control mice.


Immunoblot analysis of full length and N - terminal SREBP1c and SREBP2 protein levels in GC muscle of 4 - month - old HSA - Cre and control mice ( n = 7 - 4 ) . The ratio of the densitometric assay of the N - term and Full length forms is presented .

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

Immunoblot analysis of full length and N-terminal SREBP1c and SREBP2 protein levels in GC muscle of 4-month-old HSA-Cre and control mice (n=7-4). The ratio of the densitometric assay of the N-term and Full length forms is presented.


Metabolomics profile of GC muscle of 4 - month - old HSA - Cre and control mice presented as heatmap visualization and hierarchical clustering analysis of

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

Metabolomics profile of GC muscle of 4-month-old HSA-Cre and control mice presented as heatmap visualization and hierarchical clustering analysis of compounds


Fold change of phospholipid precursors in GC muscles of 4 - month - old HSA - Cre and control mice measured by liquid - chromatography ( LC ) tandem mass spectrometry ( MS / MS ) ( n = 7 - 4 , * 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

Fold change of phospholipid precursors in GC muscles of 4-month-old HSA-Cre and control mice measured by liquid-chromatography (LC) tandem mass spectrometry (MS/MS) (n=7-4, *P<0.05).


Fold change of Lactate / Pyruvate , Pyruvate / AcetylCoA , Pyruvate / Citrate , ATP / AMP , and ATP / ADP ratios in GC muscles of 4 month - old HSA - Cre and control mice ( n = 7 - 4 , * P < 0 . 05 ) .

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

Fold change of Lactate/Pyruvate, Pyruvate/AcetylCoA, Pyruvate/Citrate, ATP/AMP, and ATP/ADP ratios in GC muscles of 4 month-old HSA-Cre and control mice (n=7-4, *P<0.05).


UPR activation was analysed by immunoblotting with indicated antibodies in GC muscles of 4 - month - old HSA - Cre and control mice ( n = 7 - 4 ) . Quantification by densitometric analyses of Fgf21 , Bip , sXbp1 Atf6 cleaved form , p - Eif2a and Chop protein levels is presented as a graph . Data are normalized to Gapdh and expressed as a fold change relative to the control mice .

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

UPR activation was analysed by immunoblotting with indicated antibodies in GC muscles of 4-month-old HSA-Cre and control mice (n=7-4). Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, p-Eif2a and Chop protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice.


Quantitative RT - PCR measurement of Fgf21 , Gdf15 , Bip / Grp78 , hsp90b1 , and Edem1 in GC muscles from 4 - month - old HSA - Cre and control mice .

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

Quantitative RT-PCR measurement of Fgf21, Gdf15, Bip/Grp78, hsp90b1, and Edem1 in GC muscles from 4-month-old HSA-Cre and control mice.


EM analysis of EDL fibers of HSA - Cre mice revealed the presence of areas in which the non - junctional SR is more vesiculated and fragmented compared to control ( black arrows ) . Remodeling of the SR is also accompanied by modification in the positioning of mitochondria ( white arrows ) . A representative Calcium Release Unit ( CRU ) is indicated ( empty arrow ) . Scale bars : 0 . 50 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

EM analysis of EDL fibers of HSA-Cre mice revealed the presence of areas in which the non-junctional SR is more vesiculated and fragmented compared to control (black arrows). Remodeling of the SR is also accompanied by modification in the positioning of mitochondria (white arrows). A representative Calcium Release Unit (CRU) is indicated (empty arrow). Scale bars: 0.50 μm.


The relative mtDNA copy number was determined by the ratio of mitochondrial DNA - encoded Cytochrome B ( CytB ) and nuclear - encoded NADH dehydrogenase ( ubiquinone ) flavoprotein 1 ( Ndufv1 ) genes in GC and TA from HSA - Cre and control mice .

O O O O O O O O O O O O O O O O O O O O O O O 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 mtDNA copy number was determined by the ratio of mitochondrial DNA-encoded Cytochrome B (CytB) and nuclear-encoded NADH dehydrogenase (ubiquinone) flavoprotein 1 (Ndufv1) genes in GC and TA from HSA-Cre and control mice.


Mitochondria - encoded gene and Nuclear - encoded gene expression levels by quantitative RT - PCR in GC muscles from 4 - month - old HSA - Cre and control mice .

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

Mitochondria-encoded gene and Nuclear-encoded gene expression levels by quantitative RT-PCR in GC muscles from 4-month-old HSA-Cre and control mice.


Electron micrographs of HSA - Cre EDL muscles of 3 - month - old mice showed that mitochondria are almost exclusively positioned at the two sides of Z lines in EDL fibers from control mice ( black arrows ) . On the other hand , in Lipin1 deficient EDL fibers mitochondria are more often clustered and display electron - dense contact sites ( white arrows ) . Scale bars : 0 . 20 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Electron micrographs of HSA-Cre EDL muscles of 3-month-old mice showed that mitochondria are almost exclusively positioned at the two sides of Z lines in EDL fibers from control mice (black arrows). On the other hand, in Lipin1 deficient EDL fibers mitochondria are more often clustered and display electron-dense contact sites (white arrows). Scale bars: 0.20 μm.


Ultrastructural analysis of HSA - Cre EDL muscles revealed the presence of numerous autophagic vacuoles of different morphologies ( autophagosomes and lysosomes , black arrows ; multilamellar bodies , white arrow ) containing amorphous material and possibly degraded mitochondria ( M ) . Scale bars : 0 . 50

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

Ultrastructural analysis of HSA-Cre EDL muscles revealed the presence of numerous autophagic vacuoles of different morphologies (autophagosomes and lysosomes, black arrows; multilamellar bodies, white arrow) containing amorphous material and possibly degraded mitochondria (M). Scale bars: 0.50 μm


Representative immunoblots from protein extract of GC muscles of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice , with the indicated antibodies .

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

Representative immunoblots from protein extract of GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice, with the indicated antibodies.


Quantitative RT - PCR measurement of Fgf21 and GDF15 expression in GC muscle of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice .

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

Quantitative RT-PCR measurement of Fgf21 and GDF15 expression in GC muscle of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.


Histological analysis of TA muscles of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice by Oil Red O staining to detect the neutral lipids . Scale bars : 40 μm .

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

Histological analysis of TA muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice by Oil Red O staining to detect the neutral lipids. Scale bars: 40 μm.


TAG level measurement in GC muscles of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice .

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

TAG level measurement in GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.


In vivo force measurements performed on GC muscles of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice .

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

In vivo force measurements performed on GC muscles of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice.


Representative staining of IgG positive fibers on frozen sections of TA of 6 - month - old control , HSA - Cre untreated and HSA - Cre TUDCA - treated mice . The sections were stained with a secondary fluorescent anti - mouse IgG antibody . Quantification of the number of necrotic fibers over the total number of fibers .

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

Representative staining of IgG positive fibers on frozen sections of TA of 6-month-old control, HSA-Cre untreated and HSA-Cre TUDCA-treated mice. The sections were stained with a secondary fluorescent anti-mouse IgG antibody. Quantification of the number of necrotic fibers over the total number of fibers.


Representative immunoblots from protein extract of GC muscles of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice , with the indicated antibodies .

Representative immunoblots from protein extract of GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice, with the indicated antibodies.


Quantitative RT - PCR measurement of Fgf21 and GDF15 expression in GC muscle of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice .

Quantitative RT-PCR measurement of Fgf21 and GDF15 expression in GC muscle of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.


Histological analysis of TA muscles of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice by Oil Red O staining to detect the neutral lipids .

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

Histological analysis of TA muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice by Oil Red O staining to detect the neutral lipids.


TAG level measurement in GC muscles of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice .

TAG level measurement in GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.


In vivo force measurements performed on GC muscles of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice .

In vivo force measurements performed on GC muscles of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice.


Representative staining of IgG positive fibers on frozen sections of TA of 4 - month - old control , HSA - Cre untreated and HSA - Cre Bezafibrate - treated mice . The sections were stained with a secondary fluorescent anti - mouse IgG antibody . Quantification of the number of necrotic fibers over the total number of fibers .

Representative staining of IgG positive fibers on frozen sections of TA of 4-month-old control, HSA-Cre untreated and HSA-Cre Bezafibrate-treated mice. The sections were stained with a secondary fluorescent anti-mouse IgG antibody. Quantification of the number of necrotic fibers over the total number of fibers.


( a , b ) Immunoblot analysis of LAMP - 2A in total lysates ( a ) and quantitative PCR analysis of Lamp2a mRNA ( b ) of naive CD4 + T cells and TH1 and TH2 cells following activation with anti - CD3 and anti - CD28 . β - actin ( a ) serves as a loading control throughout ; mRNA results ( b ) are presented relative to those of resting cells .

(a,b) Immunoblot analysis of LAMP-2A in total lysates (a) and quantitative PCR analysis of Lamp2a mRNA (b) of naive CD4+ T cells and TH1 and TH2 cells following activation with anti-CD3 and anti-CD28. β-actin (a) serves as a loading control throughout; mRNA results (b) are presented relative to those of resting cells.


( c ) Immunofluorescence microscopy of LAMP - 2A ( green ) and nuclei ( stained with the DNA - binding dye DAPI ; blue ) in resting TH1 cells ( Rest ) and TH1 cells activated for 24 h as in a , b ( Act ) ( left ) , and quantification of lysosomes in cells as at left ( right ) . Original magnification , × 630 ; scale bar , 10 μm . * P = 0 . 013 ( t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence microscopy of LAMP-2A (green) and nuclei (stained with the DNA-binding dye DAPI; blue) in resting TH1 cells (Rest) and TH1 cells activated for 24 h as in a,b (Act) (left), and quantification of lysosomes in cells as at left (right). Original magnification, ×630; scale bar, 10 μm. *P = 0.013 (t-test).


( d ) Electron microscopy ( left ) of resting TH1 cells and TH1 cells activated for 24 h as in a , b ( top row ) ; below , enlargement of areas outlined above , showing individual lysosomes ( arrows ) . Original magnification ( top row ) , × 12 , 000 ; scale bars ( bottom row ) , 1 μm . Right , morphometric analysis of cells as at left . * P = 0 . 037 and * * P = 0 . 034 ( t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Electron microscopy (left) of resting TH1 cells and TH1 cells activated for 24 h as in a,b (top row); below, enlargement of areas outlined above, showing individual lysosomes (arrows). Original magnification (top row), ×12,000; scale bars (bottom row), 1 μm. Right, morphometric analysis of cells as at left. *P = 0.037 and **P = 0.034 (t-test).


( e ) Distribution of KFERQ - PA - mCherry - 1 in TH1 cells transiently transfected with a CMA reporter vector , photoactivated , and left unstimulated ( Rest ) or stimulated with anti - CD3 and anti - CD28 in the presence ( 3 - MA ) or absence ( vehicle ( veh ) ) of 3 - methyladenine ( left ) ; arrows indicate mCherry + puncta . Original magnification , × 630 . Right , quantification of the results for the cells at left . * P 0 . 05 ( analysis of variance with Tukey ' s post - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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) Distribution of KFERQ-PA-mCherry-1 in TH1 cells transiently transfected with a CMA reporter vector, photoactivated, and left unstimulated (Rest) or stimulated with anti-CD3 and anti-CD28 in the presence (3-MA) or absence (vehicle (veh)) of 3-methyladenine (left); arrows indicate mCherry+ puncta. Original magnification, ×630. Right, quantification of the results for the cells at left. *P 0.05 (analysis of variance with Tukey's post-test).


( f ) Immunoblot analysis of hsc70 in lysosomal fractions of resting ( Lys - R ) and stimulated ( Lys - A ) TH1 cells and in cytosolic fractions of TH1 cells activated as in a , b ( Cyto ) ; LAMP - 2A and LAMP - 1 serve as controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of hsc70 in lysosomal fractions of resting (Lys-R) and stimulated (Lys-A) TH1 cells and in cytosolic fractions of TH1 cells activated as in a,b (Cyto); LAMP-2A and LAMP-1 serve as controls.


( g ) Immunoblot analysis of LAMP - 2A in resting TH1 cells and TH1 cells activated in the presence ( NAc ) or absence ( Veh ) of N - acetylcysteine . ( h ) Quantitative PCR analysis of Lamp2a mRNA in cells as in g ( results presented as in b ) . * P = 0 . 007 ( t test ) .

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

(g) Immunoblot analysis of LAMP-2A in resting TH1 cells and TH1 cells activated in the presence (NAc) or absence (Veh) of N-acetylcysteine. (h) Quantitative PCR analysis of Lamp2a mRNA in cells as in g (results presented as in b). *P = 0.007 (t test).


( i ) Luciferase activity in TH1 cells transiently transfected with a vector containing a luciferase reporter under the control of the Lamp2 proximal promoter and stimulated for 0 , 12 or 24 h with plate - bound anti - CD3 and anti - CD28 ; results are presented relative to those of resting cells ( time 0 ) .

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

(i) Luciferase activity in TH1 cells transiently transfected with a vector containing a luciferase reporter under the control of the Lamp2 proximal promoter and stimulated for 0, 12 or 24 h with plate-bound anti-CD3 and anti-CD28; results are presented relative to those of resting cells (time 0).


( j ) Luciferase activity in TH1 cells transiently transfected as in i and left unstimulated ( control ( Ctrl ) ) or stimulated for 16 h with 50 μM paraquat ( Pq ) or 1 mM H2O2 ; results are presented in relative light units ( RLU ) .

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

(j) Luciferase activity in TH1 cells transiently transfected as in i and left unstimulated (control (Ctrl)) or stimulated for 16 h with 50 μM paraquat (Pq) or 1 mM H2O2; results are presented in relative light units (RLU).


( k ) Immunoblot analysis of LAMP - 2A in total lysates of resting TH1 cells ( − ) and TH1 cells activated for 24 h as in a , b ( + ) , transfected with a control nontargeting siRNA ( with a scrambled sequence ( Scr ) ) or siRNA specific for mRNA encoding Duox1 ( either of two siRNAs : Dx ( 1 ) and Dx ( 2 ) ) or RISP ( Uq ( 1 ) ) .

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

(k) Immunoblot analysis of LAMP-2A in total lysates of resting TH1 cells (−) and TH1 cells activated for 24 h as in a,b (+), transfected with a control nontargeting siRNA (with a scrambled sequence (Scr)) or siRNA specific for mRNA encoding Duox1 (either of two siRNAs: Dx(1) and Dx(2)) or RISP (Uq(1)).


( l ) Immunoblot analysis of LAMP - 2A in lysates of resting or activated TH1 cells in the presence ( right half ) or absence ( left half ) of cyclosporine A ( CsA ) .

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

(l) Immunoblot analysis of LAMP-2A in lysates of resting or activated TH1 cells in the presence (right half) or absence (left half) of cyclosporine A (CsA).


( m ) Chromatin - immunoprecipitation analysis of the recruitment of NFAT1 to the promoter of Lamp2 or Adad1 ( negative control ) ( left ) or to Il2 ( positive control ) ( right ) in resting and activated TH1 cells ; results are presented relative to input chromatin precipitated with anti - NFAT1 .

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

(m) Chromatin-immunoprecipitation analysis of the recruitment of NFAT1 to the promoter of Lamp2 or Adad1 (negative control) (left) or to Il2 (positive control) (right) in resting and activated TH1 cells; results are presented relative to input chromatin precipitated with anti-NFAT1.


( a ) Flow cytometry of T cells transduced with lentivirus expressing GFP and nontargeting control shRNA ( Scr ) or Lamp2a mRNA - specific shRNA ( shL2A ) ( left ) , and immunoblot analysis of LAMP - 2A in sorted GFP + T cells ( right ) . Numbers above bracketed lines ( left ) indicate percent GFP + cells .

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

(a) Flow cytometry of T cells transduced with lentivirus expressing GFP and nontargeting control shRNA (Scr) or Lamp2a mRNA-specific shRNA (shL2A) (left), and immunoblot analysis of LAMP-2A in sorted GFP+ T cells (right). Numbers above bracketed lines (left) indicate percent GFP+ cells.


( b ) Enzyme - linked immunosorbent assay of IL - 2 and interferon - γ ( IFN - γ ) in TH1 cells transduced as in a and then left unstimulated ( Rest ) or stimulated for 24 h with anti - CD3 and anti - CD28 ( Act ) . * P = 0 . 038 and * * P = 0 . 031 ( t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Enzyme-linked immunosorbent assay of IL-2 and interferon-γ (IFN-γ) in TH1 cells transduced as in a and then left unstimulated (Rest) or stimulated for 24 h with anti-CD3 and anti-CD28 (Act). *P = 0.038 and **P = 0.031 (t-test).


( c ) Proliferation in TH1 cells transduced as in a and left unstimulated or stimulated for 24 h as in b , assessed by incorporation of the thymidine analog BrdU and presented as absorbance at 495 nm ( A495 ) . * P = 0 . 013 ( t - test ) .

(c) Proliferation in TH1 cells transduced as in a and left unstimulated or stimulated for 24 h as in b, assessed by incorporation of the thymidine analog BrdU and presented as absorbance at 495 nm (A495). *P = 0.013 (t-test).


( d ) Quantification of annexin V - positive ( AnnV + ) cells among resting or stimulated TH1 cells transduced as in a .

O 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) Quantification of annexin V-positive (AnnV+) cells among resting or stimulated TH1 cells transduced as in a.


( a ) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions isolated from resting cells TH1 cells and TH1 cells activated for 24 h with anti - CD3 and anti - CD28 ; LAMP - 1 serves as a control .

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

(a) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions isolated from resting cells TH1 cells and TH1 cells activated for 24 h with anti-CD3 and anti-CD28; LAMP-1 serves as a control.


( b ) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions of TH1 cells activated for 24 h as in a with ( + ) or without ( − ) inhibition of lysosomal proteases during the final 3 h with NH4Cl and leupeptin ( Lys inh ) ; LAMP - 1 serves as a control .

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

(b) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions of TH1 cells activated for 24 h as in a with (+) or without (−) inhibition of lysosomal proteases during the final 3 h with NH4Cl and leupeptin (Lys inh); LAMP-1 serves as a control.


( c ) Immunoblot analysis of Itch and RCAN1 in resting TH1 cells and TH1 cells activated for 24 h as in a in the presence ( NAc ) or absence ( Veh ) of N - acetylcysteine .

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

(c) Immunoblot analysis of Itch and RCAN1 in resting TH1 cells and TH1 cells activated for 24 h as in a in the presence (NAc) or absence (Veh) of N-acetylcysteine.


( d ) Immunoblot analysis of lysates of TH1 cells transfected with GFP - Itch or GFP - mu - Itch ( top ) or with Myc - RCAN1 or Myc - mu - RCAN1 ( bottom ) and cotransfected with a GFP expression plasmid ( to assess transfection efficiency ) , then activated for 24 h as in a .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of lysates of TH1 cells transfected with GFP-Itch or GFP-mu-Itch (top) or with Myc-RCAN1 or Myc-mu-RCAN1 (bottom) and cotransfected with a GFP expression plasmid (to assess transfection efficiency), then activated for 24 h as in a.


( e , f ) Proliferation ( e ) and IL - 2 production ( f ) of TH1 cells transfected with plasmid expressing GFP ( Ctrl ) or plasmids as in c and left unstimulated ( Rest ) or activated for 48 h ( e ) or 24 h ( f ) as in a ( assessed as in Fig . 2c ( e ) or Fig . 2b ( f ) ) . * P = 0 . 019 and * * P = 0 . 0034 ( e ) or * P = 0 . 046 and * * P = 0 . 044 ( f ) ( t test ) . Data are representative of four ( a , b ) , two ( c ) or three ( d ) independent experiments or are from three independent experiments ( e , f ; mean and s . e . m . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Proliferation (e) and IL-2 production (f) of TH1 cells transfected with plasmid expressing GFP (Ctrl) or plasmids as in c and left unstimulated (Rest) or activated for 48 h (e) or 24 h (f) as in a (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.019 and **P = 0.0034 (e) or *P = 0.046 and **P = 0.044 (f) (t test). Data are representative of four (a,b), two (c) or three (d) independent experiments or are from three independent experiments (e,f; mean and s.e.m.).


( a ) Immunoblot analysis of LAMP - 2A in CD4 + T cells ( left ) and liver , kidneys , thymus and lungs ( right ) of L2A - cKO mice ( KO ) and their control littermates ( Ctrl ) .

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

(a) Immunoblot analysis of LAMP-2A in CD4+ T cells (left) and liver, kidneys, thymus and lungs (right) of L2A-cKO mice (KO) and their control littermates (Ctrl).


( b ) Quantitative PCR analysis of Lamp2a , Lamp2b and Lamp2c mRNA in T cells from L2A - cKO mice ( right ) ; results are presented relative to those of their control littermates ( left ) , set as 1 .

O O O O O O O O O O O O O O O O O O O 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) Quantitative PCR analysis of Lamp2a, Lamp2b and Lamp2c mRNA in T cells from L2A-cKO mice (right); results are presented relative to those of their control littermates (left), set as 1.


( c ) Immunoblot analysis of the autophagy related protein LC3 in its cytosolic form ( LC3 - I ) and its autophagosome membrane - associated form ( LC3 - II ) in total lysates of CD4 + T cells obtained from L2A - cKO mice or their control littermates and then left unstimulated ( Rest ) or activated for 24 h with anti - CD3 and anti - CD218 ( Act ) , cultured for the final 4 h in the presence ( + ) or absence ( − ) of NH4Cl and leupeptin ( NH4Cl + Leup ) .

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

(c) Immunoblot analysis of the autophagy related protein LC3 in its cytosolic form (LC3-I) and its autophagosome membrane-associated form (LC3-II) in total lysates of CD4+ T cells obtained from L2A-cKO mice or their control littermates and then left unstimulated (Rest) or activated for 24 h with anti-CD3 and anti-CD218 (Act), cultured for the final 4 h in the presence (+) or absence (−) of NH4Cl and leupeptin (NH4Cl+Leup).


( d ) Spleens from an L2A - cKO mouse and its control littermate ( left ) , and flow cytometry of peripheral T cells from such mice ( right ) . Numbers in quadrants ( right ) indicate percent CD4 − CD8 + T cells ( top left ) or CD4 + CD8 − T cells ( bottom right ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Spleens from an L2A-cKO mouse and its control littermate (left), and flow cytometry of peripheral T cells from such mice (right). Numbers in quadrants (right) indicate percent CD4−CD8+ T cells (top left) or CD4+CD8− T cells (bottom right).


( e , f ) Proliferation ( e ) and IL - 2 production ( f ) by CD4 + T cells isolated from the spleen of L2A - cKO mice and their control littermates and stimulated ex vivo for 48 h with anti - CD3 and anti - CD28 ( assessed as in Fig . 2c ( e ) or Fig . 2b ( f ) ) . * P = 0 . 0062 ( e ) or 0 . 006 ( f ) ( t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Proliferation (e) and IL-2 production (f) by CD4+ T cells isolated from the spleen of L2A-cKO mice and their control littermates and stimulated ex vivo for 48 h with anti-CD3 and anti-CD28 (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.0062 (e) or 0.006 (f) (t-test).


( g ) Immunoblot analysis of LAMP - 2A , Itch and RCAN1 in total lysates of CD4 + T cells obtained from L2A - cKO mice ( n = 3 ( KO1 , KO2 and KO3 ) ) and their control littermates ( n = 3 ) and activated for 24 h as in e , f . * , nonspecific band .

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

(g) Immunoblot analysis of LAMP-2A, Itch and RCAN1 in total lysates of CD4+ T cells obtained from L2A-cKO mice (n = 3 (KO1, KO2 and KO3)) and their control littermates (n = 3) and activated for 24 h as in e,f. *, nonspecific band.


( h ) Quantitative PCR analysis of Rcan1 and Itch mRNA in CD4 + T cells isolated from L2A - cKO mice and their control littermates and activated for 24 h as in e , f ; results are presented relative to those of resting cells .

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

(h) Quantitative PCR analysis of Rcan1 and Itch mRNA in CD4+ T cells isolated from L2A-cKO mice and their control littermates and activated for 24 h as in e,f; results are presented relative to those of resting cells.


( i , j ) Recall responses to OVA ( 323 - 339 ) in draining lymph nodes isolated from L2A - cKO mice and their control littermates after immunization with OVA ( 323 - 339 ) , assessed as proliferation ( i ) and IL - 2 production ( j ) ( as in e , f ) . * P = 0 . 0054 ( i ) or 0 . 0052 ( j ) ( t - test ) .

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

(i,j) Recall responses to OVA(323-339) in draining lymph nodes isolated from L2A-cKO mice and their control littermates after immunization with OVA(323-339), assessed as proliferation (i) and IL-2 production (j) (as in e,f). *P = 0.0054 (i) or 0.0052 (j) (t-test).


( k , l ) Recall responses to L . monocytogenes in CD4 + T cells isolated from L2A - cKO mice and their control littermates ( n = 6 per group ) previously uninfected ( UI ) or previously infected with L . monocytogenes ( Inf ) , assessed as IFN - γ production ( k ) and proliferation ( l ) ( as in e , f ) . * P = 0 . 032 ( k ) or 0 . 02 ( l ) ( t - test ) .

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

(k,l) Recall responses to L. monocytogenes in CD4+ T cells isolated from L2A-cKO mice and their control littermates (n = 6 per group) previously uninfected (UI) or previously infected with L. monocytogenes (Inf), assessed as IFN-γ production (k) and proliferation (l) (as in e,f). *P = 0.032 (k) or 0.02 (l) (t-test).


( m ) L . monocytogenes in spleen and liver of L2A - cKO mice and their control littermates ( n = 3 per genotype ) immunized with L . monocytogenes and then reinfected with a high dose of L . monocytogenes . CFU , colony - forming units . * P = 0 . 008 and * * P = 0 . 001 ( t - test ) .

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

(m) L. monocytogenes in spleen and liver of L2A-cKO mice and their control littermates (n = 3 per genotype) immunized with L. monocytogenes and then reinfected with a high dose of L. monocytogenes. CFU, colony-forming units. *P = 0.008 and **P = 0.001 (t-test).


( a ) Immunoblot analysis of LAMP - 2A in CD4 + T cells obtained from 4 - month - old or 22 - month old mice , then left resting or stimulated for 24 h with anti - CD3 plus anti - CD28 .

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

(a) Immunoblot analysis of LAMP-2A in CD4+ T cells obtained from 4-month-old or 22-month old mice, then left resting or stimulated for 24 h with anti-CD3 plus anti-CD28.


( b ) Immunoblot analysis of RCAN1 and Itch in CD4 + T cells obtained from 4 - month - old or 22 - month old mice ( n = 3 per group ) , then stimulated for 24 h as in a ( left ) , and quantification of protein in 22 - month old mice , presented relative to that of 4 - month - old mice , set as 100 % . * P = 0 . 044 ( t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of RCAN1 and Itch in CD4+ T cells obtained from 4-month-old or 22-month old mice (n = 3 per group), then stimulated for 24 h as in a (left), and quantification of protein in 22-month old mice, presented relative to that of 4-month-old mice, set as 100%. *P = 0.044 (t-test).


( c ) Immunoblot analysis of LAMP - 2A in total lysates of naive and memory CD4 + T cells left resting or activated for 24 h as in a .

O O O O O O O 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) Immunoblot analysis of LAMP-2A in total lysates of naive and memory CD4+ T cells left resting or activated for 24 h as in a.


( d ) Immunoblot analysis ( bottom ) of LAMP - 2A in total lysates of naive and memory CD4 + T cells obtained from 4 - or 22 - month - old mice and left resting ( R ) or activated for 24 h as in a ( A ) ; above , quantification from results below ( n = 3 mice per group ) . * P = 0 . 009 ( t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis (bottom) of LAMP-2A in total lysates of naive and memory CD4+ T cells obtained from 4- or 22-month-old mice and left resting (R) or activated for 24 h as in a (A); above, quantification from results below (n = 3 mice per group). *P = 0.009 (t-test).


( e ) Electrophoretic mobility - shift assay of the binding of NFAT ( N ) and free probe ( FP ) in nuclear extracts of CD4 + T cells isolated from 3 - , 12 - or 22 - month - old mice and activated for 4 h as in a .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Electrophoretic mobility-shift assay of the binding of NFAT (N) and free probe (FP) in nuclear extracts of CD4+ T cells isolated from 3-, 12- or 22-month-old mice and activated for 4 h as in a.


( f ) ROS production in CD4 + T cells isolated from 4 - or 22 - month - old mice ( n = 3 per group ) and left resting or activated for 12 h as in a , assessed by flow cytometry and presented as the mean fluorescence intensity ( MFI ) of a fluorogenic probe for oxidative stress ( CellROX Green ) .

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

(f) ROS production in CD4+ T cells isolated from 4- or 22-month-old mice (n = 3 per group) and left resting or activated for 12 h as in a, assessed by flow cytometry and presented as the mean fluorescence intensity (MFI) of a fluorogenic probe for oxidative stress (CellROX Green).


( g - i ) Immunoblot analysis of LAMP - 2A ( g ) and analysis of the proliferation ( h ) and IL - 2 production ( i ) of TH1 cells obtained from 4 - or 22 - month - old mice and transduced with retrovirus expressing GFP alone ( RV - GFP ) or human LAMP - 2A and GFP ( RV - LAMP2A ) , sorted by GFP expression , cultured for 2 weeks and then stimulated for 24 - 48 h as in a ( assessed as in Fig . 2c ( h ) or Fig . 2b ( i ) ) . * P 0 . 05 ( analysis of variance with Tukey ' s post - test ) .

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

(g-i) Immunoblot analysis of LAMP-2A (g) and analysis of the proliferation (h) and IL-2 production (i) of TH1 cells obtained from 4- or 22-month-old mice and transduced with retrovirus expressing GFP alone (RV-GFP) or human LAMP-2A and GFP (RV-LAMP2A), sorted by GFP expression, cultured for 2 weeks and then stimulated for 24-48 h as in a (assessed as in Fig. 2c (h) or Fig. 2b (i)). *P 0.05 (analysis of variance with Tukey's post-test).


( j ) Immunoblot analysis of LAMP - 2A in total lysates of CD4 + T cells obtained from two human donors ( A and B ) and left resting or activated for 24 or 48 h as in a ( above lanes ) .

(j) Immunoblot analysis of LAMP-2A in total lysates of CD4+ T cells obtained from two human donors (A and B) and left resting or activated for 24 or 48 h as in a (above lanes).


( k ) Real - time PCR analysis of LAMP2A mRNA in naive and memory CD4 + T cells obtained from young human donors ( 26 ± 2 years of age ; n = 3 ) or old human donors ( 72 ± 6 years of age ; n = 6 ) and activated for 24 h as in a ; results were calculated by the change - in - cycle - threshold method ( 2 − ΔCt ) relative to those of the gene encoding β - actin . * P = 0 . 047 and * * P = 0 . 0033 ( Mann - Whitney ) .

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

(k) Real-time PCR analysis of LAMP2A mRNA in naive and memory CD4+ T cells obtained from young human donors (26 ± 2 years of age; n = 3) or old human donors (72 ± 6 years of age; n = 6) and activated for 24 h as in a; results were calculated by the change-in-cycle-threshold method (2−ΔCt) relative to those of the gene encoding β-actin. *P = 0.047 and **P = 0.0033 (Mann-Whitney).


A , B . Western blot showing the absence of HUWE1 protein in 293T ( A ) and HeLa ( B ) cells subjected to CRISPR / Cas9 - mediated HUWE1 - deletion .

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

A, B. Western blot showing the absence of HUWE1 protein in 293T (A) and HeLa (B) cells subjected to CRISPR/Cas9-mediated HUWE1-deletion.


C , D . HUWE1 - knockout 293T ( C ) and HeLa ( D ) cells show increased DNA breaks in the absence of exogenous DNA damage treatment . Results from the Alkaline Comet Assay are shown . The " n " numbers of comet tails analyzed ( pooled from two independent experiments ) , as well as the mean , are indicated on the graphs . HUWE1 - knockout HeLa cells did not show increases breakage in the Neutral Comet assay ( Fig EV1B ) , indicating that the majority of breaks observed in cycling HUWE1 - knockout cells are not double strand breaks , but rather single strand breaks and other types of lesions .

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

C, D. HUWE1-knockout 293T (C) and HeLa (D) cells show increased DNA breaks in the absence of exogenous DNA damage treatment. Results from the Alkaline Comet Assay are shown. The "n" numbers of comet tails analyzed (pooled from two independent experiments), as well as the mean, are indicated on the graphs. HUWE1-knockout HeLa cells did not show increases breakage in the Neutral Comet assay (Fig EV1B), indicating that the majority of breaks observed in cycling HUWE1-knockout cells are not double strand breaks, but rather single strand breaks and other types of lesions.


E , F . Increased S - phase arrest in HUWE1 - knockout 293T ( E ) and HeLa ( F ) cells . Cycling cells were incubated with BrdU and subjected to BrdU / PI bi - dimensional flow cytometry . Representative flow cytometry profiles are presented in Fig EV1D - F . Bars represent the fold increase in the percentage of cells with S - phase DNA content ( between 2N and 4N ) but negative for BrdU staining . Bars represent the average of three independent experiments , with error bars showing SD . The p - values are 0 . 0091 ( E ) and 0 . 0007 ( F ) .

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

E, F. Increased S-phase arrest in HUWE1-knockout 293T (E) and HeLa (F) cells. Cycling cells were incubated with BrdU and subjected to BrdU/PI bi-dimensional flow cytometry. Representative flow cytometry profiles are presented in Fig EV1D-F. Bars represent the fold increase in the percentage of cells with S-phase DNA content (between 2N and 4N) but negative for BrdU staining. Bars represent the average of three independent experiments, with error bars showing SD. The p-values are 0.0091 (E) and 0.0007 (F).


G , H . The DNA fiber assay shows reduced replication tract length in HUWE1 - knockout 293T ( G ) and HeLa ( H ) cells in the absence of exogenous DNA damage treatment . The " n " numbers of fibers analyzed ( pooled from three independent experiments ) , as well as the mean + / - SEM , are indicated on the graphs . P - values are 9 . 8x10 - 22 ( G ) and 1 . 0x10 - 10 ( H ) .

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

G, H. The DNA fiber assay shows reduced replication tract length in HUWE1-knockout 293T (G) and HeLa (H) cells in the absence of exogenous DNA damage treatment. The "n" numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. P-values are 9.8x10-22 (G) and 1.0x10-10 (H).


A . HUWE1 levels are efficiently downregulated by two different HUWE1 siRNA oligonucleotides in 8988T cells . B . Quantification of HUWE1 expression following siRNA treatment . Band intensity was quantified using ImageJ software and normalized to Tubulin loading control . The average of three experiments is shown . Error bars indicate SD .

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

A. HUWE1 levels are efficiently downregulated by two different HUWE1 siRNA oligonucleotides in 8988T cells. B. Quantification of HUWE1 expression following siRNA treatment. Band intensity was quantified using ImageJ software and normalized to Tubulin loading control. The average of three experiments is shown. Error bars indicate SD.


C - E . Cell cycle analyses by flow cytometry using BrdU / PI double staining show increased replication arrest in HUWE1 - depleted 8988T cells in the absence of exogenous DNA damage treatment . C . Representative flow cytometry profiles of control and HUWE1 - knockdown cells . R1 - labeled region indicates mid and late S - phase cells ( BrdU - positive , > 2N DNA content ) , while R2 - labeled region indicates S - phase arrested cells ( BrdU - negative , DNA content between 2N and 4N ) .

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

C-E. Cell cycle analyses by flow cytometry using BrdU/PI double staining show increased replication arrest in HUWE1-depleted 8988T cells in the absence of exogenous DNA damage treatment. C. Representative flow cytometry profiles of control and HUWE1-knockdown cells. R1-labeled region indicates mid and late S-phase cells (BrdU-positive, >2N DNA content), while R2-labeled region indicates S-phase arrested cells (BrdU-negative, DNA content between 2N and 4N).


C - E . Cell cycle analyses by flow cytometry using BrdU / PI double staining show increased replication arrest in HUWE1 - depleted 8988T cells in the absence of exogenous DNA damage treatment . C . Representative flow cytometry profiles of control and HUWE1 - knockdown cells . R1 - labeled region indicates mid and late S - phase cells ( BrdU - positive , > 2N DNA content ) , while R2 - labeled region indicates S - phase arrested cells ( BrdU - negative , DNA content between 2N and 4N ) . D . Quantification of S - phase cells . Percentage of cells in R1 region is shown . Bars represent the average of three independent experiments . Error bars indicate SD . P - value is 0 . 0014 . E . Quantification of S - phase arrested cells . Bars represent the fold increase in the percentage of cells in R2 region , normalized to siControl - treated cells . The average of three independent experiments is shown . Error bars indicate SD . P - value is 0 . 0135 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E. Cell cycle analyses by flow cytometry using BrdU/PI double staining show increased replication arrest in HUWE1-depleted 8988T cells in the absence of exogenous DNA damage treatment. C. Representative flow cytometry profiles of control and HUWE1-knockdown cells. R1-labeled region indicates mid and late S-phase cells (BrdU-positive, >2N DNA content), while R2-labeled region indicates S-phase arrested cells (BrdU-negative, DNA content between 2N and 4N). D. Quantification of S-phase cells. Percentage of cells in R1 region is shown. Bars represent the average of three independent experiments. Error bars indicate SD. P-value is 0.0014. E. Quantification of S-phase arrested cells. Bars represent the fold increase in the percentage of cells in R2 region, normalized to siControl-treated cells. The average of three independent experiments is shown. Error bars indicate SD. P-value is 0.0135.


F . DNA fiber experiment showing reduced replication tract length in HUWE1 - depleted HeLa cells in the absence of exogenous DNA damage treatment . The " n " numbers of fibers analyzed ( pooled from two independent experiments ) , as well as the mean , are indicated on the graphs .

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

F. DNA fiber experiment showing reduced replication tract length in HUWE1-depleted HeLa cells in the absence of exogenous DNA damage treatment. The "n" numbers of fibers analyzed (pooled from two independent experiments), as well as the mean, are indicated on the graphs.


G , H . Clonogenic assay showing that HUWE1 knockdown in 8988T cells are sensitive to UV ( G ) , and HU ( H ) . As controls , both cells transfected with non - targeting siRNA ( siControl ) , and non - transfected cells , were used . Note that the siHUWE1 # 2 oligonucleotide shows a stronger downregulation of HUWE1 , and confers increased sensitivity compared to siHUWE1 # 1 . The average of nine experiments is shown . Error bars represent SEM . P - values ( representing the statistical difference between the samples at the highest dose treatment ) are : 4 . 55x10 - 9 ( G ) and 1 . 8x10 - 8 ( H ) .

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

G, H. Clonogenic assay showing that HUWE1 knockdown in 8988T cells are sensitive to UV (G), and HU (H). As controls, both cells transfected with non-targeting siRNA (siControl), and non- transfected cells, were used. Note that the siHUWE1#2 oligonucleotide shows a stronger downregulation of HUWE1, and confers increased sensitivity compared to siHUWE1#1. The average of nine experiments is shown. Error bars represent SEM. P-values (representing the statistical difference between the samples at the highest dose treatment) are: 4.55x10-9 (G) and 1.8x10-8 (H).


I . DNA fiber experiments showing decreased replication tract length following UV - B treatment ( 30J / m2 ) in HUWE1 - depleted HeLa cells . The " n " numbers of fibers analyzed ( pooled from three independent experiments ) , as well as the mean + / - SEM , are indicated on the graphs . P - value is 2 . 11x10 - 20 .

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

I. DNA fiber experiments showing decreased replication tract length following UV-B treatment (30J/m2) in HUWE1-depleted HeLa cells. The "n" numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. P-value is 2.11x10-20.


A . iPOND experiment showing that HUWE1 localizes to replication forks in 293T cells . Binding to nascent DNA was increased after UV exposure . Because of the difficulties in removing all the crosslinks in high molecular weightproteins , HUWE1 is detected as a high molecular weight smear . A control experiment using the HUWE1 - knockout cells ( shown in the right side panel ) confirmed the specificity of the HUWE1 signal .

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

A. iPOND experiment showing that HUWE1 localizes to replication forks in 293T cells. Binding to nascent DNA was increased after UV exposure. Because of the difficulties in removing all the crosslinks in high molecular weightproteins, HUWE1 is detected as a high molecular weight smear. A control experiment using the HUWE1-knockout cells (shown in the right side panel) confirmed the specificity of the HUWE1 signal.


B . Immunofluorescence experiment showing that HUWE1 localizes to chromatin foci in HeLa cells exposed to replication fork stalling agents HU ( 2mM for 16h ) and UV ( 40J / m2 , analyzed 2h later ) .

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

B. Immunofluorescence experiment showing that HUWE1 localizes to chromatin foci in HeLa cells exposed to replication fork stalling agents HU (2mM for 16h) and UV (40J/m2, analyzed 2h later).


C . Co - immunofluorescence experiments showing co - localization of HUWE1 and PCNA in chromatin foci . U2OS cells were analyzed 2h after exposure to UV ( 40J / m2 ) . Quantifications of co - localization , and additional micrographs are presented in Fig EV2A - D .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Co-immunofluorescence experiments showing co-localization of HUWE1 and PCNA in chromatin foci. U2OS cells were analyzed 2h after exposure to UV (40J/m2). Quantifications of co-localization, and additional micrographs are presented in Fig EV2A-D.


B . Anti - PCNA immunoprecipitation , showing that HUWE1 co - precipitates .

B. Anti-PCNA immunoprecipitation, showing that HUWE1 co-precipitates.


C . Reciprocal experiment showing that PCNA co - precipitates with HUWE1 in anti - HUWE1 immunoprecipitation .

C. Reciprocal experiment showing that PCNA co-precipitates with HUWE1 in anti-HUWE1 immunoprecipitation.


D . As control , endogenous HUWE1 was depleted by siRNA treatment , and extracts were subjected to anti - HUWE1 immunoprecipitation . The HUWE1 blot shows that much less HUWE1 is precipitated by anti - HUWE1 antibodies from HUWE1 - knockdown cells than control , as expected . PCNA is co - precipitated by anti - HUWE1 antibodies from control , but not HUWE1 - depleted cells , showing that the interaction is specific and not due to unspecific binding of PCNA to anti - HUWE1

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. As control, endogenous HUWE1 was depleted by siRNA treatment, and extracts were subjected to anti-HUWE1 immunoprecipitation. The HUWE1 blot shows that much less HUWE1 is precipitated by anti-HUWE1 antibodies from HUWE1-knockdown cells than control, as expected. PCNA is co-precipitated by anti-HUWE1 antibodies from control, but not HUWE1-depleted cells, showing that the interaction is specific and not due to unspecific binding of PCNA to anti-HUWE1 antibodies


. E , F . HUWE1 co - precipitates with PCNA in anti - PCNA immunoprecipitation from extracts of MCF7 ( E ) and 8988T ( F ) cells , showing that the HUWE1 - PCNA interaction can be detected in different cell lines .

O O O O O O O O O O O O O O O O O O O O O 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. HUWE1 co-precipitates with PCNA in anti-PCNA immunoprecipitation from extracts of MCF7 (E) and 8988T (F) cells, showing that the HUWE1-PCNA interaction can be detected in different cell lines.


G . Co - immunoprecipitation experiment from control and RAD18 - knockout 293T cells , showing that the HUWE1 - PCNA interaction is not affected by loss of PCNA ubiquitination . RAD18 - knockout cells were obtained by CRISPR / Cas9 technology . The RAD18 and PCNA blots show loss of RAD18 , and of PCNA ubiquitination respectively .

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

G. Co-immunoprecipitation experiment from control and RAD18-knockout 293T cells, showing that the HUWE1-PCNA interaction is not affected by loss of PCNA ubiquitination. RAD18-knockout cells were obtained by CRISPR/Cas9 technology. The RAD18 and PCNA blots show loss of RAD18, and of PCNA ubiquitination respectively.


H . Myc - HUWE1C - ter fragment was transfected in 293T cells . Anti - PCNA immunoprecipitation showed that the C - terminal HUWE1 fragment interacts with PCNA .

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

H. Myc-HUWE1C-ter fragment was transfected in 293T cells. Anti-PCNA immunoprecipitation showed that the C-terminal HUWE1 fragment interacts with PCNA.


I . Coomassie staining of recombinant GST - PCNA expressed and purified from bacteria .

I. Coomassie staining of recombinant GST-PCNA expressed and purified from bacteria.


J . GST - pulldowns using GST - PCNA or GST - empty as control , and extracts of 293T cells transfected with Myc - HUWE1C - ter , showing interaction of this fragment with recombinant PCNA .

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

J. GST-pulldowns using GST-PCNA or GST-empty as control, and extracts of 293T cells transfected with Myc-HUWE1C-ter, showing interaction of this fragment with recombinant PCNA.


A , B . GST - pulldowns showing that HUWE1 PIP - box mutants do not interact with PCNA . A . Recombinant GST - tagged HUWE1C - ter , either wild - type or PIP - box mutant variants ( QVFF and VFF - the indicated residues , critical for the PIP - box , were mutated to A ) , were purified from bacteria ( see Coomassiestaining ) and employed for GST - pulldowns using extracts of 293T cells . PCNA co - precipitated with wild - type , but not PIP - box mutants . B . Reciprocal GST - pulldown using recombinant GST - PCNA and extracts of 293T cells overexpressing Myc - HUWE1C - ter . Wild - type , but not the PIP - box mutant ( VFF ) HUWE1 fragment bound to GST - PCNA .

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

A, B. GST-pulldowns showing that HUWE1 PIP-box mutants do not interact with PCNA. A. Recombinant GST-tagged HUWE1C-ter, either wild-type or PIP-box mutant variants (QVFF and VFF -the indicated residues, critical for the PIP-box, were mutated to A), were purified from bacteria (see Coomassiestaining) and employed for GST-pulldowns using extracts of 293T cells. PCNA co-precipitated with wild-type, but not PIP-box mutants. B. Reciprocal GST-pulldown using recombinant GST-PCNA and extracts of 293T cells overexpressing Myc-HUWE1C-ter. Wild-type, but not the PIP-box mutant (VFF) HUWE1 fragment bound to GST-PCNA.


C , D . Reciprocal co - immunoprecipitationexperiments showing that PIP - box mutation abolishes PCNA interaction . C . 293T cells were transfected with Myc - HUWE1C - ter variants , and subjected to anti - PCNA ( C ) or anti - Myc ( D ) immunoprecipitation . Only wild - type , but not PIP - box mutant ( VFF ) HUWE1 interacted with PCNA in both experimental setups .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D. Reciprocal co-immunoprecipitationexperiments showing that PIP-box mutation abolishes PCNA interaction. C. 293T cells were transfected with Myc-HUWE1C-ter variants, and subjected to anti-PCNA (C) or anti-Myc (D) immunoprecipitation. Only wild-type, but not PIP-box mutant (VFF) HUWE1 interacted with PCNA in both experimental setups.


E . Mutation of the PIP - box in full - length HUWE1 also blocks PCNA interaction . Full - length Myc - tagged HUWE1 ( wild - type or PIP - mutant ) was transfected in 293T cells . Following Myc - immunoprecipitation , endogenous PCNA was detected in wild - type , but not PIP - box mutant ( FF ) complexes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Mutation of the PIP-box in full-length HUWE1 also blocks PCNA interaction. Full-length Myc-tagged HUWE1 (wild-type or PIP-mutant) was transfected in 293T cells. Following Myc-immunoprecipitation, endogenous PCNA was detected in wild-type, but not PIP-box mutant (FF) complexes.


A . Stable expression of Myc - tagged wild - type , PIP - box mutant , and catalytic inactive HUWE1 variants in HUWE1 - knockout 293T cells . The Western blot shows that expression of the Myc - tagged variants stably transfected in the knockout cell lines , is similar to the endogenous HUWE1 expression .

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

A. Stable expression of Myc-tagged wild-type, PIP-box mutant, and catalytic inactive HUWE1 variants in HUWE1-knockout 293T cells. The Western blot shows that expression of the Myc-tagged variants stably transfected in the knockout cell lines, is similar to the endogenous HUWE1 expression.


B . Myc - tag immunoprecipitation using extracts of the corrected cell lines described above . As expected , PCNA co - precipitates with wild - type , but not PIP - box mutant HUWE1 ( FF ) .

O O O O O O O O O O O O O O O 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. Myc-tag immunoprecipitation using extracts of the corrected cell lines described above. As expected, PCNA co-precipitates with wild-type, but not PIP-box mutant HUWE1 (FF).


C . iPOND experiment using HUWE1 - knockout 293T cells , corrected with wild - type or PIP - mutant HUWE1 . The PIP - mutant shows reduced crosslinking to nascent DNA , indicated defective recruitment to replication forks .

O O O O O O O O O O O O O O O O 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. iPOND experiment using HUWE1-knockout 293T cells, corrected with wild-type or PIP-mutant HUWE1. The PIP-mutant shows reduced crosslinking to nascent DNA, indicated defective recruitment to replication forks.


D . Clonogenic assay showing that stable expression of wild - type HUWE1 , but not of the PIP box mutant or the catalytic mutant , corrects the HU sensitivity of the HUWE1 - knockout 293T cells . Shown is the average of 3 independent experiments - / + SD . If not indicated otherwise , the p - values shown specify the statistical significance relative to 293T ( for the 0 . 5mM HU condition ) . P - values are : 0 . 0003 ( 293T vs HUWE1 - / - ; 0 . 157 ( 293T vs HUWE1 - / - + WT ) ; 0 . 0045 ( 293T vs HUWE1 - / - + FF ) ; 0 . 0006 ( 293T vs HUWE1 - / - + C4341A ; 0 . 047 ( HUWE1 - / - + WT vs HUWE1 - / - + FF ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Clonogenic assay showing that stable expression of wild-type HUWE1, but not of the PIP box mutant or the catalytic mutant, corrects the HU sensitivity of the HUWE1-knockout 293T cells. Shown is the average of 3 independent experiments -/+SD. If not indicated otherwise, the p-values shown specify the statistical significance relative to 293T (for the 0.5mM HU condition). P-values are: 0.0003 (293T vs HUWE1-/-; 0.157 (293T vs HUWE1-/- +WT); 0.0045 (293T vs HUWE1-/- +FF); 0.0006 (293T vs HUWE1-/- +C4341A; 0.047 (HUWE1-/- +WT vs HUWE1-/- +FF).


E . The S - phase arrest phenotype is also rescued by wild - type , but not PIP - mutant or catalytic mutant HUWE1 . S - phase arrested cells were quantified using the BrdU / PI bi - dimensional flow cytometry assay . Shown is the average of 3 independent experiments - / + SD . The p - values shown specify the statistical significance relative to 293T ( 0 . 018 , 0 . 957 , 0 . 015 , and 0 . 024 respectively ) . A quantification of the same data , showing the percentage of cells arrested in S - phase , is shown in Fig EV3A .

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

E. The S-phase arrest phenotype is also rescued by wild-type, but not PIP-mutant or catalytic mutant HUWE1. S-phase arrested cells were quantified using the BrdU/PI bi-dimensional flow cytometry assay. Shown is the average of 3 independent experiments -/+SD. The p-values shown specify the statistical significance relative to 293T (0.018, 0.957, 0.015, and 0.024 respectively). A quantification of the same data, showing the percentage of cells arrested in S-phase, is shown in Fig EV3A.


F . Normal replication tract length , quantified using the DNA fiber assay , is restored by expressing wild - type , but not PIP - mutant HUWE1 , in the HUWE1 - knockout 293T cells . The " n " numbers of fibers analyzed ( pooled from three independent experiments ) , as well as the mean + / - SEM , are indicated on the graphs . If not indicated otherwise , the p - values shown specify the statistical significance relative to 293T . P - values are : 1 . 47x10 - 11 ( 293T vs HUWE - / - samples ) , 0 . 094 ( 293T vs HUWE - / - + WT samples ) , 1 . 40x10 - 7 ( 293T vs HUWE - / - + FF samples ) , 1 . 22x10 - 7 ( HUWE - / - vs HUWE - / - + WT samples ) , 0 . 679 ( HUWE - / - vs HUWE - / - + FF samples ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Normal replication tract length, quantified using the DNA fiber assay, is restored by expressing wild-type, but not PIP-mutant HUWE1, in the HUWE1-knockout 293T cells. The "n" numbers of fibers analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. If not indicated otherwise, the p-values shown specify the statistical significance relative to 293T. P-values are: 1.47x10-11 (293T vs HUWE-/- samples), 0.094 (293T vs HUWE-/- +WT samples), 1.40x10-7 (293T vs HUWE-/- +FF samples), 1.22x10-7 (HUWE-/- vs HUWE-/- +WT samples), 0.679 (HUWE-/- vs HUWE-/- +FF samples).


G . Alkaline Comet Assay showing that wild - type , but not the FF mutant can correct the breakage phenotype of HUWE1 - knockout 293T cells . The " n " numbers of comet tails analyzed ( pooled from three independent experiments ) , as well as the mean + / - SEM , are indicated on the graphs . The p - values indicated are : 4 . 18x10 - 43 ( 293T vs HUWE - / - samples ) , 6 . 89x10 - 41 ( HUWE1 - / - + WT vs FF samples ) , and 5 . 97x10 - 50 ( HUWE1 - / - + WT vs C4341A samples ) .

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

G. Alkaline Comet Assay showing that wild-type, but not the FF mutant can correct the breakage phenotype of HUWE1-knockout 293T cells. The "n" numbers of comet tails analyzed (pooled from three independent experiments), as well as the mean +/- SEM, are indicated on the graphs. The p-values indicated are: 4.18x10-43 (293T vs HUWE-/- samples), 6.89x10-41 (HUWE1-/- +WT vs FF samples), and 5.97x10-50 (HUWE1-/- +WT vs C4341A samples).


A , B . The impact of HUWE1 on H2AX phosphorylation and ubiquitination , under normal conditions ( A ) or upon induction of replication stress ( 2mM HU for 18h , or 2h after exposure to 40J / m2 UV ) . Control or HUWE1 - knockout HeLa cells were analyzed .

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

A, B. The impact of HUWE1 on H2AX phosphorylation and ubiquitination, under normal conditions (A) or upon induction of replication stress (2mM HU for 18h, or 2h after exposure to 40J/m2 UV). Control or HUWE1-knockout HeLa cells were analyzed.


C . In vitro ubiquitination assay showing that recombinant HUWE1C - ter can mono - ubiquitinate H2AX .

C. In vitro ubiquitination assay showing that recombinant HUWE1C-ter can mono-ubiquitinate H2AX.


D . Wild - type , but not the PIP - mutant can correct the defective H2AX ubiquitination in HUWE1 - knockout 293T cells .

O 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. Wild-type, but not the PIP-mutant can correct the defective H2AX ubiquitination in HUWE1-knockout 293T cells.


. E . Chromatin immunoprecipitation showing that H2AX binding to the common fragile site FRA3B is reduced in HUWE1 - knockout 8988T cells . Cells were treated with 600nM aphidicolin for 24h . Binding was quantified relative to input material . Shown is the average of 4 experiments - / + SD . P - value is 4 . 4x10 - 6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Chromatin immunoprecipitation showing that H2AX binding to the common fragile site FRA3B is reduced in HUWE1-knockout 8988T cells. Cells were treated with 600nM aphidicolin for 24h. Binding was quantified relative to input material. Shown is the average of 4 experiments -/+ SD. P-value is 4.4x10-6.


F . Western blots showing that several H2AX ubiquitin ligases participate in H2AX ubiquitination following replication stress . HUWE1 , RNF168 , and BMI1 were knocked - down in HeLa cells . The efficiency of the knockdown is shown in Fig EV4 . Cells were treated with 600nM aphidicolin for 24h , 2mM HU for 24h , or analyzed 2h after exposure to 40J / m2 UV .

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

F. Western blots showing that several H2AX ubiquitin ligases participate in H2AX ubiquitination following replication stress. HUWE1, RNF168, and BMI1 were knocked-down in HeLa cells. The efficiency of the knockdown is shown in Fig EV4. Cells were treated with 600nM aphidicolin for 24h, 2mM HU for 24h, or analyzed 2h after exposure to 40J/m2 UV.


G . Chromatin immunoprecipitation from HeLa cells , showing that knockdown of H2AX ubiquitin ligases can reduce H2AX binding to FRA3B . Cells were treated with 600nM aphidicolin for 24h . Binding was quantified relative to input material . Shown is the average of 3 experiments - / + SD . The p - values shown indicate the statistical significance relative to siControl ( 0 . 0016 , 0 . 0005 , and 0 . 0009 respectively ) .

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

G. Chromatin immunoprecipitation from HeLa cells, showing that knockdown of H2AX ubiquitin ligases can reduce H2AX binding to FRA3B. Cells were treated with 600nM aphidicolin for 24h. Binding was quantified relative to input material. Shown is the average of 3 experiments -/+ SD. The p-values shown indicate the statistical significance relative to siControl (0.0016, 0.0005, and 0.0009 respectively).


H . Chromatin fractionation experiments showing that HUWE1 - knockout HeLa cells , treated with 2mM HU for 24h , fail to efficiently recruit BRCA1 and BRCA2 proteins to DNA . In contrast , 53BP1 chromatin association is not induced by HU treatment , and not affected by HUWE1 knockout , and thus can serve as loading control . Shown are blots of whole cell extract ( WCE ) samples , representing input material , and of chromatin pellet samples .

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

H. Chromatin fractionation experiments showing that HUWE1-knockout HeLa cells, treated with 2mM HU for 24h, fail to efficiently recruit BRCA1 and BRCA2 proteins to DNA. In contrast, 53BP1 chromatin association is not induced by HU treatment, and not affected by HUWE1 knockout, and thus can serve as loading control. Shown are blots of whole cell extract (WCE) samples, representing input material, and of chromatin pellet samples.


( A ) Growth curves of S . pombe cultures in minimal medium without ( EMM ) or with antimycin A ( EMM + antA ) . Cultures were supplemented with either the complete amino - acid mix ( all AA mix , black ) , without any amino acids ( no AA , red ) , with arginine only ( R , blue ) , or with individual amino acids other than arginine ( individual AAs , grey ) .

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

(A) Growth curves of S. pombe cultures in minimal medium without (EMM) or with antimycin A (EMM + antA). Cultures were supplemented with either the complete amino-acid mix (all AA mix, black), without any amino acids (no AA, red), with arginine only (R, blue), or with individual amino acids other than arginine (individual AAs, grey).


( B ) Growth curves as in ( A ) for cultures supplemented with complete amino - acid mix ( black ) , without any amino acids ( red ) , or amino - acid mix without arginine ( green ) .

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

(B) Growth curves as in (A) for cultures supplemented with complete amino-acid mix (black), without any amino acids (red), or amino-acid mix without arginine (green).


( C ) Scatter plot of normalised colony sizes of cells grown on EMM against normalised colony sizes of cells grown on EMM supplemented with arginine ( EMM + R ) ( Dataset file 1 ) . Mutants that passed our filtering ( Methods ) and whose colony - size ratios were 30 % bigger when supplemented with arginine are shown in red ; mutants encoding known genes of the arginine biosynthesis pathway are shown with blue circles .

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

(C) Scatter plot of normalised colony sizes of cells grown on EMM against normalised colony sizes of cells grown on EMM supplemented with arginine (EMM+R) (Dataset file 1). Mutants that passed our filtering (Methods) and whose colony-size ratios were 30% bigger when supplemented with arginine are shown in red; mutants encoding known genes of the arginine biosynthesis pathway are shown with blue circles.


( E ) Box plot of mean colony size ratios of mutants grown with vs without arginine for all mutants ( grey ) and respiratory deficient mutants ( orange , based on previous screens ( Malecki & Bähler , 2016 ) ( Welch ' s t - test pval * * < 0 . 002 ) . The same analysis was performed for mutants grown on EMM ( left ; 4 biological repeats , Dataset file 1 ) or on EMM supplemented with antimycin A ( right ; 2 biological repeats , Dataset file 2 ) . Boxplots were created using the R boxplot command with default settings . The central band indicates the median , the upper and lower limits of the box indicate first and third quartiles , and the whiskers show the most extreme data , limited to 1 . 5 - time interquartile range .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Box plot of mean colony size ratios of mutants grown with vs without arginine for all mutants (grey) and respiratory deficient mutants (orange, based on previous screens (Malecki & Bähler, 2016) (Welch's t-test pval** < 0.002). The same analysis was performed for mutants grown on EMM (left; 4 biological repeats, Dataset file 1) or on EMM supplemented with antimycin A (right; 2 biological repeats, Dataset file 2). Boxplots were created using the R boxplot command with default settings. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.


( A ) Normalised concentration values of 16 amino acids in cells grown in EMM without ( grey ) or with ( blue ) antimycin A . For each condition , four independent samples were analysed . Concentrations are presented relative to the mean value for the given amino acid in EMM medium . * Ornithine ( Orn ) is abbreviated as O . Boxplots were created using R boxplot command with default settings . The central band indicates the median , the upper and lower limits of the box indicate first and third quartiles , and the whiskers show the most extreme data , limited to 1 . 5 - time interquartile range .

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

(A) Normalised concentration values of 16 amino acids in cells grown in EMM without (grey) or with (blue) antimycin A. For each condition, four independent samples were analysed. Concentrations are presented relative to the mean value for the given amino acid in EMM medium. *Ornithine (Orn) is abbreviated as O. Boxplots were created using R boxplot command with default settings. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.


( B ) Amino - acid concentrations before and after addition of antimycin A to cells exponentially growing in EMM . Left : heat map showing concentration changes ( log2 fold change ) of each measured amino acid during the time course relative to time point 0 ( before antimycin A addition ) . Right : simplified scheme of amino - acid biosynthetic pathways . Amino acids for which any biosynthetic enzyme has an annotated mitochondrial location are highlighted in yellow . * Ornithine ( Orn ) is abbreviated as O .

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

(B) Amino-acid concentrations before and after addition of antimycin A to cells exponentially growing in EMM. Left: heat map showing concentration changes (log2 fold change) of each measured amino acid during the time course relative to time point 0 (before antimycin A addition). Right: simplified scheme of amino-acid biosynthetic pathways. Amino acids for which any biosynthetic enzyme has an annotated mitochondrial location are highlighted in yellow. *Ornithine (Orn) is abbreviated as O.


( C ) Left panels : cell growth in EMM without ( red ) or with ( blue ) arginine supplementation for five selected respiratory mutants as indicated ( graphs selected from Fig EV3 ) . Right panels : normalised intracellular amino - acid concentrations as in ( A ) for the same 5 respiratory mutants ( blue ) and wild - type control cells ( grey ) . For each mutant and the wild type strain , four independent samples were analysed . * Ornithine ( Orn ) is abbreviated as O . Boxplot features as in ( A ) .

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

(C) Left panels: cell growth in EMM without (red) or with (blue) arginine supplementation for five selected respiratory mutants as indicated (graphs selected from Fig EV3). Right panels: normalised intracellular amino-acid concentrations as in (A) for the same 5 respiratory mutants (blue) and wild-type control cells (grey). For each mutant and the wild type strain, four independent samples were analysed. *Ornithine (Orn) is abbreviated as O. Boxplot features as in (A).


( A ) Normalised amino - acid concentrations in cells grown in EMM without ( grey ) or with arginine ( red ) . For each condition , four independent samples were analysed . Concentrations are presented relative to the mean value for the given amino acid in EMM medium . * Ornithine ( Orn ) is abbreviated as O . The central band indicates the median , the upper and lower limits of the box indicate first and third quartiles , and the whiskers show the most extreme data , limited to 1 . 5 - time interquartile range .

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

(A) Normalised amino-acid concentrations in cells grown in EMM without (grey) or with arginine (red). For each condition, four independent samples were analysed. Concentrations are presented relative to the mean value for the given amino acid in EMM medium. *Ornithine (Orn) is abbreviated as O. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.


( C ) Growth curves of strains deleted for car1 , car2 or car3 , and a wild - type control . Strains were grown in EMM ( black ) , EMM with antimycin A ( red ) , EMM with arginine ( light blue ) or EMM with both arginine and antimycin A ( dark blue ) as indicated .

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

(C) Growth curves of strains deleted for car1, car2 or car3, and a wild-type control. Strains were grown in EMM (black), EMM with antimycin A (red), EMM with arginine (light blue) or EMM with both arginine and antimycin A (dark blue) as indicated.


( D ) Growth curves of strains deleted for car2 ( top ) or car3 ( bottom ) . Strains were grown in EMM ( black ) , EMM with antimycin A ( red ) , EMM with arginine and antimycin A ( dark blue ) , EMM with mix of all amino acids and antimycin A ( bright blue ) , and EMM with antimycin A and each individual amino acid from the mix ( grey ) .

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

(D) Growth curves of strains deleted for car2 (top) or car3 (bottom). Strains were grown in EMM (black), EMM with antimycin A (red), EMM with arginine and antimycin A (dark blue), EMM with mix of all amino acids and antimycin A (bright blue), and EMM with antimycin A and each individual amino acid from the mix (grey).


( E ) Growth curves of wild - type control ( WT ) and car2 deletion strain ( Δcar2 ) . Strains were grown in EMM media with antimycin A and with either a mix of all amino acids ( + AA mix ) or different combinations of lysine ( K ) , glutamine ( Q ) , glutamic acid ( E ) , or arginine ( R ) as indicated . Dashed line : time point where wild - type strain saturates in EMM with amino - acid mix .

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

(E) Growth curves of wild-type control (WT) and car2 deletion strain (Δcar2). Strains were grown in EMM media with antimycin A and with either a mix of all amino acids (+AA mix) or different combinations of lysine (K), glutamine (Q), glutamic acid (E), or arginine (R) as indicated. Dashed line: time point where wild-type strain saturates in EMM with amino-acid mix.


( A ) Changes in transcript abundance 30 and 120 min after addition of antimycin A to exponentially growing cells without ( EMM ) or with arginine ( EMM + R ) . Transcripts annotated as up - or down - regulated Core Environmental Stress Response ( CESR ) are indicated in red and blue , respectively . All data is normalised to EMM time point 0 .

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

(A) Changes in transcript abundance 30 and 120 min after addition of antimycin A to exponentially growing cells without (EMM) or with arginine (EMM+R). Transcripts annotated as up- or down-regulated Core Environmental Stress Response (CESR) are indicated in red and blue, respectively. All data is normalised to EMM time point 0.


( B ) Graphs as in ( A ) showing expression profiles of transcripts encoding enzymes for arginine catabolism ( blue ) or arginine biosynthesis ( red ) .

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

(B) Graphs as in (A) showing expression profiles of transcripts encoding enzymes for arginine catabolism (blue) or arginine biosynthesis (red).


( C ) Heat map of expression changes for transcripts repressed over 2 - fold at 30 min in cells supplemented with arginine ( EMM + R ) relative to cells without supplementation ( EMM ) . Values for timepoints 0 and 30 min after antimycin A addition are shown for cells grown in EMM , EMM with arginine and ssp1 deletion mutant grown in EMM . The data are normalised to EMM timepoint 0 .

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

(C) Heat map of expression changes for transcripts repressed over 2-fold at 30 min in cells supplemented with arginine (EMM +R) relative to cells without supplementation (EMM). Values for timepoints 0 and 30 min after antimycin A addition are shown for cells grown in EMM, EMM with arginine and ssp1 deletion mutant grown in EMM. The data are normalised to EMM timepoint 0.


( D ) Heat map of expression changes of the same genes as in ( B ) after one hour of inhibiting TOR kinase with torin1 or caffeine / rapamycin treatment relative to cell before treatment ; cells were grown in rich media - YES ( data from ( Rodríguez - López et al . , 2019 ) )

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

(D) Heat map of expression changes of the same genes as in (B) after one hour of inhibiting TOR kinase with torin1 or caffeine/rapamycin treatment relative to cell before treatment; cells were grown in rich media - YES (data from (Rodríguez-López et al., 2019))


B ) Volcano plot of quantified proteins . Log2 fold change was determined as the difference between the mean LFQ intensity of the four replicates of telomeric to control sequence and p - values were calculated with a Welch t - test . Proteins above the threshold p = 0 . 05 , s0 = 1 , c = 0 . 5 ( dashed line ) are considered enriched . Abbreviations : LFQ : Label free

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plot of quantified proteins. Log2 fold change was determined as the difference between the mean LFQ intensity of the four replicates of telomeric to control sequence and p-values were calculated with a Welch t-test. Proteins above the threshold p=0.05, s0=1, c=0.5 (dashed line) are considered enriched. Abbreviations: LFQ: Label free quantification