( h ) Wild - type N2 worms or daf - 2 mutants were synchronized to L1 by hypochlorite treatment and placed on nematode growth medium plates with or without bacteria expressing DAF - 16 double - stranded RNA . After five days worms were lysed in imidazole and analysed for glutamine synthetase activity . ( g , h ) Shown are the means of five independent plates for each condition . Uncropped images of blots are shown in Supplementary Fig . S7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wild-type N2 worms or daf-2 mutants were synchronized to L1 by hypochlorite treatment and placed on nematode growth medium plates with or without bacteria expressing DAF-16 double-stranded RNA. After five days worms were lysed in imidazole and analysed for glutamine synthetase activity. (g,h) Shown are the means of five independent plates for each condition. Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) Ba / F3 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT in the presence of mIL - 3 . Cell lysates were analysed for glutamine synthetase ( GS ) activity in an enzyme assay ( left ) . In addition , the expression of glutamine synthetase , p27 and actin was determined using western blot ( right ) . Shown are the mean ± s . e . m . ( n = 3 ) and representative blots of these experiments . * * P0 . 01 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Cell lysates were analysed for glutamine synthetase (GS) activity in an enzyme assay (left). In addition, the expression of glutamine synthetase, p27 and actin was determined using western blot (right). Shown are the mean ± s.e.m. (n=3) and representative blots of these experiments. **P0.01.


( b ) Ba / F3 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT for 16 h in the presence of mIL - 3 together with MSO , as indicated . Cell lysates were analysed for glutamine synthetase activity in an enzyme assay ( left ) . Furthermore the expression levels of glutamine synthetase , p27 and actin were determined using western blot ( right ) . Shown are the mean ± s . e . m . ( n = 3 ) and representative blots of these experiments . * P0 . 05 , * * P0 . 01 .

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 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

(b) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT for 16 h in the presence of mIL-3 together with MSO, as indicated. Cell lysates were analysed for glutamine synthetase activity in an enzyme assay (left). Furthermore the expression levels of glutamine synthetase, p27 and actin were determined using western blot (right). Shown are the mean ±  s.e.m. (n=3) and representative blots of these experiments. *P0.05, **P0.01.


( c ) Ba / F3 cells expressing myrPI ( 3 ) K - ER were cytokine - starved overnight . The next day , cells were washed in PBS and put in a medium without serum , with or without 4 - OHT . At the times indicated , medium samples were taken and analysed for amino acid levels by high - performance liquid chromatography . Cells were lysed and equal amounts of protein were analysed by western blotting for levels of phospho - PKB ( S473 ) , phospho - FOXO3 ( T32 ) and actin ( inset ) . Shown are the mean of relative amino acid levels , compared with t = 0 ( n = 2 ) , and representative blots of these experiments .

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

(c) Ba/F3 cells expressing myrPI(3)K-ER were cytokine-starved overnight. The next day, cells were washed in PBS and put in a medium without serum, with or without 4-OHT. At the times indicated, medium samples were taken and analysed for amino acid levels by high-performance liquid chromatography. Cells were lysed and equal amounts of protein were analysed by western blotting for levels of phospho-PKB (S473), phospho-FOXO3 (T32) and actin (inset). Shown are the mean of relative amino acid levels, compared with t=0 (n=2), and representative blots of these experiments.


( d ) Ba / F3 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT in serum - free medium containing mIL - 3 . The medium was analysed for amino acid levels by high - performance liquid chromatography . Cells were lysed and analysed for protein levels of glutamine synthetase , p27 and actin ( inset ) . Shown are the mean ± s . e . m . of relative amino acid levels , compared with t = 0 ( n = 4 ) , and representative blots of these experiments . * * P0 . 01 . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

(d) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT in serum-free medium containing mIL-3. The medium was analysed for amino acid levels by high-performance liquid chromatography. Cells were lysed and analysed for protein levels of glutamine synthetase, p27 and actin (inset). Shown are the mean ±  s.e.m. of relative amino acid levels, compared with t=0 (n=4), and representative blots of these experiments. **P0.01. Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) DLD1 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT with or without MSO . After 24 h , the cells were starved of serum , amino acids and glucose in D - PBS containing the indicated inhibitors and stimulated with amino acids for 10 min . Cell lysates were analysed for protein levels of phospho - S6K ( Thr 389 ) and actin . Shown are representative blots of three independent experiments .

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 B-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 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

(a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT with or without MSO. After 24 h, the cells were starved of serum, amino acids and glucose in D-PBS containing the indicated inhibitors and stimulated with amino acids for 10 min. Cell lysates were analysed for protein levels of phospho-S6K (Thr 389) and actin. Shown are representative blots of three independent experiments.


( b , c ) DLD1 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT and MSO ( 0 . 25 M ) for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy . ( b ) Shown are representative pictures ( n = 4 ) . Scale bars , 100 μm . ( c ) Quantification of co - localization between mTOR and LAMP2 as seen in b . Depicted are the mean ± s . e . m . of the percentage of co - localized pixels from four experiments . * P0 . 05 , * * P0 . 01 .

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

(b,c) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO (0.25 M) for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy. (b) Shown are representative pictures (n=4). Scale bars, 100 μm. (c) Quantification of co-localization between mTOR and LAMP2 as seen in b. Depicted are the mean ±  s.e.m. of the percentage of co-localized pixels from four experiments. *P0.05, **P0.01.


( d ) DLD1 cells expressing FOXO3 ( A3 ) - ER were treated with or without 4 - OHT , MSO and BafA1 ( 200 nM ) . After 24 h , cell lysates were analysed for protein levels of LC3 and tubulin . Shown are representative blots ( n = 4 ) .

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

(d) DLD1 cells expressing FOXO3(A3)-ER were treated with or without 4-OHT, MSO and BafA1 (200 nM). After 24 h, cell lysates were analysed for protein levels of LC3 and tubulin. Shown are representative blots (n=4).


( e ) DLD1 cells expressing FOXO3 ( A3 ) - ER were transfected with glutamine synthetase ( GS ) siRNA or a non - targeting siRNA and stimulated with 4 - OHT in glutamine - free DMEM containing 0 . 1 % FBS . After 24 h , cell lysates were analysed for protein levels of glutamine synthetase , LC3 and actin . Shown are representative blots ( n = 2 )

O O O O O O 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 O O O O O O O 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) DLD1 cells expressing FOXO3(A3)-ER were transfected with glutamine synthetase (GS) siRNA or a non-targeting siRNA and stimulated with 4-OHT in glutamine-free DMEM containing 0.1% FBS. After 24 h, cell lysates were analysed for protein levels of glutamine synthetase, LC3 and actin. Shown are representative blots (n=2)


( f ) DLD1 cells expressing FOXO3 ( A3 ) - ER were transfected with glutamine synthetasesiRNA or non - template ( NT ) control siRNA and stimulated with 4 - OHT for 16 h . Cells were lysed and equal amounts of proteins were analysed for levels of p62 or glutamine synthetase . Shown are representative blots ( n = 2 ) .

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

(f) DLD1 cells expressing FOXO3(A3)-ER were transfected with glutamine synthetasesiRNA or non-template (NT) control siRNA and stimulated with 4-OHT for 16 h. Cells were lysed and equal amounts of proteins were analysed for levels of p62 or glutamine synthetase. Shown are representative blots (n=2).


( g ) DLD1 cells were stimulated with LY294002 or PKB inhibitor VIII for 24 h . Cell lysates were analysed for protein levels of LC3 and actin . Shown are representative blots ( n = 4 ) . Uncropped images of blots are shown in Supplementary Figs S7 and S8 .

O O O O O O O O B-CONTROLLED_VAR 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

(g) DLD1 cells were stimulated with LY294002 or PKB inhibitor VIII for 24 h. Cell lysates were analysed for protein levels of LC3 and actin. Shown are representative blots (n=4). Uncropped images of blots are shown in Supplementary Figs S7 and S8.


( a ) DLD1 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT and MSO for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . Cells were stained for LC3 and ER and analysed by confocal microscopy . Shown are representative pictures ( n = 3 ) . Scale bars , 20 μm . ( b ) Quantification of LC3 - positive spots as seen in a using ImageJ software . Depicted are mean ± s . e . m . of the number of LC3 - positive spots divided by the number of DAPI - positive cells ( n = 4 ) . * P0 . 05 .

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 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

(a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and ER and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm. (b) Quantification of LC3-positive spots as seen in a using ImageJ software. Depicted are mean ±  s.e.m. of the number of LC3-positive spots divided by the number of DAPI-positive cells (n=4). *P0.05.


( c ) MSCs expressing FOXO3 ( A3 ) - ER were treated with or without 4 - OHT and BafA1 ( 100 nM ) . At the indicated time points , cell lysates were analysed for protein levels of glutamine synthetase ( GS ) , LC3 , pS6 ( Ser 235 / 236 ) and actin . Shown are representative blots ( n = 2 ) .

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 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

(c) MSCs expressing FOXO3(A3)-ER were treated with or without 4-OHT and BafA1 (100 nM). At the indicated time points, cell lysates were analysed for protein levels of glutamine synthetase (GS), LC3, pS6 (Ser 235/236) and actin. Shown are representative blots (n=2).


( d ) IL - 3 was withdrawn from Ba / F3 cells in the presence or absence of BafA1 ( 100 nM ) as indicated . Cell lysates were analysed for protein levels of glutamine synthetase , LC3 and hsp90 . Shown are representative blots ( n = 2 ) .

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

(d) IL-3 was withdrawn from Ba/F3 cells in the presence or absence of BafA1 (100 nM) as indicated. Cell lysates were analysed for protein levels of glutamine synthetase, LC3 and hsp90. Shown are representative blots (n=2).


( e ) DLD1 cells were stimulated with l - glutamine ( l - Gln ) in glutamine - free DMEM containing 0 . 1 % FBS . After 24 h , cell lysates were analysed for protein levels of LC3 , p62 , p - S6K ( Thr 389 ) and actin . Shown are representative blots ( n = 3 ) .

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 O O O O O O O O

(e) DLD1 cells were stimulated with l-glutamine (l-Gln) in glutamine-free DMEM containing 0.1% FBS. After 24 h, cell lysates were analysed for protein levels of LC3, p62, p-S6K (Thr 389) and actin. Shown are representative blots (n=3).


( f ) DLD1 cells were starved and stimulated with glutamine ( 5 and 10 mM ) . Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy , and the co - localization between mTOR and LAMP2 was quantified by ImageJ . Depicted are the means of the percentage of co - localized pixels ( n = 2 ) .

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

(f) DLD1 cells were starved and stimulated with glutamine (5 and 10 mM). Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy, and the co-localization between mTOR and LAMP2 was quantified by ImageJ. Depicted are the means of the percentage of co-localized pixels (n=2).


( g ) DLD1 cells expressing FOXO3 ( A3 ) - ER were transiently transfected with GFP - WIPI - 1 and subsequently stimulated with 4 - OHT in the presence or absence of MSO in glutamine - free DMEM containing 0 . 1 % FCS or medium without amino acids for 24 h . GFP - WIPI - 1 puncta formation analysis was performed by confocal microscopy . Depicted are the percentages of cells positive for GFP - WIPI - 1 puncta . Shown are the mean ± s . e . m . ( n = 4 ) . * P0 . 05 and * * P0 . 01 . Uncropped images of blots are shown in Supplementary Fig . S8 .

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

(g) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-WIPI-1 and subsequently stimulated with 4-OHT in the presence or absence of MSO in glutamine-free DMEM containing 0.1% FCS or medium without amino acids for 24 h. GFP-WIPI-1 puncta formation analysis was performed by confocal microscopy. Depicted are the percentages of cells positive for GFP-WIPI-1 puncta. Shown are the mean ±  s.e.m. (n=4). *P0.05 and **P0.01. Uncropped images of blots are shown in Supplementary Fig. S8.


( a ) DLD1 cells expressing FOXO3 ( A3 ) - ER were transiently transfected with GFP - ULK2 and stimulated with 4 - OHT and MSO for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . Cells were stained for LC3 and analysed by confocal microscopy . Shown are representative pictures ( n = 3 ) . Scale bars , 20 μm .

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 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

(a) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-ULK2 and stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm.


( b ) DLD1 cells expressing FOXO3 ( A3 ) - ER were transiently transfected with GFP - WIPI - 1 and subsequently treated with 4 - OHT in the presence or absence of MSO in glutamine - free DMEM containing 0 . 1 % FCS . After 24 h , cells were analysed for GFP and p62 expression by confocal microscopy . Shown are representative pictures ( n = 3 ) . Scale bar , 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

(b) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-WIPI-1 and subsequently treated with 4-OHT in the presence or absence of MSO in glutamine-free DMEM containing 0.1% FCS. After 24 h, cells were analysed for GFP and p62 expression by confocal microscopy. Shown are representative pictures (n=3). Scale bar, 20 μm.


( c ) DLD1 cells expressing FOXO3 ( A3 ) - ER were treated with 4 - OHT and MSO in glutamine - free DMEM containing 0 . 1 % FCS . After 24 h cells were analysed for endogenous Atg12 puncta formation by confocal microscopy . Shown are the mean ± s . d . ( n = 3 ) . * P0 . 05 .

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 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

(c) DLD1 cells expressing FOXO3(A3)-ER were treated with 4-OHT and MSO in glutamine-free DMEM containing 0.1% FCS. After 24 h cells were analysed for endogenous Atg12 puncta formation by confocal microscopy. Shown are the mean ± s.d. (n=3). *P0.05.


( d ) DLD1 cells expressing FOXO ( A3 ) - ER were transfected with glutamine synthetase ( GS ) siRNA or non - template ( NT ) control siRNA , and stimulated with 4 - OHT for 24 h with or without 3 - methyladenine ( 3 - MA ) . Apoptosis was determined by FACS analysis after labelling cells with annexin V ( AxV ) - phycoerythrin and DAPI . The graph shows relative fold versus control samples , mean ± s . e . m . ( n = 3 ) . * P0 . 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O

(d) DLD1 cells expressing FOXO(A3)-ER were transfected with glutamine synthetase (GS) siRNA or non-template (NT) control siRNA, and stimulated with 4-OHT for 24 h with or without 3-methyladenine (3-MA). Apoptosis was determined by FACS analysis after labelling cells with annexin V (AxV)-phycoerythrin and DAPI. The graph shows relative fold versus control samples, mean ±  s.e.m. (n=3). *P0.05.


A - E Scatter plots showing the association of each of the p - tau biomarkers with age in the Aβ - negative ( A - ; blue ; n = 250 ) and the Aβ - positive ( A + ; red ; n = 131 ) groups . The solid lines indicate the regression line and the 95 % confidence intervals for each of the groups . For each group , the standardized regression coefficients ( β ) and the P - values were computed using a linear model adjusting for sex . Additionally , we computed the ' Age x Aβ status ' interaction term . Abbreviations : CSF , cerebrospinal fluid ; Mid , mid - region ; N , N - terminal ; p - tau , phosphorylated tau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E Scatter plots showing the association of each of the p-tau biomarkers with age in the Aβ-negative (A-; blue; n = 250) and the Aβ-positive (A+; red; n = 131) groups. The solid lines indicate the regression line and the 95% confidence intervals for each of the groups. For each group, the standardized regression coefficients (β) and the P-values were computed using a linear model adjusting for sex. Additionally, we computed the 'Age x Aβ status' interaction term. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; N, N-terminal; p-tau, phosphorylated tau.


( A , B , C , D , E ) Dot and box - plot comparing each of the p - tau biomarker between the Aβ - negative ( A - ; blue ; n = 250 ) and the Aβ - positive ( A + ; red ; n = 131 ) groups . Aβ positivity was defined as CSF Aβ42 / 40 ratio < 0 . 071 .

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

(A, B, C, D, E) Dot and box-plot comparing each of the p-tau biomarker between the Aβ-negative (A-; blue; n = 250) and the Aβ-positive (A+; red; n =131) groups. Aβ positivity was defined as CSF Aβ42/40 ratio < 0.071.


( F , G , H , I , J ) Scatter plots depicting the changes between each p - tau biomarker as a function of CSF Aβ42 / 40 . The horizontal - axes directions were inverted ; lower CSF Aβ42 / 40 ratio reflects higher Aβ pathology . For each Aβ status group , we computed the standardized regression coefficients ( β ) and the P - values , adjusted for age and sex . The solid lines indicate the regression line and the 95 % confidence intervals for each of the Aβ status groups . The dashed green lines indicate the CSF Aβ42 / 40 cutoff . Participants were also colour - coded based on the Aβ PET CL scale ( ≤ 12CL , black ; > 12CL , turquoise ; Aβ PET non - available , 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 B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

(F, G, H, I, J) Scatter plots depicting the changes between each p-tau biomarker as a function of CSF Aβ42/40. The horizontal-axes directions were inverted; lower CSF Aβ42/40 ratio reflects higher Aβ pathology. For each Aβ status group, we computed the standardized regression coefficients (β) and the P-values, adjusted for age and sex. The solid lines indicate the regression line and the 95% confidence intervals for each of the Aβ status groups. The dashed green lines indicate the CSF Aβ42/40 cutoff. Participants were also colour-coded based on the Aβ PET CL scale (≤12CL, black; >12CL, turquoise; Aβ PET non-available, grey).


( A , B , C , D , E ) Dot and box - plot comparing each of the p - tau biomarker between the Aβ negative ( A - ; blue ; n = 287 ) and the Aβ positive ( A + ; red ; n = 42 ) groups . Aβ positivity was defined with Aβ PET visual read .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C, D, E) Dot and box-plot comparing each of the p-tau biomarker between the Aβ negative (A-; blue; n = 287) and the Aβ positive (A+; red; n = 42) groups. Aβ positivity was defined with Aβ PET visual read.


( F , G , H , I , J ) Scatter plots depicting the changes between each p - tau biomarker as a function of Aβ PET Centiloids ( CL ) . The standardized regression coefficients ( β ) and the P - values were computed using a linear model adjusting for age and sex . The solid lines indicate the regression line and the 95 % confidence intervals . The dashed green lines indicate the CL12 and CL30 cutoffs . Participants were also colour - coded based on the CSF Aβ42 / 40 ratio ( A - , black ; A + , turquoise ) .

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

(F, G, H, I, J) Scatter plots depicting the changes between each p-tau biomarker as a function of Aβ PET Centiloids (CL). The standardized regression coefficients (β) and the P-values were computed using a linear model adjusting for age and sex. The solid lines indicate the regression line and the 95% confidence intervals. The dashed green lines indicate the CL12 and CL30 cutoffs. Participants were also colour-coded based on the CSF Aβ42/40 ratio (A-, black; A+, turquoise).


( K , L , M , N , O ) Dot and box - plots depicting comparison between each of the p - tau biomarker between Centiloid scale groups : ( i ) ≤ 12CL ( blue ; n = 278 ) , ( ii ) 12 - 30CL ( subthreshold Aβ pathology group ; grey ; n = 28 ) , ( iii ) > 30CL ( red ; n = 25 ) . The box - plots depict the median ( horizontal bar ) , interquartile range ( IQR , hinges ) and 1 . 5 x IQR ( whiskers ) .

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(K, L, M, N, O) Dot and box-plots depicting comparison between each of the p-tau biomarker between Centiloid scale groups: (i) ≤12CL (blue; n = 278), (ii) 12 - 30CL (subthreshold Aβ pathology group; grey; n = 28), (iii) >30CL (red; n = 25). The box-plots depict the median (horizontal bar), interquartile range (IQR, hinges) and 1.5 x IQR (whiskers).


A - C ROC analysis was performed to test the accuracy to discriminate between Aβ - positive ( A + ) from Aβ - negative ( A - ) individuals . Aβ positivity was defined as CSF Aβ42 / 40 < 0 . 071 ( A ) , Aβ PET positive visual read ( B ) or Aβ PET Centiloid ( CL ) > 12 ( C ) . Abbreviations : CSF , cerebrospinal fluid ; Mid , mid - region ; NfL , neurofilament light ; N , N - terminal ; p - tau , phosphorylated tau ; t - tau , total tau .

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A-C ROC analysis was performed to test the accuracy to discriminate between Aβ-positive (A+) from Aβ-negative (A-) individuals. Aβ positivity was defined as CSF Aβ42/40 < 0.071 (A), Aβ PET positive visual read (B) or Aβ PET Centiloid (CL) > 12 (C). Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; NfL, neurofilament light; N, N-terminal; p-tau, phosphorylated tau; t-tau, total tau.


A - D Dot and box - plot showing the levels of each p - tau biomarker in each of the AT groups . Aβ - positive ( A + ) was defined by a CSF Aβ42 / 40 < 0 . 071 and Tau - positive ( T + ) by an Elecsys CSF Mid - p - tau181 > 24 pg / ml . The box - plots depict the median ( horizontal bar ) , interquartile range ( IQR , hinges ) and 1 . 5 x IQR ( whiskers ) . The horizontal dashed line indicates the median of the p - tau biomarker in the A - T - group . Abbreviations : CSF , cerebrospinal fluid ; Mid , mid - region ; N , N - terminal ; p - tau , phosphorylated tau .

O O O O O O O O O O O O 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 O O 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-D Dot and box-plot showing the levels of each p-tau biomarker in each of the AT groups. Aβ-positive (A+) was defined by a CSF Aβ42/40 < 0.071 and Tau-positive (T+) by an Elecsys CSF Mid-p-tau181 > 24 pg/ml. The box-plots depict the median (horizontal bar), interquartile range (IQR, hinges) and 1.5 x IQR (whiskers). The horizontal dashed line indicates the median of the p-tau biomarker in the A-T- group. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; N, N-terminal; p-tau, phosphorylated tau.


The graphs represent the z - scores changes of each CSF biomarker as a function of CSF Aβ42 / 40 ratio ( as proxy of disease progression ) using a robust local weighted regression method . The z - scores were calculated using the mean and the SD of each CSF biomarker in the A - T - group as a reference . The solid lines depict the trajectory of each CSF biomarker . The dashed lines depict the trajectories of the plasma biomarkers . The vertical black dashed line indicates the CSF Aβ42 / 40 cutoff for A + . Note that the CSF p - tau biomarkers reach the 2 z - scores ( depicted with an horizontal dashed line ) with the following sequence : Mid - p - tau231 , N - p - tau181 , N - p - tau217 , Mid - p - tau181 and t - tau . Abbreviations : CSF , cerebrospinal fluid ; Mid , mid - region ; NfL , Neurofilament light ; N , N - terminal ; p - tau , phosphorylated tau ; t - tau , total tau .

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 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

The graphs represent the z-scores changes of each CSF biomarker as a function of CSF Aβ42/40 ratio (as proxy of disease progression) using a robust local weighted regression method. The z-scores were calculated using the mean and the SD of each CSF biomarker in the A-T- group as a reference. The solid lines depict the trajectory of each CSF biomarker. The dashed lines depict the trajectories of the plasma biomarkers. The vertical black dashed line indicates the CSF Aβ42/40 cutoff for A+. Note that the CSF p-tau biomarkers reach the 2 z-scores (depicted with an horizontal dashed line) with the following sequence: Mid-p-tau231, N-p-tau181, N-p-tau217, Mid-p-tau181 and t-tau. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; NfL, Neurofilament light; N, N-terminal; p-tau, phosphorylated tau; t-tau, total tau.


E . Ectopic expression of CG14044 in the wing pouch ( GFP area indicated by the dotted line ) induces caspase activation and DNA fragmentation ( TUNEL ) in the larval disc , melanization in the pupal wing ( arrow head ) and a structure defect in the adult wing . Data information : Scale bars , 100 μm in E ( cleaved caspase ) and 20 μm in E ( TUNEL ) and 500 μm in E ( pupae and adult wing ) .

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E. Ectopic expression of CG14044 in the wing pouch (GFP area indicated by the dotted line) induces caspase activation and DNA fragmentation (TUNEL) in the larval disc, melanization in the pupal wing (arrow head) and a structure defect in the adult wing. Data information: Scale bars, 100 μm in E (cleaved caspase) and 20 μm in E (TUNEL) and 500 μm in E (pupae and adult wing).


H . Deletion of the BH3 motif suppresses Sayonara - induced caspase activation , which was detected by the caspase sensor GC3Ai . I . Quantification of GC3Ai positive areas with Synr WT and the BH3 motif deletion mutant . Data information : Statistical significance was determined using one - way ANOVA with Dunnett ' s post hoc test . Scale bars , 100 μm

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H. Deletion of the BH3 motif suppresses Sayonara-induced caspase activation, which was detected by the caspase sensor GC3Ai. I. Quantification of GC3Ai positive areas with Synr WT and the BH3 motif deletion mutant. Data information: Statistical significance was determined using one-way ANOVA with Dunnett's post hoc test. Scale bars, 100 μm in


A - D . The Synr - induced wing structural defect is suppressed by knockdown of Debcl , Buffy or DCP1 . Data information : Scale bars , 500 μm in

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A-D. The Synr-induced wing structural defect is suppressed by knockdown of Debcl, Buffy or DCP1. Data information: Scale bars, 500 μm in A


F . Synr - induced caspase activation is suppressed by knockdown of Debcl . G . GC3Ai signals are suppressed by knockdown of Debcl or Buffy Data information : Scale bars , 50 μm in F and

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F. Synr-induced caspase activation is suppressed by knockdown of Debcl. G. GC3Ai signals are suppressed by knockdown of Debcl or Buffy Data information: Scale bars, 50 μm in F and G


H - I . Lysates from HEK293T cells that express Flag - Debcl or Flag - Buffy were incubated with GST or GST - Synr bound to glutathione - Sepharose . Flag - Debcl or Flag - Buffy is pulled down with GST - Synr but not with GST .

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H-I. Lysates from HEK293T cells that express Flag-Debcl or Flag-Buffy were incubated with GST or GST-Synr bound to glutathione-Sepharose. Flag-Debcl or Flag-Buffy is pulled down with GST-Synr but not with GST.


J . Deletion of neither the coiled coil domain nor the BH3 motif suppresses binding Flag - Debcl to GST - Synr .

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J. Deletion of neither the coiled coil domain nor the BH3 motif suppresses binding Flag-Debcl to GST-Synr.


O . Debcl can be photo - crosslinked with Synr at BPA that corresponds to Y124 and F141 . BPA at Y340 was used as a negative control . Red arrowheads indicate covalently crosslinked products . P . Buffy is also photo - crosslinked with Synr at BPA that corresponds to Y124 and F141 . Red arrowheads indicate covalently crosslinked products .

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O. Debcl can be photo-crosslinked with Synr at BPA that corresponds to Y124 and F141. BPA at Y340 was used as a negative control. Red arrowheads indicate covalently crosslinked products. P. Buffy is also photo-crosslinked with Synr at BPA that corresponds to Y124 and F141. Red arrowheads indicate covalently crosslinked products.


R . HA - Debcl is detected after two series of immunoprecipitation . The first immunoprecipitation was performed for Flag - Buffy , followed by Flag peptide - mediated elution and secondary precipitation for Myc - Synr . HA - Debcl can be detected after this sequential immunoprecipitation .

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R. HA-Debcl is detected after two series of immunoprecipitation. The first immunoprecipitation was performed for Flag-Buffy, followed by Flag peptide-mediated elution and secondary precipitation for Myc-Synr. HA-Debcl can be detected after this sequential immunoprecipitation.


A . Ectopic expression of Synr induces autolysosome accumulation , which was detected by Lysotracker , in the wing disc . B . Ectopic expression of Synr also induces autophagosome accumulation , which was detected by mCherry - atg8a . The dotted square regions are magnified in the right pictures . Data information : Scale bars , 100 μm in A and H , 50 μm in

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A. Ectopic expression of Synr induces autolysosome accumulation, which was detected by Lysotracker, in the wing disc. B. Ectopic expression of Synr also induces autophagosome accumulation, which was detected by mCherry-atg8a. The dotted square regions are magnified in the right pictures. Data information: Scale bars, 100 μm in A and H, 50 μm in B


D - F . Knockdown of Atg2 or Atg8a suppresses the wing structural defect that is induced by Synr . Data information : Scale bars , 500 μm in D .

D-F. Knockdown of Atg2 or Atg8a suppresses the wing structural defect that is induced by Synr. Data information: Scale bars, 500 μm in D.


Synr - induced caspase activation is suppressed by inhibition of autophagy ( atg2 , atg8a RNAis ) in a similar manner to apoptosis inhibition ( Debcl , Dcp - 1 RNAis ) . Data information : Scale bars , 100 μm

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Synr-induced caspase activation is suppressed by inhibition of autophagy (atg2, atg8a RNAis) in a similar manner to apoptosis inhibition (Debcl, Dcp-1 RNAis). Data information: Scale bars, 100 μm in


A . Inhibition of Synr suppresses the wing defect that is induced by p53 , a potent apoptosis inducer . B . Inhibition of Synr suppresses p53 - induced caspase activation . Data information : Statistical significance was determined using a two - tailed unpaired t - test ( B ) , Scale bar , 500 μm in

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A. Inhibition of Synr suppresses the wing defect that is induced by p53, a potent apoptosis inducer. B. Inhibition of Synr suppresses p53-induced caspase activation. Data information: Statistical significance was determined using a two-tailed unpaired t-test (B), Scale bar, 500 μm in A


D . Inhibition of Synr in the midgut enterocytes enhances resistance to starvation . E . The synr mutant also exhibits enhanced resistance to starvation . Data information : Statistical significance was determined using a log - rank ( Mantel - Cox ) test ( D - E ) .

D. Inhibition of Synr in the midgut enterocytes enhances resistance to starvation. E. The synr mutant also exhibits enhanced resistance to starvation. Data information: Statistical significance was determined using a log-rank (Mantel-Cox) test (D-E).


( b ) in N2 wild type ( black diamonds ) and ung - 1 mutants ( red squares ) , ( c ) following the depletion of EXO - 3 ( red triangles ) , APN - 1 ( blue circles ) or control RNAi ( L4440 , black diamonds ) in the N2 wild - type background , and ( d ) in msh - 2 ( ok2410 ) ( orange triangles ) , msh - 6 ( pk2504 ) ( green circles ) and mlh - 1 ( ok1917 ) ( grey squares ) mutants .

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(b) in N2 wild type (black diamonds) and ung-1 mutants (red squares), (c) following the depletion of EXO-3 (red triangles), APN-1 (blue circles) or control RNAi (L4440, black diamonds) in the N2 wild-type background, and (d) in msh-2(ok2410) (orange triangles), msh-6(pk2504) (green circles) and mlh-1(ok1917) (grey squares) mutants.


( e ) Western blottings showing expression of MSH - 6 protein in the wild type strain grown on E . coli expressing control ( N2 ) or the indicated RNAi , as well as in msh - 6 ( pk2504 ) and msh - 2 ( ok2410 ) mutants ( top panel ) , and expression of MSH - 2 : : GFP fusion protein in transgenic worms grown on E . coli expressing RNAi for MSH - 2 , MSH - 6 or empty vector control as indicated ( lower panel ) . Actin was used as the loading control .

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(e) Western blottings showing expression of MSH-6 protein in the wild type strain grown on E. coli expressing control (N2) or the indicated RNAi, as well as in msh-6(pk2504) and msh-2(ok2410) mutants (top panel), and expression of MSH-2::GFP fusion protein in transgenic worms grown on E. coli expressing RNAi for MSH-2, MSH-6 or empty vector control as indicated (lower panel). Actin was used as the loading control.


( f ) F1 survival after depletion of MSH - 2 and MSH - 6 in the mlh - 1 ( ok1917 ) mutant . ( g , h ) F1 survival after depletion of the AP endonucleases EXO - 3 and APN - 1 in ( g ) the msh - 6 ( pk2504 ) or ( h ) msh - 2 ( ok2410 ) mutant background . Kruskal - Wallis one - way analysis on ranks ( P = 0 . 963 and P = 0 . 985 in h and g , respectively ) . Mann - Whitney rank - sum test failed to identify a difference in median survival after depleting EXO - 3 in the msh - 6 mutant ( g , P = 0 . 929 ) . ( i ) F1 survival following depletion of the indicated genes in exo - 3 ( tm4374 ) mutants . ( j ) F1 survival in msh - 6 ( pk2504 ) , exo - 3 ( tm4374 ) and exo - 3 ; msh - 6 double mutants . ( k ) F1 survival in exo - 3 ( tm4374 ) , mlh - 1 ( ok1917 ) and exo - 3 ; mlh - 1 double mutants . All survival curves ( b - d , f - k ) show the mean ± s . d . for each data point from three independent experiments .

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(f) F1 survival after depletion of MSH-2 and MSH-6 in the mlh-1(ok1917) mutant. (g,h) F1 survival after depletion of the AP endonucleases EXO-3 and APN-1 in (g) the msh-6(pk2504) or (h) msh-2(ok2410) mutant background. Kruskal-Wallis one-way analysis on ranks (P=0.963 and P=0.985 in h and g, respectively). Mann-Whitney rank-sum test failed to identify a difference in median survival after depleting EXO-3 in the msh-6 mutant (g, P=0.929). (i) F1 survival following depletion of the indicated genes in exo-3(tm4374) mutants. (j) F1 survival in msh-6(pk2504), exo-3(tm4374) and exo-3;msh-6 double mutants. (k) F1 survival in exo-3(tm4374), mlh-1(ok1917) and exo-3;mlh-1 double mutants. All survival curves (b-d,f-k) show the mean±s.d. for each data point from three independent experiments.


( a ) Immunofluorescence images showing anti - RPA - 1 - positive foci in response to 5 - FU after depleting the indicated genes by RNAi or empty vector control RNAi ( L4440 ; scale bars , 5 μm ) .

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(a) Immunofluorescence images showing anti-RPA-1-positive foci in response to 5-FU after depleting the indicated genes by RNAi or empty vector control RNAi (L4440; scale bars, 5 μm).


b ) Quantification of the fraction ( % ) of 5 - FU - treated embryos that contain RPA - 1 - positive foci .

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b) Quantification of the fraction (%) of 5-FU-treated embryos that contain RPA-1-positive foci.


( c ) Quantification of the fraction ( % ) of 5 - FU - treated embryos that contain RPA - 1 - positive foci in N2 , mlh - 1 ( ok1917 ) and exo - 1 ( tm1842 ) mutants fed control ( L4440 ) or RNAi targeting APN - 1 ( apn - 1 ) .

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(c) Quantification of the fraction (%) of 5-FU-treated embryos that contain RPA-1-positive foci in N2, mlh-1(ok1917) and exo-1(tm1842) mutants fed control (L4440) or RNAi targeting APN-1 (apn-1).


( d ) Immunofluorescence showing CHK - 1 phosphorylation at Ser139 in response to 5 - FU in N2 but not in msh - 6 ( pk2504 ) mutants or in N2 after apn - 1 ( RNAi ) ( scale bars , 5 μm ) . ( e ) Quantification of the fraction ( % ) of 5 - FU - treated embryos with phospho - CHK - 1 foci .

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(d) Immunofluorescence showing CHK-1 phosphorylation at Ser139 in response to 5-FU in N2 but not in msh-6(pk2504) mutants or in N2 after apn-1(RNAi) (scale bars, 5 μm). (e) Quantification of the fraction (%) of 5-FU-treated embryos with phospho-CHK-1 foci.


( f ) Western blottings showing H1X . 101 levels in N2 and msh - 6 mutants , treated ( + ) or not ( − ) with 5 - FU . Actin was used as loading control . ( g ) Quantification of H1X . 101 levels relative to actin in control versus 5 - FU - treated worms . ( b , c , e , g ) Bar graphs show the mean ± s . d . from three independent experiments .

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(f) Western blottings showing H1X.101 levels in N2 and msh-6 mutants, treated (+) or not (−) with 5-FU. Actin was used as loading control. (g) Quantification of H1X.101 levels relative to actin in control versus 5-FU-treated worms. (b,c,e,g) Bar graphs show the mean±s.d. from three independent experiments.


a ) Autophagy induction in response to 5 - FU measured in a GFP : : LGG - 1 reporter strain fed control RNAi ( L4440 ) or RNAi targeting the indicated genes ( scale bars , 10 μm ) .

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a) Autophagy induction in response to 5-FU measured in a GFP::LGG-1 reporter strain fed control RNAi (L4440) or RNAi targeting the indicated genes (scale bars, 10 μm).


( b ) Immunofluorescence showing anti - VPS - 34 staining in dissected embryos in the absence or presence of 5 - FU ( scale bar , 5 μm ) .

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(b) Immunofluorescence showing anti-VPS-34 staining in dissected embryos in the absence or presence of 5-FU (scale bar, 5 μm).


( c ) Western blot analysis of embryonic extracts with and without the addition of 5 - FU or the autophagy inhibitor 3 - MA , detecting the ~ 40 - kDa GFP : : LGG - 1 fusion protein and the cleaved ~ 25 - kDa product .

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(c) Western blot analysis of embryonic extracts with and without the addition of 5-FU or the autophagy inhibitor 3-MA, detecting the ~40-kDa GFP::LGG-1 fusion protein and the cleaved ~25-kDa product.


( d ) F1 survival measured after depletion of BEC - 1 ( red triangles ) and ATG - 7 ( blue squares ) as compared with worms fed control RNAi ( black diamonds ) . The survival curve shows the mean ± s . d . for each data point from three independent experiments .

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(d) F1 survival measured after depletion of BEC-1 (red triangles) and ATG-7 (blue squares) as compared with worms fed control RNAi (black diamonds). The survival curve shows the mean±s.d. for each data point from three independent experiments.


( e ) Induction of autophagy ( % GFP - positive embryos ) following depletion of the indicated genes .

(e) Induction of autophagy (% GFP-positive embryos) following depletion of the indicated genes.


( f ) Immunofluorescence showing anti - VPS - 34 staining in N2 and atl - 1 ( tm853 ) embryos in the absence or presence of 5 - FU ( scale bar , 5 μm ) .

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(f) Immunofluorescence showing anti-VPS-34 staining in N2 and atl-1(tm853) embryos in the absence or presence of 5-FU (scale bar, 5 μm).


( g ) The fraction ( as % of untreated control ) of embryos with VPS - 34 - positive foci following 5 - FU treatment .

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(g) The fraction (as % of untreated control) of embryos with VPS-34-positive foci following 5-FU treatment.


( h ) Induction of autophagy ( % GFP - positive embryos ) in control ( GFP : : LGG - 1 ) and atm - 1 ( gk186 ) mutants ( atm - 1 ; GFP : : LGG - 1 ) . ( e , g , h ) Bar graphs represent mean ± s . d . from three independent experiments .

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(h) Induction of autophagy (% GFP-positive embryos) in control (GFP::LGG-1) and atm-1(gk186) mutants (atm-1; GFP::LGG-1). (e,g,h) Bar graphs represent mean±s.d. from three independent experiments.


a ) Induction of autophagy in response to 5 - FU in the GFP : : LGG - 1 reporter strain fed on RNAi targeting the indicated DNA repair genes ( scale bars , 10 μm ) .

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a) Induction of autophagy in response to 5-FU in the GFP::LGG-1 reporter strain fed on RNAi targeting the indicated DNA repair genes (scale bars, 10 μm).


( b ) The fraction ( as % of untreated control ) of hermaphrodites having embryos with excessive GFP expression following 5 - FU treatment . Autophagy induction in independent experiments ( circles ) and the mean ( line ) are presented .

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(b) The fraction (as % of untreated control) of hermaphrodites having embryos with excessive GFP expression following 5-FU treatment. Autophagy induction in independent experiments (circles) and the mean (line) are presented.


( c ) F1 survival after control ( black diamonds ) or bec - 1 RNAi ( red squares ) in N2 and msh - 6 ( pk2504 ) mutants . The survival curve shows the mean ± s . d . for each data point from three independent experiments .

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(c) F1 survival after control (black diamonds) or bec-1 RNAi (red squares) in N2 and msh-6(pk2504) mutants. The survival curve shows the mean±s.d. for each data point from three independent experiments.


( a ) Expression of ATF - 2 : : GFP in 5 - FU - treated hermaphrodites fed control or RNAi targeting the indicated genes ( scale bars , 20 μm ) . ( b ) The number of animals harbouring embryos with excessive ATF - 2 : : GFP expression was scored in at least 150 animals and given as the fraction ( % ) of the total .

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(a) Expression of ATF-2::GFP in 5-FU-treated hermaphrodites fed control or RNAi targeting the indicated genes (scale bars, 20 μm). (b) The number of animals harbouring embryos with excessive ATF-2::GFP expression was scored in at least 150 animals and given as the fraction (%) of the total.


( c ) N2 or msh - 6 ( pk2504 ) embryos were continuously monitored under differential interference contrast microscopy ( scale bars , 5 μm ) from the three - cell stage until completion of the four - cell stage . Magnified images of representative nuclei are shown . ( d ) The fraction of embryos with visible , enlarged nucleoli ( arrows ) after 5 - FU treatment was scored . Bar graphs represent mean ± s . d . from three independent experiments .

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(c) N2 or msh-6(pk2504) embryos were continuously monitored under differential interference contrast microscopy (scale bars, 5 μm) from the three-cell stage until completion of the four-cell stage. Magnified images of representative nuclei are shown. (d) The fraction of embryos with visible, enlarged nucleoli (arrows) after 5-FU treatment was scored. Bar graphs represent mean±s.d. from three independent experiments.


( a ) Western blots showing autophagy induction by monitoring LC3 levels in whole - cell extracts from U2OS cells treated or not with 5 - FU alone or in combination with Mx for the indicated time . Staurosporin was used as a positive control for apoptosis induction shown by the appearance of cleaved fragments of caspase - 3 ( 17 and 19 kDa ) and poly ( ADP - ribose ) polymerase 1 ( PARP1 ; ΔPARP , 85 kDa ) . α − Actin was used as loading control . ( b ) Quantification of LC3 - II levels in cells treated as in a . Intensities of the lower LC3 band ( LC3 - II ) was normalized to those of actin .

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(a) Western blots showing autophagy induction by monitoring LC3 levels in whole-cell extracts from U2OS cells treated or not with 5-FU alone or in combination with Mx for the indicated time. Staurosporin was used as a positive control for apoptosis induction shown by the appearance of cleaved fragments of caspase-3 (17 and 19 kDa) and poly (ADP-ribose) polymerase 1 (PARP1; ΔPARP, 85 kDa). α−Actin was used as loading control. (b) Quantification of LC3-II levels in cells treated as in a. Intensities of the lower LC3 band (LC3-II) was normalized to those of actin.


. ( c ) Autophagy induction demonstrated by accumulation of LC3 - positive puncta in U2OS cells treated or not with 5 - FU alone or in combination with Mx for 48 h . The cells were fixed and prepared for immunofluorescence of LC3 ( green ) . DAPI staining is shown in blue . Scale bar , 20 μm . ( d ) Quantification of LC3 - positive puncta in cells treated as in c . In each separate experiment , 200 - 1 , 600 cells were scored per condition .

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. (c) Autophagy induction demonstrated by accumulation of LC3-positive puncta in U2OS cells treated or not with 5-FU alone or in combination with Mx for 48 h. The cells were fixed and prepared for immunofluorescence of LC3 (green). DAPI staining is shown in blue. Scale bar, 20 μm. (d) Quantification of LC3-positive puncta in cells treated as in c. In each separate experiment, 200-1,600 cells were scored per condition.


e ) Western blots showing absence of LC3 - II accumulation in U2OS cells transfected with siRNA against MSH - 2 . The same blot probed with anti - MSH - 2 antibodies confirm efficient knockdown . ( f ) Quantification of LC3 - II levels in cells transfected with control or MSH - 2 siRNA with or without FU treatment for 24 h .

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e) Western blots showing absence of LC3-II accumulation in U2OS cells transfected with siRNA against MSH-2. The same blot probed with anti-MSH-2 antibodies confirm efficient knockdown. (f) Quantification of LC3-II levels in cells transfected with control or MSH-2 siRNA with or without FU treatment for 24 h.


( g ) Immunofluorescence showing reduced accumulation of LC3 puncta in U2OS cells transfected with siRNA against MSH - 2 . ( h ) Quantification of LC3 - positive puncta in cells treated as in g .

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(g) Immunofluorescence showing reduced accumulation of LC3 puncta in U2OS cells transfected with siRNA against MSH-2. (h) Quantification of LC3-positive puncta in cells treated as in g.


( i ) Quantification of normalized RPS3 protein level ( black bars ) or RPS3 mRNA level ( white bars ) in U2OS cells after 48 h 5 - FU treatment . RPS3 mRNA levels were determined by quantitative real - time reverse transcriptase - PCR .

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(i) Quantification of normalized RPS3 protein level (black bars) or RPS3 mRNA level (white bars) in U2OS cells after 48 h 5-FU treatment. RPS3 mRNA levels were determined by quantitative real-time reverse transcriptase-PCR.


A , PTPN3 promotes TGF - β - induced CAGA - luc reporter gene activity . HaCaT cells were transfected with expression plasmids for CAGA - luc reporter gene , Renilla - luc reporter gene and PTPN3 as indicated and treated with TGF - β ( 2 ng / mL , 8 h ) . Relative luciferase activity was measured Data are shown as mean ± SEM ; n = 3 . Statistical analysis was performed using two - tailed Student ' s t - test . * * * P < 0 . 001 .

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A, PTPN3 promotes TGF-β-induced CAGA-luc reporter gene activity. HaCaT cells were transfected with expression plasmids for CAGA-luc reporter gene, Renilla-luc reporter gene and PTPN3 as indicated and treated with TGF-β (2 ng/mL, 8 h). Relative luciferase activity was measured Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. ***P < 0.001.


, Knockdown of PTPN3 decreases TGF - β - induced CAGA - luc reporter gene activity . HaCaT cells were transfected with siRNA against PTPN3 or control siRNA . 24 h post - transfection , expression plasmids for CAGA - luc reporter and Renilla - luc reporter were transfected as indicated . Cells were harvested for relative luciferase assay after another 24 h with TGF - β treatment ( 2 ng / mL , 8 h ) . Data are shown as mean ± SEM ; n = 3 . Statistical analysis was performed using two - tailed Student ' s t - test . * * * P < 0 . 001 .

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, Knockdown of PTPN3 decreases TGF-β-induced CAGA-luc reporter gene activity. HaCaT cells were transfected with siRNA against PTPN3 or control siRNA. 24 h post-transfection, expression plasmids for CAGA-luc reporter and Renilla-luc reporter were transfected as indicated. Cells were harvested for relative luciferase assay after another 24 h with TGF-β treatment (2 ng/mL, 8 h). Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. ***P < 0.001.


C , Knockdown of PTPN3 blocks TGF - β - induced p21 transcription . HaCaT cells were transfected with two independent siRNAs against PTPN3 and treated with TGF - β ( 2 ng / mL ) for 8 h before harvested . Total mRNA was analyzed by qRT - PCR using primers specific to p21 . Data are shown as mean ± SEM ; n = 3 . Statistical analysis was performed using two - tailed Student ' s t - test . * * P < 0 . 01 , * * * P < 0 . 001 .

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C, Knockdown of PTPN3 blocks TGF-β-induced p21 transcription. HaCaT cells were transfected with two independent siRNAs against PTPN3 and treated with TGF-β (2 ng/mL) for 8 h before harvested. Total mRNA was analyzed by qRT-PCR using primers specific to p21. Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. **P<0.01, ***P < 0.001.


D , Knockdown of PTPN3 blocks TGF - β - induced PAI - 1 gene transcription . Experiment was done with qRT - PCR primers specific to PAI - 1 . Data are shown as mean ± SEM ; n = 3 . Statistical analysis was performed using two - tailed Student ' s t - test . * * * P < 0 . 001 .

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D, Knockdown of PTPN3 blocks TGF-β-induced PAI-1 gene transcription. Experiment was done with qRT-PCR primers specific to PAI-1. Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. ***P < 0.001.


E , Knockdown of PTPN3 abolishes TGF - β - induced p21 transcription . HaCaT cells were transfected with siRNA and then treated with TGF - β ( 2 ng / mL ) for 0 , 4 , 8 or 24 h as indicated . qRT - PCR was carried out Data are shown as mean ± SEM ; n = 3 .

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E, Knockdown of PTPN3 abolishes TGF-β-induced p21 transcription. HaCaT cells were transfected with siRNA and then treated with TGF-β (2 ng/mL) for 0, 4, 8 or 24 h as indicated. qRT-PCR was carried out Data are shown as mean ± SEM; n = 3.


F , Knockdown of PTPN3 abolishes TGF - β - induced transcription of PAI - 1 . Experiment was done Data are shown as mean ± SEM ; n = 3 .

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F, Knockdown of PTPN3 abolishes TGF-β-induced transcription of PAI-1. Experiment was done Data are shown as mean ± SEM; n = 3.


G , Depletion of PTPN3 abolishes TGF - β - induced target protein expression . HaCaT cells with stable knockdown of PTPN3 were treated with TGF - β ( 2 ng / mL ) for 6 h before harvested . Cells were harvested for Western blotting with anti - p21 , anti - PAI - 1 , anti - PTPN3 and anti - β - Actin antibodies .

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G, Depletion of PTPN3 abolishes TGF-β-induced target protein expression. HaCaT cells with stable knockdown of PTPN3 were treated with TGF-β (2 ng/mL) for 6 h before harvested. Cells were harvested for Western blotting with anti-p21, anti-PAI-1, anti-PTPN3 and anti-β-Actin antibodies.


H , PTPN3 enhances TGF - β - induced target protein expression . HaCaT cells stably expressing HA - PTPN3 were stimulated with 2 ng / mL of TGF - β for 6 h and then harvested for cell lysates . Protein levels were examined by Western blotting with appropriate antibodies as indicated .

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H, PTPN3 enhances TGF-β-induced target protein expression. HaCaT cells stably expressing HA-PTPN3 were stimulated with 2 ng/mL of TGF-β for 6 h and then harvested for cell lysates. Protein levels were examined by Western blotting with appropriate antibodies as indicated.


I , Depletion of PTPN3 attenuates TGF - β global gene responses . Venn diagram shows the number of differential expression genes ( DEGs ) with TGF - β treatment in control or PTPN3 - depleted HaCaT cells by RNA - Seq analysis . The overlap represents TGF - β target genes that are commonly regulated in both cell lines . HaCaT cells transfected with siControl or siPTPN3 were harvested for RNA - Seq after treated with or without TGF - β ( 2 ng / mL , 8 h ) .

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I, Depletion of PTPN3 attenuates TGF-β global gene responses. Venn diagram shows the number of differential expression genes (DEGs) with TGF-β treatment in control or PTPN3-depleted HaCaT cells by RNA-Seq analysis. The overlap represents TGF-β target genes that are commonly regulated in both cell lines. HaCaT cells transfected with siControl or siPTPN3 were harvested for RNA-Seq after treated with or without TGF-β (2 ng/mL, 8 h).


J , Loss of PTPN3 markedly decreases TGF - β - dependent upregulation or downregulation of target genes . Heat map of DEGs in control HaCaT cells ( siControl ) or HaCaT cells depleted of PTPN3 ( siPTPN3 ) . Log2FC data represent fold change of TGF - β ( 2 ng / mL ) treatment over vehicle for 8 h .

J, Loss of PTPN3 markedly decreases TGF-β-dependent upregulation or downregulation of target genes. Heat map of DEGs in control HaCaT cells (siControl) or HaCaT cells depleted of PTPN3 (siPTPN3). Log2FC data represent fold change of TGF-β (2 ng/mL) treatment over vehicle for 8 h.


K , Heat map of Log2FC of select TGF - β target genes .

K, Heat map of Log2FC of select TGF-β target genes.


B , Both PTPN3 and PTPN3 ( L232R ) , but not the phosphatase - dead mutants PTPN3 ( D811A ) and PTPN3 ( C842S ) , dephosphorylate P - EGFR ( Y1173 ) . Wildtype PTPN3 or its mutants were co - transfected with EGFR into HEK293T cells as indicated . 24 h later , cells were harvested for Western blotting . Levels of P - EGFR ( Y1173 ) , EGFR , PTPN3 and its mutants were examined by appropriate antibodies .

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B, Both PTPN3 and PTPN3 (L232R), but not the phosphatase-dead mutants PTPN3 (D811A) and PTPN3 (C842S), dephosphorylate P-EGFR (Y1173). Wildtype PTPN3 or its mutants were co-transfected with EGFR into HEK293T cells as indicated. 24 h later, cells were harvested for Western blotting. Levels of P-EGFR (Y1173), EGFR, PTPN3 and its mutants were examined by appropriate antibodies.


C , PTPN3 and PTPN3 ( D811A ) enhance TGF - β - induced target protein expression . HaCaT cells stably expressing HA - PTPN3 or HA - PTPN3 ( D811A ) were stimulated with 2 ng / mL of TGF - β for 6 h and then harvested for cell lysates . Protein levels were examined by Western blotting with appropriate antibodies as indicated .

C, PTPN3 and PTPN3 (D811A) enhance TGF-β-induced target protein expression. HaCaT cells stably expressing HA-PTPN3 or HA-PTPN3 (D811A) were stimulated with 2 ng/mL of TGF-β for 6 h and then harvested for cell lysates. Protein levels were examined by Western blotting with appropriate antibodies as indicated.


D , PTPN3 and its catalytically inactive mutants PTPN3 ( D811A ) and PTPN3 ( C842S ) equally enhance TGF - β - induced reporter gene activity . HaCaT cells were transfected with expression plasmids for each PTPN3 variant and CAGA - luc reporter and Renilla - luc reporter . Cells were harvested for relative luciferase assay after TGF - β ( 2 ng / mL , 8 h ) treatment . Data are shown as mean ± SEM ; n = 3 . Statistical analysis was performed using two - tailed Student ' s t - test . * * * P < 0 . 001 .

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D, PTPN3 and its catalytically inactive mutants PTPN3 (D811A) and PTPN3 (C842S) equally enhance TGF-β-induced reporter gene activity. HaCaT cells were transfected with expression plasmids for each PTPN3 variant and CAGA-luc reporter and Renilla-luc reporter. Cells were harvested for relative luciferase assay after TGF-β (2 ng/mL, 8 h) treatment. Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. ***P < 0.001.


E , PTPN3 FERM domain enhances TGF - β - induced reporter gene activity as potently as wildtype PTPN3 . HaCaT cells were transfected with expression plasmids for PTPN3 truncations and CAGA - luc reporter , and internal control Renilla - luc reporter . Cells were harvested for relative luciferase assay with TGF - β treatment ( 2 ng / mL , 8 h ) . Data are shown as mean ± SEM ; n = 3 . Statistical analysis was performed using two - tailed Student ' s t - test . * * P < 0 . 01 , * * * P < 0 . 001 . NS , non - significant .

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E, PTPN3 FERM domain enhances TGF-β-induced reporter gene activity as potently as wildtype PTPN3. HaCaT cells were transfected with expression plasmids for PTPN3 truncations and CAGA-luc reporter, and internal control Renilla-luc reporter. Cells were harvested for relative luciferase assay with TGF-β treatment (2 ng/mL, 8 h). Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. **P<0.01, ***P<0.001. NS, non-significant.


A , PTPN3 and PTPN3 ( D811A ) stabilize TβRI . TβRI and PTPN3 were detected by Western blotting with appropriate antibodies as indicated in HaCaT cells stably expressing PTPN3 or PTPN3 ( D811A ) .

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A, PTPN3 and PTPN3 (D811A) stabilize TβRI. TβRI and PTPN3 were detected by Western blotting with appropriate antibodies as indicated in HaCaT cells stably expressing PTPN3 or PTPN3 (D811A).


B , shRNA PTPN3 - mediated reduction of endogenous TβRI level is restored by MG132 . HaCaT cells expressing shRNA PTPN3 - 1 , shRNA PTPN3 - 2 or control shRNA were treated with MG132 ( 20 μM ) for 4 h . Cell lysates were analyzed by Western blotting with anti - TβRI , anti - PTPN3 and anti - GAPDH antibodies .

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B, shRNA PTPN3-mediated reduction of endogenous TβRI level is restored by MG132. HaCaT cells expressing shRNA PTPN3-1, shRNA PTPN3-2 or control shRNA were treated with MG132 (20 μM) for 4 h. Cell lysates were analyzed by Western blotting with anti-TβRI, anti-PTPN3 and anti-GAPDH antibodies.


C , PTPN3 rescues the degradation of TβRI induced by Smurf2 . HEK293T cells were transfected with the indicated plasmids . Cells were harvested after 24 h post - transfection . Western blotting was done with the indicated antibodies .

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C, PTPN3 rescues the degradation of TβRI induced by Smurf2. HEK293T cells were transfected with the indicated plasmids. Cells were harvested after 24 h post-transfection. Western blotting was done with the indicated antibodies.


D , PTPN3 and PTPN3 ( D811A ) attenuate ubiquitination of TβRI . HEK293T cells were transfected with the indicated plasmids . 24 h after transfection , IP and immunoblotting analysis were performed with the anti - FLAG antibody . Levels of TβRI ubiquitination and other proteins were examined by Western blotting with the indicated antibodies .

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D, PTPN3 and PTPN3 (D811A) attenuate ubiquitination of TβRI. HEK293T cells were transfected with the indicated plasmids. 24 h after transfection, IP and immunoblotting analysis were performed with the anti-FLAG antibody. Levels of TβRI ubiquitination and other proteins were examined by Western blotting with the indicated antibodies.


E , PTPN3 blocks Smurf2 - mediated ubiquitination of TβRI . HEK293T cells were transfected with the indicated plasmids , and treated with MG132 ( 20 μM , 4 h ) before harvest . TβRI IP was performed with the anti - HA antibody , and TβRI ubiquitination was determined by Western blotting with the anti - Myc antibody . Protein levels were examined by Western blotting with the indicated antibodies .

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E, PTPN3 blocks Smurf2-mediated ubiquitination of TβRI. HEK293T cells were transfected with the indicated plasmids, and treated with MG132 (20 μM, 4 h) before harvest. TβRI IP was performed with the anti-HA antibody, and TβRI ubiquitination was determined by Western blotting with the anti-Myc antibody. Protein levels were examined by Western blotting with the indicated antibodies.


F , Depletion of Smurf2 reverses the shPTPN3 - induced degradation of TβRI . HEK293T cells were transfected with expression plasmids for shPTPN3 , shSmurf2 and HA - TβRI . 48 h post - transfection , cells were harvested for immunoblotting analysis . TβRI level was determined by Western blotting with the anti - HA antibody . Levels of other proteins were done by Western blotting with the indicated antibodies .

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F, Depletion of Smurf2 reverses the shPTPN3-induced degradation of TβRI. HEK293T cells were transfected with expression plasmids for shPTPN3, shSmurf2 and HA-TβRI. 48 h post-transfection, cells were harvested for immunoblotting analysis. TβRI level was determined by Western blotting with the anti-HA antibody. Levels of other proteins were done by Western blotting with the indicated antibodies.


G , Dominant negative Smurf2 mutant C716A prevents shPTPN3 - induced TβRI degradation . HaCaT cells stably expressing shPTPN3 were transfected with FLAG - Smurf2 ( C716A ) . Cells were harvested for Western blotting with anti - TβRI , anti - FLAG , anti - PTPN3 and anti - GAPDH antibodies .

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G, Dominant negative Smurf2 mutant C716A prevents shPTPN3-induced TβRI degradation. HaCaT cells stably expressing shPTPN3 were transfected with FLAG-Smurf2 (C716A). Cells were harvested for Western blotting with anti-TβRI, anti-FLAG, anti-PTPN3 and anti-GAPDH antibodies.


A , PTPN3 interacts with TβRI at endogenous levels . HaCaT cells were harvested after TGF - β treatment ( 2 ng / mL , 1 h ) and then subjected to IP by PTPN3 antibody or control IgG . PTPN3 and TβRI were detected by Western blotting .

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A, PTPN3 interacts with TβRI at endogenous levels. HaCaT cells were harvested after TGF-β treatment (2 ng/mL, 1 h) and then subjected to IP by PTPN3 antibody or control IgG. PTPN3 and TβRI were detected by Western blotting.


B , Both PTPN3 and PTPN3 ( D811A ) interact with TβRI . FLAG - TβRI was co - transfected with HA - PTPN3 or HA - PTPN3 ( D811A ) into HEK293T cells . Cell lysates were harvested and subjected to IP with FLAG antibody . PTPN3 , PTPN3 ( D811A ) and TβRI were detected by Western blotting with appropriate antibodies .

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B, Both PTPN3 and PTPN3 (D811A) interact with TβRI. FLAG-TβRI was co-transfected with HA-PTPN3 or HA-PTPN3 (D811A) into HEK293T cells. Cell lysates were harvested and subjected to IP with FLAG antibody. PTPN3, PTPN3 (D811A) and TβRI were detected by Western blotting with appropriate antibodies.


C , The FERM domain of PTPN3 is necessary for its binding to TβRI . FLAG - TβRI and HA - PTPN3 ( wildtype or truncations ) were co - transfected into HEK293T cells . Cells were harvested after 24 h and subjected to IP by HA antibody . TβRI , PTPN3 and PTPN3 truncations were detected by Western blotting with appropriate antibodies .

C, The FERM domain of PTPN3 is necessary for its binding to TβRI. FLAG-TβRI and HA-PTPN3 (wildtype or truncations) were co-transfected into HEK293T cells. Cells were harvested after 24 h and subjected to IP by HA antibody. TβRI, PTPN3 and PTPN3 truncations were detected by Western blotting with appropriate antibodies.


E , Knockdown of endogenous PTPN3 promotes the Smad7 - TβRI association . HEK293T cells were transfected with the indicated plasmids and treated with MG132 ( 20 µM ) for 4 h before harvested . SFB - Smad7 proteins were retrieved with the Streptavidin pulldown . Levels of TβRI and other proteins were examined by Western blotting with the indicated antibodies .

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E, Knockdown of endogenous PTPN3 promotes the Smad7-TβRI association. HEK293T cells were transfected with the indicated plasmids and treated with MG132 (20 µM) for 4 h before harvested. SFB-Smad7 proteins were retrieved with the Streptavidin pulldown. Levels of TβRI and other proteins were examined by Western blotting with the indicated antibodies.


F , PTPN3 and PTPN3 ( D811A ) abolish the interaction between TβRI and Smurf2 in HEK293T cells . HEK293T cells were co - transfected with plasmids as indicated . 20 h later , cells were treated with MG132 ( 20 μM ) for 4 h , followed by IP and Western blotting .

F, PTPN3 and PTPN3 (D811A) abolish the interaction between TβRI and Smurf2 in HEK293T cells. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 μM) for 4 h, followed by IP and Western blotting.


A , PTPN3 wildtype , phosphatase - dead D811A and ICC - derived mutant L232R exhibit similar patterns of subcellular localization and co - localization with TβRI . HaCaT cells were transfected with HA - TβRI and Flag - PTPN3 ( wildtype or its mutants ) . 24 h later , cells were harvested for immunofluorescence with the indicated antibodies . Scale bar = 10 μm .

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A, PTPN3 wildtype, phosphatase-dead D811A and ICC-derived mutant L232R exhibit similar patterns of subcellular localization and co-localization with TβRI. HaCaT cells were transfected with HA-TβRI and Flag-PTPN3 (wildtype or its mutants). 24 h later, cells were harvested for immunofluorescence with the indicated antibodies. Scale bar = 10 μm.


B , PTPN3 ( L232R ) does not increase the TβRI protein level . TβRI and PTPN3 were determined by Western blotting in HaCaT cells stably expressing PTPN3 , PTPN3 ( D811A ) or PTPN3 ( L232R ) .

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B, PTPN3 (L232R) does not increase the TβRI protein level. TβRI and PTPN3 were determined by Western blotting in HaCaT cells stably expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R).


C , PTPN3 ( L232R ) fails to rescue the Smurf2 - mediated degradation of TβRI . HEK293T cells were transfected with FLAG - TβRI , Myc - Smurf2 and the increasing anount of PTPN3 ( wildtype and mutants ) . Cells were harvested 24 h post - transfection . Protein levels were examined by Western blotting with the indicated antibodies .

C, PTPN3 (L232R) fails to rescue the Smurf2-mediated degradation of TβRI. HEK293T cells were transfected with FLAG- TβRI, Myc-Smurf2 and the increasing anount of PTPN3 (wildtype and mutants). Cells were harvested 24 h post-transfection. Protein levels were examined by Western blotting with the indicated antibodies.


D , PTPN3 ( L232R ) does not block the interaction between TβRI and Smurf2 . HEK293T cells were co - transfected with plasmids as indicated . 20 h later , cells were treated with MG132 ( 20 μM ) for 4 h and analyzed by IP - Western blotting .

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D, PTPN3 (L232R) does not block the interaction between TβRI and Smurf2. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 μM) for 4 h and analyzed by IP-Western blotting.


E , Ectopic expression of PTPN3 wildtype and PTPN3 ( D811A ) , but not PTPN3 ( L232R ) , completely abolishes the Smad7 - TβRI interaction . HEK293T cells were co - transfected with plasmids as indicated . 20 h later , cells were treated with MG132 ( 20 µM ) for 4 h , followed by IP and Western blotting .

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E, Ectopic expression of PTPN3 wildtype and PTPN3 (D811A), but not PTPN3 (L232R), completely abolishes the Smad7-TβRI interaction. HEK293T cells were co-transfected with plasmids as indicated. 20 h later, cells were treated with MG132 (20 µM) for 4 h, followed by IP and Western blotting.


F , PTPN3 and PTPN3 ( D811A ) , but not PTPN3 ( L232R ) , attenuate ubiquitination of TβRI . HEK293T cells were transfected with the indicated plasmids . 24 h after transfection , IP - Western blotting analysis was performed with anti - Flag antibody IP and followed by Western blotting with the indicated antibodies .

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F, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), attenuate ubiquitination of TβRI. HEK293T cells were transfected with the indicated plasmids. 24 h after transfection, IP-Western blotting analysis was performed with anti-Flag antibody IP and followed by Western blotting with the indicated antibodies.


A , PTPN3 and PTPN3 ( D811A ) , but not PTPN3 ( L232R ) , enhance Smad2 and Smad3 phosphorylation . HaCaT cells were stimulated with TGF - β ( 2 ng / mL ) for 1 h . Levels of P - Smad2 , P - Smad3 , Smad2 , Smad3 , PTPN3 and GAPDH were detected by Western blotting .

A, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), enhance Smad2 and Smad3 phosphorylation. HaCaT cells were stimulated with TGF-β (2 ng/mL) for 1 h. Levels of P-Smad2, P-Smad3, Smad2, Smad3, PTPN3 and GAPDH were detected by Western blotting.


B , PTPN3 ( L232R ) inhibits TGF - β - induced reporter gene activity . HepG2 cells were transfected with expression plasmids for PTPN3 , PTPN3 mutants and CAGA - luc reporter , and Renilla - luc reporter . Cells were harvested for luciferase assay after 8 h of TGF - β ( 2 ng / mL ) treatment . Data are shown as mean ± SEM ; n = 3 . Statistical analysis was performed using two - tailed Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 .

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B, PTPN3 (L232R) inhibits TGF-β-induced reporter gene activity. HepG2 cells were transfected with expression plasmids for PTPN3, PTPN3 mutants and CAGA-luc reporter, and Renilla-luc reporter. Cells were harvested for luciferase assay after 8 h of TGF-β (2 ng/mL) treatment. Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. *P<0.05, **P<0.01.


C , PTPN3 and PTPN3 ( D811A ) , but not PTPN3 ( L232R ) , enhance TGF - β - induced growth inhibitory effect . HepG2 cells stably expressing PTPN3 , PTPN3 ( D811A ) or PTPN3 ( L232R ) were treated with or without TGF - β ( 1 ng / mL ) for indicated days . Cells were analyzed for cell proliferation by MTS assay . Data are shown as mean ± SEM ; n = 3 .

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C, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), enhance TGF-β-induced growth inhibitory effect. HepG2 cells stably expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R) were treated with or without TGF-β (1 ng/mL) for indicated days. Cells were analyzed for cell proliferation by MTS assay. Data are shown as mean ± SEM; n = 3.


D , Depletion of PTPN3 attenuates TGF - β - induced growth inhibitory effect . HepG2 cells stably expressing shPTPN3 were treated with or without TGF - β ( 1 ng / mL ) for indicated days . Cells were analyzed for cell proliferation by using MTS assay . Data are shown as mean ± SEM ; n = 3 .

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D, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect. HepG2 cells stably expressing shPTPN3 were treated with or without TGF-β (1 ng/mL) for indicated days. Cells were analyzed for cell proliferation by using MTS assay. Data are shown as mean ± SEM; n = 3.


E , PTPN3 ( L232R ) attenuates TGF - β - induced growth inhibition on colony formation in HepG2 cells . Cells were treated with or without TGF - β ( 1 ng / mL ) treatment for 10 days . Clonogenic assay Quantification of the results in clonogenic assays was analyzed by Image J . Data are shown as mean ± SEM ; n = 3 .

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E, PTPN3 (L232R) attenuates TGF-β-induced growth inhibition on colony formation in HepG2 cells. Cells were treated with or without TGF-β (1 ng/mL) treatment for 10 days. Clonogenic assay Quantification of the results in clonogenic assays was analyzed by Image J. Data are shown as mean ± SEM; n = 3.


F , Depletion of PTPN3 attenuates TGF - β - induced growth inhibitory effect in HepG2 cells . Cells with stable knockdown of PTPN3 were treated with or without TGF - β ( 1 ng / mL ) for 10 days . Clonogenic assay Quantification of the results in clonogenic assays were analyzed by Image J . Knockdown efficiency of PTPN3 in HepG2 cells were analyzed by Western blotting with the anti - PTPN3 antibody . Data are shown as mean ± SEM ; n = 3 . G , Depletion of PTPN3 attenuates TGF - β - induced growth inhibitory effect in Huh7 cells . Experiments and analysis were similarly done as in ( F ) . Data are shown as mean ± SEM ; n = 3 .

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F, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect in HepG2 cells. Cells with stable knockdown of PTPN3 were treated with or without TGF-β (1 ng/mL) for 10 days. Clonogenic assay Quantification of the results in clonogenic assays were analyzed by Image J. Knockdown efficiency of PTPN3 in HepG2 cells were analyzed by Western blotting with the anti-PTPN3 antibody. Data are shown as mean ± SEM; n = 3. G, Depletion of PTPN3 attenuates TGF-β-induced growth inhibitory effect in Huh7 cells. Experiments and analysis were similarly done as in (F). Data are shown as mean ± SEM; n = 3.


A , PTPN3 , PTPN3 ( D811A ) and PTPN3 ( L232R ) are similarly expressed in Huh7 stable cell lines . Expression levels of indicated proteins were detected with appropriate antibodies in Western blotting .

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A, PTPN3, PTPN3 (D811A) and PTPN3 (L232R) are similarly expressed in Huh7 stable cell lines. Expression levels of indicated proteins were detected with appropriate antibodies in Western blotting.


B , PTPN3 and PTPN3 ( D811A ) , but not PTPN3 ( L232R ) , attenuates tumorigenesis . Luciferase - harboring Huh7 tumor cells ( 2 × 106 cells / mouse ) expressing PTPN3 , PTPN3 ( D811A ) or PTPN3 ( L232R ) or empty vector ( served as negative control ) were subcutaneously injected into 5 - week - old nude mice . Twenty days after injection , mice were analyzed by bioluminescence using Xenogen IVIS imaging system . Tumors expressing luciferase is indicated by radiance bar ( p / sec / cm2 / sr ) . C , Quantitation of bioluminescence with data from ( B ) . Data are shown as mean ± SEM ; n = 5 .

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B, PTPN3 and PTPN3 (D811A), but not PTPN3 (L232R), attenuates tumorigenesis. Luciferase-harboring Huh7 tumor cells (2 × 106 cells/mouse) expressing PTPN3, PTPN3 (D811A) or PTPN3 (L232R) or empty vector (served as negative control) were subcutaneously injected into 5-week-old nude mice. Twenty days after injection, mice were analyzed by bioluminescence using Xenogen IVIS imaging system. Tumors expressing luciferase is indicated by radiance bar (p/sec/cm2/sr). C, Quantitation of bioluminescence with data from (B). Data are shown as mean ± SEM; n = 5.


D , Depletion of PTPN3 accelerates tumorigenesis . HepG2 cells stably expressing shPTPN3 or shControl were subcutaneously injected into nude mice . Fifty days after cell implantation , tumors were dissected and photographed .

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D, Depletion of PTPN3 accelerates tumorigenesis. HepG2 cells stably expressing shPTPN3 or shControl were subcutaneously injected into nude mice. Fifty days after cell implantation, tumors were dissected and photographed.


E , Depletion of PTPN3 increased tumor sizes . Tumor volume was measured at indicated days post - implantation . Data are shown as mean ± SEM ; n = 6 for each group . F , Depletion of PTPN3 increased tumor weight . Weight from all tumors was recorded from both control and shPTPN3 groups . Data are shown as mean ± SEM ; n = 6 for each group .

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E, Depletion of PTPN3 increased tumor sizes. Tumor volume was measured at indicated days post-implantation. Data are shown as mean ± SEM; n = 6 for each group. F, Depletion of PTPN3 increased tumor weight. Weight from all tumors was recorded from both control and shPTPN3 groups. Data are shown as mean ± SEM; n = 6 for each group.


, PTPN3 depletion impairs TGF - β signaling in tumors . Levels of PTPN3 , GAPDH and TGF - β signaling associated proteins , such as TβRI , P - Smad2 , P - Smad3 , Smad2 / 3 , Smad4 , PAI - 1 , c - Myc and p15 in tumors were analyzed by Western blotting analysis .

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, PTPN3 depletion impairs TGF-β signaling in tumors. Levels of PTPN3, GAPDH and TGF-β signaling associated proteins, such as TβRI, P-Smad2, P-Smad3, Smad2/3, Smad4, PAI-1, c-Myc and p15 in tumors were analyzed by Western blotting analysis.


H , Clinical significance of PTPN3 expression in LIHC based on TCGA database . In the boxplots , Y - axis represents the expression level of PTPN3 in normal and tumor samples . The boxes show the median ( horizontal line in the box ) ± 1 quartile ; the upper and lower box limits represent the upper and lower quartiles , respectively . The whiskers ( vertical lines ) extend from the hinge to the smallest or larget value within 1 . 5 interquartile range from the box boundaries .

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H, Clinical significance of PTPN3 expression in LIHC based on TCGA database. In the boxplots, Y-axis represents the expression level of PTPN3 in normal and tumor samples. The boxes show the median (horizontal line in the box) ± 1 quartile; the upper and lower box limits represent the upper and lower quartiles, respectively. The whiskers (vertical lines) extend from the hinge to the smallest or larget value within 1.5 interquartile range from the box boundaries.


( b ) Flow cytometry - based stratification of cells in different cell cycle phases and in G0 ( quiescence , 2n DNA - and low RNA content ) , shown for SKOV3 cells in serum starvation conditions at 8 and 96 hours , respectively . Cells were detached , fixed with ethanol and stained with Hoechst 33342 and Pyronin Y prior to flow cytometry analysis .

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(b) Flow cytometry-based stratification of cells in different cell cycle phases and in G0 (quiescence, 2n DNA- and low RNA content), shown for SKOV3 cells in serum starvation conditions at 8 and 96 hours, respectively. Cells were detached, fixed with ethanol and stained with Hoechst 33342 and Pyronin Y prior to flow cytometry analysis.


( c ) Distribution of cell cycle phases in six cell lines exposed to quiescence - inducing conditions ( see panel a ) for 8 and 96 hours , respectively . Each stacked bar plot reports the fraction of cells in G0 , G1 , S and G2 / M phases , estimated using the assay depicted in panel b .

(c) Distribution of cell cycle phases in six cell lines exposed to quiescence-inducing conditions (see panel a) for 8 and 96 hours, respectively. Each stacked bar plot reports the fraction of cells in G0, G1, S and G2/M phases, estimated using the assay depicted in panel b.


( d ) Cell confluence of adherent cell cultures in quiescence - inducing conditions ( i . e . serum starvation and glutamine limitation ) was monitored using time - lapse microscopy in a plate reader ( TECAN Spark 10M ) in 1 . 5 - hour intervals . Contact inhibited cells no longer grow in monolayers and could not be accurately imaged with this approach . Data points and shaded areas indicate mean ± standard deviation ( SD ) over three biological replicates . Approximately 9500 , 4500 , 19500 , 6700 , 19500 and 9500 cells / well were seeded for glutamine limitation and serum starvation of A549 , CCD1070Sk , HCT116 , HFL1 , MCF7 and SKOV3 cells , respectively .

(d) Cell confluence of adherent cell cultures in quiescence-inducing conditions (i.e. serum starvation and glutamine limitation) was monitored using time-lapse microscopy in a plate reader (TECAN Spark 10M) in 1.5-hour intervals. Contact inhibited cells no longer grow in monolayers and could not be accurately imaged with this approach. Data points and shaded areas indicate mean ± standard deviation (SD) over three biological replicates. Approximately 9500, 4500, 19500, 6700, 19500 and 9500 cells/well were seeded for glutamine limitation and serum starvation of A549, CCD1070Sk, HCT116, HFL1, MCF7 and SKOV3 cells, respectively.


( b ) Growth phenotypic changes induced by TMZ treatment in A549 and HCT116 cells grown in full growth medium ( RPMI - 1640 with 5 % dialyzed FBS ) . Cell confluence as a proxy of cell numbers was monitored using automated time - lapse microscopy in a plate reader ( TECAN Spark 10M ) . Data points and shaded areas indicate mean ± SD over three biological replicates . ( c ) Growth phenotypic changes induced by TMZ treatment in quiescence - inducing conditions ( serum starvation ) in A549 and HCT116 cancer cells . Growth was monitored via cell confluence as described in panel b . At 96 hours , cells were stimulated to exit quiescence by replacing serum - free media with fresh growth medium without TMZ ( regrowth ) . Data points and shaded areas indicate mean ± SD over three biological replicates .

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(b) Growth phenotypic changes induced by TMZ treatment in A549 and HCT116 cells grown in full growth medium (RPMI-1640 with 5% dialyzed FBS). Cell confluence as a proxy of cell numbers was monitored using automated time-lapse microscopy in a plate reader (TECAN Spark 10M). Data points and shaded areas indicate mean ± SD over three biological replicates. (c) Growth phenotypic changes induced by TMZ treatment in quiescence-inducing conditions (serum starvation) in A549 and HCT116 cancer cells. Growth was monitored via cell confluence as described in panel b. At 96 hours, cells were stimulated to exit quiescence by replacing serum-free media with fresh growth medium without TMZ (regrowth). Data points and shaded areas indicate mean ± SD over three biological replicates.


( d ) Viability of quiescence - induced cells treated with TMZ , or with TMZ plus 10 µM etomoxir was assessed using a fluorescence imaging assay involving two fluorescent dyes : Hoechst 33342 ( all nuclei ) and propidium iodide ( dead cells ) . Data points and error bars indicate mean ± SD over three biological replicates .

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(d) Viability of quiescence-induced cells treated with TMZ, or with TMZ plus 10 µM etomoxir was assessed using a fluorescence imaging assay involving two fluorescent dyes: Hoechst 33342 (all nuclei) and propidium iodide (dead cells). Data points and error bars indicate mean ± SD over three biological replicates.


( e ) Dynamic changes in the intracellular levels of citrate in A549 and HCT116 cells induced to enter quiescence by serum starvation with or without TMZ . Fold - changes were estimated relative to steady - state levels in full medium Data points and error bars indicate mean ± SD over three biological replicates .

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(e) Dynamic changes in the intracellular levels of citrate in A549 and HCT116 cells induced to enter quiescence by serum starvation with or without TMZ. Fold-changes were estimated relative to steady-state levels in full medium Data points and error bars indicate mean ± SD over three biological replicates.