Steady state of different metabolites obtained from metabolomics were used to derive activity curves for ( A ) Adenylate Kinase ( AK ) ( B ) Uridylate Kinase ( PyrH ) ( C ) Adenylosuccinate Lyase ( PurB ) ( D ) GAR Transformylase ( PurN ) . AK , PyrH PurB appear to follow hyperbolic MM kinetics in vivo , while PurN shows Hill - like dependence with cooperativity . In panel ( A ) , red data points were acquired during growth in acid supplemented M9 medium in absence of external AMP are derived from WT various stabilizing destabilizing point mutations of AK ( Adkar et al . , 2019 ) as well as DHFR mutants used in present study , while black points were derived exclusively from cultures of AK mutants supplemented with 1mM external AMP ( Adkar et al . , 2019 ) . Data points for other panels ( B - D ) are derived from mutant DHFR strains in present study . Since SAICAR , AICAR , GAR FGAR were undetectable in WT , data points in ( C - D ) are relative to those observed in I91L + W133V mutant following 4 hours of growth at 40 ° C . Data points in panels represent metabolite for biological replicates without averaging .

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

Steady state levels of different metabolites obtained from metabolomics were used to derive activity curves for (A) Adenylate Kinase (AK) (B) Uridylate Kinase (PyrH) (C) Adenylosuccinate Lyase (PurB) and (D) GAR Transformylase (PurN). AK, PyrH and PurB appear to follow hyperbolic MM kinetics in vivo, while PurN shows Hill-like dependence with cooperativity. In panel (A), the red data points were acquired during growth in amino acid supplemented M9 medium in the absence of external AMP and are derived from WT and various stabilizing and destabilizing point mutations of AK (Adkar et al., 2019) as well as DHFR mutants used in the present study, while black points were derived exclusively from cultures of AK mutants supplemented with 1mM external AMP (Adkar et al., 2019). Data points for all other panels (B-D) are derived from mutant DHFR strains in the present study. Since SAICAR, AICAR, GAR and FGAR levels were undetectable in WT, all data points in (C-D) are relative to those observed in I91L+W133V mutant following 4 hours of growth at 40°C. Data points in all panels represent metabolite levels for individual biological replicates without averaging.


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

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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

(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 in wild type grown on E . coli expressing control ( N2 ) or indicated RNAi , as well as in msh - 6 ( pk2504 ) msh - 2 ( ok2410 ) mutants ( top panel ) , expression of MSH - 2 : : GFP fusion 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 loading control .

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

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

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

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

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

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

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

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


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

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

(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 ) of fraction ( % ) of 5 - FU - treated with phospho - CHK - 1 foci .

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

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

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

(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 fed control RNAi ( L4440 ) or RNAi targeting indicated genes ( scale bars , 10 μm ) .

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

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 in dissected in absence or presence of 5 - FU ( scale bar , 5 μm ) .

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

(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 of embryonic extracts with without addition of 5 - FU or autophagy inhibitor 3 - MA , detecting ~ 40 - kDa GFP : : LGG - 1 fusion ~ 25 - kDa product .

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

(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 ) ATG - 7 ( blue squares ) as compared with worms fed control RNAi ( black diamonds ) . survival curve shows mean ± s . d . for each data point from three independent experiments .

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

(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 ) following depletion of indicated genes .

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


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

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

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

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

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


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

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

(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 GFP : : LGG - 1 reporter fed on RNAi targeting indicated repair genes ( scale bars , 10 μm ) .

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

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

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

(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 msh - 6 ( pk2504 ) mutants . survival curve shows mean ± s . d . for each data point from three independent experiments .

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

(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 indicated genes ( scale bars , 20 μm ) . ( b ) number of harbouring with excessive ATF - 2 : : GFP expression was scored in at least 150 given as fraction ( % ) of total .

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

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

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 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

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

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

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

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

. (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 transfected with siRNA against MSH - 2 . same blot probed with anti - MSH - 2 antibodies confirm efficient knockdown . ( f ) of LC3 - II in transfected with control or MSH - 2 siRNA with or without FU treatment for 24 h .

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

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 in U2OS transfected with siRNA against MSH - 2 . ( h ) of LC3 - positive in treated as in g .

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

(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 ) of normalized RPS3 level ( black bars ) or RPS3 mRNA level ( bars ) in U2OS after 48 h 5 - FU treatment . RPS3 mRNA 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.


( B ) When transposon insertion libraries subjected to ampicillin resistance selection , a substantial increase in transposon insertion frequency in pstSCA genes was observed in strains containing folding sensor ( pBR322 bla : : Im7 L53A I54A ) relative to a containing just pBR322 . Transposon insertion frequency increased 21 - , 24 - , 8 - fold in pstS , pstA , pstC genes , respectively , compared to insertion frequency found with no ampicillin selection .

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(B) When transposon insertion libraries subjected to ampicillin resistance selection, a substantial increase in transposon insertion frequency in the pstSCA genes was observed in strains containing the protein folding sensor (pBR322 bla::Im7 L53A I54A) relative to a strain containing just pBR322. Transposon insertion frequency increased 21-, 24-, and 8-fold in the pstS, pstA, and pstC genes, respectively, compared to the insertion frequency found with no ampicillin selection.


( A ) To verify that disruption of pst genes is sufficient to increase ampicillin resistance of strains containing pBR322 bla : : Im7 L53A I54A folding biosensor to which of genes in pst operon are sufficient for this effect , pBR322 pBR322 bla : : Im7 L53A I54A were transformed into strains containing in - frame deletion mutants of each of pst genes ( pstS , pstC , pstA , respectively ) . deletion mutants of each of these pst genes showed higher ampicillin resistance than did WT MG1655 in presence of pBR322 bla : : Im7 L53A I54A , but did not shower higher ampicillin resistance in presence of pBR322 .

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(A) To verify that disruption of the pst genes is sufficient to increase ampicillin resistance of strains containing the pBR322 bla::Im7 L53A I54A folding biosensor and to test which of the genes in the pst operon are sufficient for this effect, pBR322 and pBR322 bla::Im7 L53A I54A were transformed into strains containing in-frame deletion mutants of each of the pst genes (pstS, pstC, and pstA, respectively). The deletion mutants of each of these pst genes showed higher ampicillin resistance than did the WT strain MG1655 in the presence of pBR322 bla::Im7 L53A I54A, but did not shower higher ampicillin resistance in the presence of pBR322.


( B ) of β - lactamase ( Bla ) β - lactamase - Im7 L53A I54A were detected by Western blotting using a polyclonal antibody recognizing β - lactamase in WT MG1655 in isogenic strains containing insertions in pstSCA genes ( CL248 ( pstS : : Tn5 ) , CL249 ( pstC : : Tn5 ) , CL250 ( pstA : : Tn5 ) ) . Maltose binding ( MBP ) was used as a control .

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(B) Levels of β-lactamase (Bla) and β-lactamase-Im7 L53A I54A were detected by Western blotting using a polyclonal antibody recognizing β-lactamase in the WT strain MG1655 and in isogenic strains containing insertions in the pstSCA genes (CL248 (pstS::Tn5), CL249 (pstC::Tn5), CL250 (pstA::Tn5)). Maltose binding protein (MBP) was used as a control.


( C ) β - lactamase enzymatic activities present in periplasmic extracts of Im7 folding biosensor were monitored in pst WT pstSCA mutant strains using a spectrophotometric . Data are represented as mean ± from three technical replicates ( unpaired t - , * * P < 0 . 01 , * * * P < 0 . 001 comparison to MG1655 ; ns , not significant ) .

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(C) β-lactamase enzymatic activities present in periplasmic extracts of the Im7 folding biosensor were monitored in pst WT and pstSCA mutant strains using a spectrophotometric assay. Data are represented as mean ± SEM from three technical replicates (unpaired t-test,** P<0.01, *** P<0.001 comparison to MG1655; ns, not significant).


( D ) To if of Im7 L53A I54A itself , in absence of fusion , were also higher in pstA mutant , expression of Im7 L53A I54A from IPTG - inducible plasmid pCDFTrc was induced using IPTG concentrations ranging from 0 - 5 mM . Periplasmic fractions were isolated examined by SDS - PAGE subsequent Coomassie to determine overall expression patterns . Im7 L53A I54A were determined by Western blotting using an antibody for Im7 . MBP was used as a loading control . Im7 L53A I54A was increased in pstA relative to WT control . Coomassie revealed that a number of periplasmic , including UgpB , were also induced in pstA .

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(D) To test if levels of the Im7 L53A I54A protein itself, in the absence of the fusion, were also higher in the pstA mutant, expression of the Im7 L53A I54A protein from the IPTG-inducible plasmid pCDFTrc was induced using IPTG concentrations ranging from 0-5 mM. Periplasmic fractions were isolated and examined by SDS-PAGE and subsequent Coomassie staining to determine overall protein expression patterns. Im7 L53A I54A levels were determined by Western blotting using an antibody for Im7. MBP was used as a loading control. Im7 L53A I54A was increased in the pstA strain relative to the WT control strain. Coomassie staining revealed that a number of periplasmic proteins, including UgpB, were also induced in the pstA strain.


( A ) MG1655 WT , single mutants of pstA ugpB , a double mutant of pstA ugpB were transformed with pBR322 various pBR322 - folding biosensor constructs . transformants were examined by a spot titer on LB agar plates containing various ampicillin concentrations . Strains containing pBR322 bla : : Im7 L53A I54A gain considerable ampicillin resistance in presence of a pstA mutation ( compare uppermost left right panels ) . This resistance is completely lost upon transducing in a ugpB mutation ( compare next panels down ) , showing that ugpB is responsible for resistance gained by pstA mutation .

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(A) MG1655 WT, single mutants of pstA and ugpB, and a double mutant of pstA ugpB were transformed with pBR322 and various pBR322-based folding biosensor constructs. The transformants were examined by a spot titer assay on LB agar plates containing various ampicillin concentrations. Strains containing pBR322 bla::Im7 L53A I54A gain considerable ampicillin resistance in the presence of a pstA mutation (compare uppermost left and right panels). This resistance is completely lost upon transducing in a ugpB mutation (compare next panels down), showing that ugpB is responsible for the resistance gained by the pstA mutation.


( B ) Ampicillin sensitivity conferred by a ugpB deletion mutant ( CL543 ) is complemented by UgpB expression from arabinose inducible expression vector pBAD33 ugpB ( pCL1 ) .

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(B) Ampicillin sensitivity conferred by a ugpB deletion mutant (CL543) is complemented by UgpB expression from the arabinose inducible expression vector pBAD33 ugpB (pCL1).


( C ) UgpB expression is sufficient to lead to higher of Im7 L53A I54A . Im7 L53A I54A UgpB were expressed in a ugpB mutant ( CL543 ) from pCDFTrc pTrc99a vectors , respectively , both of which have an IPTG - inducible promoter . Periplasmic fractions were isolated examined by Western blotting . " - " indicates strains containing empty vector " + " indicates strains containing a recombinant plasmid encoding either Im7 L53A I54A or UgpB . MBP was used as a loading control . Data information : These experiments were repeated twice with similar results one representative is shown .

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(C) UgpB expression is sufficient to lead to higher levels of Im7 L53A I54A protein. Im7 L53A I54A and UgpB were expressed in a ugpB mutant strain (CL543) from pCDFTrc and pTrc99a vectors, respectively, both of which have an IPTG-inducible promoter. Periplasmic fractions were isolated and examined by Western blotting. "-" indicates strains containing empty vector and "+" indicates strains containing a recombinant plasmid encoding either Im7 L53A I54A or UgpB. MBP was used as a loading control. Data information: These experiments were repeated twice with similar results and one representative is shown.


( A ) UgpB overexpression confers bile salt resistance , a ugpB deletion mutant is slightly CHO sensitive .

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(A) UgpB overexpression confers bile salt resistance, and a ugpB deletion mutant is slightly CHO sensitive.


( B ) CHO - induced MDH aggregation analyzed by monitoring scattering .

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(B) CHO-induced MDH protein aggregation analyzed by monitoring light scattering.


( C ) CHO - induced MDH aggregation analyzed by a solubility using centrifugation ( cfg ) at 16 , 000 g for 20 min . supernatant ( Sup ) represents soluble fraction pellet insoluble fraction ; 12 µM MDH was used as a model aggregation - prone substrate ( see Methods ) . Addition of increasing quantities of UgpB increasingly solubilizes MDH . molar ratios of MDH : UgpB that were used ( left to right ) are as follows : 1 : 0 , 1 : 0 . 1 , 1 : 0 . 2 , 1 : 0 . 5 , 1 : 1 . 5 - fold more of pellet fraction was loaded relative to supernatant fraction .

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(C) CHO-induced MDH protein aggregation analyzed by a solubility assay using centrifugation (cfg) at 16,000 g for 20 min. The supernatant (Sup) represents the soluble fraction and the pellet the insoluble fraction; 12 µM MDH was used as a model aggregation-prone substrate (see Methods). Addition of increasing quantities of UgpB increasingly solubilizes MDH. The molar ratios of MDH:UgpB that were used (left to right) are as follows: 1:0, 1:0.1, 1:0.2, 1:0.5, and 1:1. 5-fold more of the pellet fraction was loaded relative to the supernatant fraction.


( D ) supernatant / pellet was performed using a 1 : 1 MDH : chaperone for Spy , SurA , OsmY . pellet fraction was concentrated 5 - fold .

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(D) The supernatant/pellet assay was performed using a 1:1 MDH:chaperone ratio for Spy, SurA, and OsmY. The pellet fraction was concentrated 5-fold.


( E ) 12 µM of MDH alone a 1 : 1 of either MDH : UgpB or MDH : MBP were incubated at 37 ° C for 60 min in presence of CHO then fractionated via centrifugation for use in a solubility as described above ( C ) . pellet fraction was concentrated 5 - fold . In some samples ( + G3P ) , 120 µM of G3P was added prior to addition of any other . Addition of G3P inactivates UgpB ' s ability to inhibit aggregation .

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(E) 12 µM of MDH alone and a 1:1 ratio of either MDH:UgpB or MDH:MBP were incubated at 37°C for 60 min in the presence of CHO and then fractionated via centrifugation for use in a solubility assay as described above (C) . The pellet fraction was concentrated 5-fold. In some samples (+G3P), 120 µM of G3P was added prior to the addition of any other protein. Addition of G3P inactivates UgpB's ability to inhibit protein aggregation.


( F ) 800 μg of E . coli total lysate 60 µM of UgpB / MBP were used for supernatant / pellet . 600 µM of G3P was added before adding . pellet fraction was concentrated 10 - fold . As before , addition of UgpB inhibited aggregation , but only in absence of G3P .

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(F) 800 μg of E. coli total lysate and 60 µM of UgpB/MBP were used for the supernatant/pellet assay. 600 µM of G3P was added before adding proteins. The pellet fraction was concentrated 10-fold. As before, addition of UgpB inhibited aggregation, but only in the absence of G3P.


( G ) anti - aggregation activity of tryptophan single mutant ( W169S ) double mutant ( W169S / W172S ) was examined by CHO - induced MDH aggregation . scattering values were monitored .

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(G) The anti-aggregation activity of tryptophan single mutant (W169S) and double mutant (W169S/W172S) was examined by CHO-induced MDH aggregation assay. Light scattering values were monitored.


( I ) anti - aggregation activity of UgpB variants ( E66A R374V ) was examined by CHO - induced MDH aggregation . CHO - induced MDH aggregation analyzed by monitoring scattering .

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(I) The anti-aggregation activity of the UgpB variants (E66A and R374V) was examined by CHO-induced MDH aggregation assay. CHO-induced MDH protein aggregation analyzed by monitoring light scattering.


( J ) Solubility of MDH is also monitored by supernatant / pellet . 12 µM of MDH alone a 1 : 1 of MDH : UgpB , 120 µM of G3P were used . 15 mM CHO were used for aggregation supernatant / pellet assays .

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(J) Solubility of MDH is also monitored by supernatant/pellet assay. 12 µM of MDH alone and a 1:1 ratio of MDH:UgpB proteins, 120 µM of G3P were used. 15 mM CHO were used for all the aggregation and supernatant/pellet assays.


( J ) Solubility of MDH is also monitored by supernatant / pellet . 12 µM of MDH alone a 1 : 1 of MDH : UgpB , 120 µM of G3P were used .

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(J) Solubility of MDH is also monitored by supernatant/pellet assay. 12 µM of MDH alone and a 1:1 ratio of MDH:UgpB proteins, 120 µM of G3P were used.


( A ) Acid denatures UgpB . secondary structure of 2 µM UgpB was monitored by circular dichroism ( CD ) spectroscopy in 10 mM sodium phosphate buffer at pH 2 pH 7 .

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(A) Acid denatures UgpB. The secondary structure of 2 µM UgpB was monitored by circular dichroism (CD) spectroscopy in 10 mM sodium phosphate buffer at pH 2 and pH 7.


( B ) G3P binds to acid - stripped UgpB as observed by monitoring tryptophan spectrum , either at pH7 or after transition from pH 2 to 7 ( see Methods ) . Transitions from pH 2 to 7 in presence of G3P are accompanied by a blue shift , indicating G3P binding .

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(B) G3P binds to acid-stripped UgpB as observed by monitoring the tryptophan fluorescence spectrum, either at pH7 or after transition from pH 2 to 7 (see Methods). Transitions from pH 2 to 7 in the presence of G3P are accompanied by a blue shift, indicating G3P binding.


( C ) anti - aggregation activity of UgpB is monitored by CHO - induced MDH aggregation . scattering values were monitored . anti - aggregation activity of UgpB is activated after transition from pH 2 to 7 addition of G3P ( 120 µM ) can again inactivate its chaperone activity .

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(C) The anti-aggregation activity of UgpB is monitored by CHO-induced MDH aggregation assay. Light scattering values were monitored. The anti-aggregation activity of UgpB is activated after transition from pH 2 to 7 and the addition of G3P (120 µM) can again inactivate its chaperone activity.


( D ) Addition of G3P to UgpB inhibits its anti - aggregation chaperone activity . 12 µM of MDH a 1 : 1 of UgpB : MDH were used in a CHO - induced aggregation . At 30 min , indicated concentrations of G3P were added to solutions containing MDH , MDH aggregation was monitored by scattering . Note that G3P binding to UgpB still occurs in presence of 15 mM CHO as monitored by tryptophan

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(D) Addition of G3P to UgpB inhibits its anti-aggregation chaperone activity . 12 µM of MDH and a 1:1 ratio of UgpB:MDH were used in a CHO-induced aggregation assay. At 30 min, the indicated concentrations of G3P were added to solutions containing MDH, and MDH aggregation was monitored by light scattering. Note that G3P binding to UgpB still occurs in the presence of 15 mM CHO as monitored by tryptophan fluorescence


( D ) Addition of G3P to UgpB inhibits its anti - aggregation chaperone

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(D) Addition of G3P to UgpB inhibits its anti-aggregation chaperone activity


B Growth curves of indicated strains shown as population doublings ( pds ) on y axis hours in liquid culture on x axis . Before inoculation in liquid YES medium , single underwent approximately 25 pds to produce visible colonies on solid medium . For each , averages SDs ( error bars ) of three biological replicates are shown . Arrows indicate times of harvesting corresponding pds for experiments shown in ( C ) ( D ) .

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B Growth curves of the indicated strains shown as population doublings (pds) on the y axis and hours in liquid culture on the x axis. Before inoculation in liquid YES medium, single cells underwent approximately 25 pds to produce visible colonies on solid medium. For each strain, averages and SDs (error bars) of three biological replicates are shown. Arrows indicate times of harvesting and corresponding pds for experiments shown in (C) and (D).


C Telo - PCR of telomere in wt ( ter1 + est1 + ) two independent clones ( a b ) deleted for ter1 + or est1 + grown for ~ 25 + 10 ( ~ 35 ) , ~ 25 + 14 ( ~ 39 ) ~ 25 + 18 ( ~ 43 ) pds in YES medium . PCRs were performed with subtelomeric oligonucleotide oF5 , located 620 bp upstream of first telomeric repeat , oligo ( dG ) - containing oligonucleotide odG18 . Marker ( m ) molecular weights are on left in kilobases . tels : telomere - containing Telo - PCR products .

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C Telo-PCR analysis of telomere length in wt (ter1+ and est1+) and two independent clones (a and b) deleted for ter1+ or est1+ grown for ~ 25+10 (~ 35), ~ 25+14 (~ 39) and ~ 25+18 (~ 43) pds in YES medium. PCRs were performed with the subtelomeric oligonucleotide oF5, located 620 bp upstream of the first telomeric repeat, and the oligo(dG)-containing oligonucleotide odG18. Marker (m) molecular weights are on the left in kilobases. tels: telomere-containing Telo-PCR products.


D qRT - PCR of G - rich TERRA , polyA + TERRA ARRET / αARRET in as in ( B ) . Values are normalized to ACT1 mRNA expressed as fold increase over control wt strains ( ter1 + est1 + ) grown in parallel . Bars error bars are averages SD from 4 ( ter1Δ clones ) or 3 ( est1Δ clones ) independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( relative to wt ; two - Student ' s t ) .

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D qRT-PCR analysis of G-rich TERRA, polyA+ TERRA and ARRET/αARRET levels in cells as in (B). Values are normalized to ACT1 mRNA and expressed as fold increase over control wt strains (ter1+ and est1+) grown in parallel. Bars and error bars are averages and SD from 4 (ter1Δ clones) or 3 (est1Δ clones) independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to wt; two-tailed Student's t test).


E ChIP experiments with anti - polymerase II ( RPII ) antibodies extracts from ter1 + ter1Δ grown for ~ 43 pds . Quantitative PCRs were performed using oligonucleotides oF1 + oR1 ( subtel ) or oligonucleotides amplifying a region from actively transcribed act1 + locus . Values are expressed as fold increase over ter1 + . Bars error bars are averages SD from three independent experiments . * P < 0 . 05 ( relative to ter1 + ; two - Student ' s t ) . western blot to right shows no difference in RNPII between ter1 + ter1Δ used in ChIP experiments . Ponceau Sstaining serves as loading control .

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E ChIP experiments with anti-RNA polymerase II (RPII) antibodies and extracts from ter1+ and ter1Δ cells grown for ~ 43 pds. Quantitative PCRs were performed using oligonucleotides oF1 + oR1 (subtel) or oligonucleotides amplifying a region from the actively transcribed act1+ locus. Values are expressed as fold increase over ter1+. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to ter1+; two-tailed Student's t test). The western blot to the right shows no difference in RNPII levels between ter1+ and ter1Δ cells used in ChIP experiments. Ponceau Sstaining serves as loading control.


G Western blot of fractions from ter1 + ter1Δ grown for ~ 43 pds . Total ( tot ) equivalents of soluble ( sol ) insoluble ( ins ) were loaded . Actin serves as a control for soluble , H3K9ac for - bound .

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G Western blot analysis of cellular fractions from ter1+ and ter1Δ cells grown for ~ 43 pds. Total proteins (tot) and equivalents of soluble (sol) and insoluble (ins) proteins were loaded. Actin serves as a control for soluble proteins, H3K9ac for chromatin-bound proteins.


H qRT - PCR of total TERRA polyA + TERRA in fractions as in ( G ) . Values are expressed after normalization to total extracts . Bars error bars are averages SD from 3 independent experiments .

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H qRT-PCR analysis of total TERRA and polyA+ TERRA in cellular fractions as in (G). Values are expressed after normalization to total cellular extracts. Bars and error bars are averages and SD from 3 independent experiments.


A RIP experiments were performed using anti - myc antibodies extracts from Trt1 - myc expressing strains followed by qRT - PCR to detect G - rich TERRA , polyA + TERRA , ARRET , TER1 ACT1 . Values represent fractions of input detected in immunoprecipitated material expressed as fold increase over untagged ( unt ) wt . Bars error bars are averages SD from three independent experiments . * P < 0 . 05 ( relative to unt ; two - Student ' s t ) .

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 I-MEASURED_VAR B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains followed by qRT-PCR to detect G-rich TERRA, polyA+ TERRA, ARRET, TER1 and ACT1. Values represent fractions of input RNA detected in immunoprecipitated material expressed as fold increase over untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student's t test).


B RIP experiments as in ( A ) where , prior to extraction , washed beads were resuspended in DNaseI buffer ( DNbuff ) incubated for 1 hour at 30 ° C in presence or absence of DNaseI . Values represent fractions of input polyA + TERRA detected in immunoprecipitated material expressed as fold increase over an untagged ( unt ) wt . Bars error bars are averages SD from three independent experiments . * P < 0 . 05 ( relative to unt ; two - Student ' s t ) .

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

B RIP experiments as in (A) where, prior to RNA extraction, washed beads were resuspended in DNaseI buffer (DNbuff) and incubated for 1 hour at 30° C in presence or absence of DNaseI. Values represent fractions of input polyA+ TERRA detected in immunoprecipitated material expressed as fold increase over an untagged (unt) wt strain. Bars and error bars are averages and SD from three independent experiments. * P < 0.05 (relative to unt; two-tailed Student's t test).


C RIP experiments were performed using anti - myc antibodies extracts from Trt1 - myc expressing strains ( either ter1 + or ter1Δ ) cultured in YES medium for ~ 43 pds . Values represent polyA + TERRA detected in immunoprecipitated material expressed as a fraction of input ( graph on left ) or after normalization to ACT1 in corresponding input ( on right ) , expressed as fold increase over an untagged wt ( unt ter1 + ) . Bars error bars are averages SD from three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( relative to unt ter1 + ; two - Student ' s t ) .

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C RIP experiments were performed using anti-myc antibodies and extracts from Trt1-myc expressing strains (either ter1+ or ter1Δ) cultured in YES medium for ~ 43 pds. Values represent polyA+ TERRA detected in immunoprecipitated material expressed as a fraction of the input (graph on the left) or after normalization to ACT1 RNA in the corresponding input (on the right), and expressed as fold increase over an untagged wt strain (unt ter1+). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to unt ter1+; two-tailed Student's t test).


D Western blot of Trt1 - myc in used in ( C ) . Ponceau S serves as a loading control .

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D Western blot analysis of Trt1-myc in cells used in (C). Ponceau S staining serves as a loading control.


B Genomic from wt ( CAF13 ) carrying two tiTELs ( CAF110 ) maintained in YES medium was digested with HindIII Southern blot - hybridized consecutively with indicated probes . RET refers to telomeric sequences ( tiTELs or natural telomeres , nTELs ) retained in upper part of gel ; REL refers to telomeric sequences released in lower part of gel . black arrow points to natural nmt1 + on chromosome III . Marker molecular weights are on left in kilobases .

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B Genomic DNA from wt cells (CAF13) and cells carrying two tiTELs (CAF110) maintained in YES medium was digested with HindIII and Southern blot-hybridized consecutively with the indicated probes. RET refers to telomeric sequences (tiTELs or natural telomeres, nTELs) retained in the upper part of the gel; REL refers to telomeric sequences released in the lower part of the gel. The black arrow points to the natural nmt1+ sequence on chromosome III. Marker molecular weights are on the left in kilobases.


D qRT - PCR of tiTERRA in carrying no tiTEL ( CAF13 ) , one tiTEL ( CAF545 ) or two tiTELs ( CAF110 ) cultured in EMM medium in presence or absence of Thiamine ( THI ; repressed induced condition , respectively ) for 24 h . was reverse transcribed with oC cDNA was amplified with oF3 oR3 oligonucleotides to detect only tiTERRA ( graph on left ) or with oF1 oR1 oligonucleotides to detect simultaneously tiTERRA natural TERRA ( on right ) . Values are normalized to ACT1 mRNA expressed as fold increase over CAF545 in uninduced conditions ( THI + ) . Bars error bars are averages SD from 3 independent experiments . * P < 0 . 05 , * * P < 0 . 01 ( relative to CAF545 THI + ; two - Student ' s t ) .

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D qRT-PCR analysis of tiTERRA in cells carrying no tiTEL (CAF13), one tiTEL (CAF545) or two tiTELs (CAF110) cultured in EMM medium in presence or absence of Thiamine (THI; repressed and induced condition, respectively) for 24 h. RNA was reverse transcribed with oC and cDNA was amplified with oF3 and oR3 oligonucleotides to detect only tiTERRA (graph on the left) or with oF1 and oR1 oligonucleotides to detect simultaneously tiTERRA and natural TERRA (on the right). Values are normalized to ACT1 mRNA and expressed as fold increase over CAF545 in uninduced conditions (THI+). Bars and error bars are averages and SD from 3 independent experiments. * P < 0.05, ** P < 0.01 (relative to CAF545 THI+; two-tailed Student's t test).


E Northern blot of total from wt carrying two tiTELs ( CAF110 ) treated with combinations of THI Trichostatin A ( TSA ) for 24 h . Two identical membranes were hybridized in parallel with oligonucleotides corresponding to C - rich ( oC ) or G - rich ( oG ) telomeric repeats . To confirm similar hybridization efficiencies of two oligonucleotides , northern blot membranes were simultaneously hybridized exposed along with blots of digested genomic ( upper right insets ) . After signal detection , RNAmembranes were stripped re - hybridized with U6 probes to assure equal loading . Marker molecular weights are on left in kilobases .

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

E Northern blot analysis of total RNA from wt cells and cells carrying two tiTELs (CAF110) treated with combinations of THI and Trichostatin A (TSA) for 24 h. Two identical membranes were hybridized in parallel with oligonucleotides corresponding to C-rich (oC) or G-rich (oG) telomeric repeats. To confirm similar hybridization efficiencies of the two oligonucleotides, northern blot membranes were simultaneously hybridized and exposed along with blots of digested genomic DNA (upper right insets). After signal detection, RNAmembranes were stripped and re-hybridized with U6 probes to assure equal loading. Marker molecular weights are on the left in kilobases.


A Telomere restriction fragment of genomic from wt carrying two tiTELs ( CAF110 ) grown for 24 h in EMM medium containing THI TSA as indicated . was digested with HindIII hybridized first with a nmt1 probe successively with a telomeric probe . Marker molecular weights are on left in kilobases . Numbers at bottom indicate gel lanes .

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A Telomere restriction fragment analysis of genomic DNA from wt cells and cells carrying two tiTELs (CAF110) grown for 24 h in EMM medium containing THI and TSA as indicated. DNA was digested with HindIII and hybridized first with a nmt1 probe and successively with a telomeric probe. Marker molecular weights are on the left in kilobases. Numbers at the bottom indicate gel lanes.


B Genomic from carrying two tiTELs deleted for trt1 + ( CAF113 ) was analyzed as in ( A ) . Marker molecular weights are on left in kilobases . Numbers at bottom indicate gel lanes .

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B Genomic DNA from cells carrying two tiTELs and deleted for trt1+ (CAF113) was analyzed as in (A). Marker molecular weights are on the left in kilobases. Numbers at the bottom indicate gel lanes.


C ter1 + carrying two tiTELs ( CAF110 , left panel ) or trt1Δ carrying one tiTEL ( MKSP2104 , central panel ) were cultured in EMM medium in presence of THI for 24 h ( THI + ) or in absence of THI for 24 , 48 or 72 h . In right panel , carrying two tiTELs ( CAF110 ) were treated with TSA for 24 h in presence or absence of THI . TiTEL telomeric sequences were amplified from genomic sequenced using a PacBio platform . statistical for each set of conditions is displayed above each panel . P values for each comparison were derived from Student ' s t between each condition THI + sample using Welch ' s correction when appropriate . new population of longer tiTELs appearing in trt1Δ upon prolonged transcription induction is indicated in red ( center panel ) .

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

C ter1+ cells carrying two tiTELs (CAF110, left panel) or trt1Δ cells carrying one tiTEL (MKSP2104, central panel) were cultured in EMM medium in presence of THI for 24 h (THI+) or in absence of THI for 24, 48 or 72 h. In the right panel, cells carrying two tiTELs (CAF110) were treated with TSA for 24 h in the presence or absence of THI. TiTEL telomeric sequences were amplified from genomic DNA and sequenced using a PacBio platform. The statistical analysis for each set of conditions is displayed above each panel. P values for each comparison were derived from the Student's t test between each condition and the THI+ sample using Welch's correction when appropriate. The new population of longer tiTELs appearing in trt1Δ cells upon prolonged transcription induction is indicated in red (center panel).


A qRT - PCR of G - rich polyA + tiTERRA in carrying one tiTEL expressing Trt1 - myc ( CAF610 ) cultured in EMM medium with indicated combinations of THI TSA for 24 h . Values are normalized to ACT1 mRNA expressed as fold increase over THI + TSA - samples . Bars error bars are averages SD from 4 independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ( relative to THI + TSA - ; two - Student ' s t ) .

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

A qRT-PCR analysis of G-rich and polyA+ tiTERRA in cells carrying one tiTEL and expressing Trt1-myc (CAF610) cultured in EMM medium with the indicated combinations of THI and TSA for 24 h. Values are normalized to ACT1 mRNA and expressed as fold increase over THI+ TSA- samples. Bars and error bars are averages and SD from 4 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, (relative to THI+ TSA-; two-tailed Student's t test).


B Telo - PCR of tiTELs using genomic extracted from as in ( A ) . Marker ( m ) molecular weights are on right in base pairs .

O O O O O 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 Telo-PCR analysis of tiTELs using genomic DNA extracted from cells as in (A). Marker (m) molecular weights are on the right in base pairs.


C RIP experiments performed using anti - myc antibodies extracts from as in ( A ) followed by qRT - PCR of G - rich polyA + tiTERRA . Values correspond to fraction of input expressed as fold increase over an untagged ( unt ) control tiTEL ( CAF545 ) . Bars error bars are averages SD from three independent experiments . * P < 0 . 05 , * * P < 0 . 01 ( relative to unt ; two - Student ' s t ) .

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 I-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 O O O O O O O O O O O O O O

C RIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qRT-PCR analysis of G-rich and polyA+ tiTERRA. Values correspond to fraction of input RNA expressed as fold increase over an untagged (unt) control tiTEL strain (CAF545). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01 (relative to unt; two-tailed Student's t test).


D ChIP experiments performed using anti - myc antibodies extracts from as in ( A ) followed by qPCR using tiTEL specific oligonucleotides . Values correspond to fraction of input expressed as fold increase over THI + TSA - . Unt : untagged ( CAF545 ) . Bars error bars are averages SD from at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 ( relative to THI + TSA - ; two - Student ' s t ) .

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

D ChIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qPCR using tiTEL specific oligonucleotides. Values correspond to fraction of input DNA expressed as fold increase over THI+ TSA-. Unt: untagged strain (CAF545). Bars and error bars are averages and SD from at least three independent experiments. * P < 0.05, ** P < 0.01 (relative to THI+ TSA-; two-tailed Student's t test).


A ) MALDI imaging was performed on - containing brain sections at a lateral resolution of 100µm ( M - H ) ions were analysed in negative mode with a FTICR Mass Spectrometer ( analytical range 100 - 1000Da ) . Hematoxylin - eosin ( HE ) of same sections was performed after MALDI . - derived glioma xenografts ( PDXs ) with IDH wildtype ( IDHwt ) , IDH mutant ( IDHm ) , control brain tissue ( CB ) were analyzed in a non - targeted targeted approach using regions of interest ( ROI ) of different sizes to compare ( T , yellow ) control brain ( CB , blue ) . non - targeted approach was used for statistical pair - wise comparisons between samples , while targeted approach on a selected set of metabolites allowed distribution analyses .

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A) MALDI imaging was performed on tumor-containing brain sections at a lateral resolution of 100µm and (M-H) ions were analysed in negative mode with a FTICR Mass Spectrometer (analytical range 100-1000Da). Hematoxylin-eosin (HE) staining of the same sections was performed after MALDI analysis. Patient-derived glioma xenografts (PDXs) with IDH wildtype (IDHwt), IDH mutant (IDHm), and control brain tissue (CB) were analyzed in a non-targeted and targeted approach using regions of interest (ROI) of different sizes to compare tumor (T, yellow line) and contralateral control brain (CB, blue line). The non-targeted approach was used for statistical pair-wise comparisons between samples, while the targeted approach on a selected set of metabolites allowed distribution and quantification analyses.


B ) Histological sections of 3 IDHwt PDXs ( P3 , T434 , P8 ) 3 IDHm PDXs ( ( T394 , E478 , T186 ) showing a large ROI ( boxed rectangle , > 500 pixels ) within applied for targeted of selected metabolites . Left rectangle in P3 PDX was used as control brain ( CB ) for quantifications . Middle panel shows tissue distribution of D - 2 - hydroxyglutarate ( D2HG ) by MSI , which is exclusively detected in IDHm PDXs ( D2HG in IDHwt tumours is below detection limit ) . Right panel shows tissue distribution of a key metabolite ( m / z 778 . 51 ) presenting strong accumulation in IDHm lower grade gliomas ( LGG ) versus glioblastomas ( GBM ) independent of IDH status . An - dependent color code indicates relative amount of a specific compound ( defined by m / z value ) throughout tissue section .

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B) Histological sections of 3 IDHwt PDXs (P3, T434, P8) and 3 IDHm PDXs ((T394, E478, T186) showing a large ROI (boxed rectangle, >500 pixels) within the tumor area applied for targeted quantification of selected metabolites. Left rectangle in P3 PDX was used as control contralateral brain (CB) for quantifications. Middle panel shows tissue distribution of D-2-hydroxyglutarate (D2HG) by MSI, which is exclusively detected in IDHm PDXs (D2HG in IDHwt tumours is below detection limit). Right panel shows tissue distribution of a key metabolite (m/z 778.51) presenting strong accumulation in IDHm lower grade gliomas (LGG) versus glioblastomas (GBM) independent of IDH status. An intensity-dependent color code indicates the relative amount of a specific compound (defined by m/z value) throughout the tissue section.


C ) of several high mass metabolites differentially present in IDHm PDXs ( IDHwt glioblastomas ( GBM ) in blue , IDHm GBM in orange , IDHm lower grade gliomas ( LGG ) in red , control brain ( CB ) in grey ) . Box plots represent log values of metabolite intensities measured within a large ROI ( > 500 pixels ) . Many metabolites in this mass range ( m / z 700 - 900 ) have been putatively assigned to phospholipids ( Tables EV2 - 3 ) . m / z represents mass over charge of ionized metabolites , as measured by mass spectrometer . Except where otherwise indicated , statistical significance was calculated for IDHm ( n = 3 ) versus IDHwt ( n = 3 ) PDXs using T . pv : p - values .

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C) Quantification of several high mass metabolites differentially present in IDHm PDXs (IDHwt glioblastomas (GBM) in blue, IDHm GBM in orange, IDHm lower grade gliomas (LGG) in red, contralateral control brain (CB) in grey). Box plots represent log values of metabolite intensities measured within a large ROI (>500 pixels). Many metabolites in this mass range (m/z 700-900) have been putatively assigned to phospholipids (Tables EV2-3). m/z represents the mass over charge ratio of ionized metabolites, as measured by the mass spectrometer. Except where otherwise indicated, statistical significance was calculated for IDHm (n=3) versus IDHwt (n=3) PDXs using T test. pv: p-values.


A ) MALDI imaging of indicated metabolites in IDHwt PDX ( P3 ) , IDHm PDX ( E478 ) control brain ( CB ) . Hex - P : hexose - phosphate ; AMP , ADP , ATP : adenosine mono - , di - triphosphate respectively .

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A) MALDI imaging of indicated metabolites in IDHwt PDX (P3), IDHm PDX (E478) and control brain (CB). Hex-P: hexose-phosphate; AMP, ADP, ATP: adenosine mono-, di- and triphosphate respectively.


B ) LC - MS of indicated metabolites in 6 PDXs . Relative quantities of metabolites are expressed in log scale normalized to tissue quantity used for extract preparation . IDHwt glioblastomas ( GBM ) in blue , IDHm GBM in orange , IDHm lower grade gliomas ( LGG ) in red , control brain ( CB ) in grey . Sdh7 - P : sedoheptulose - 7 - phosphate .

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B) LC-MS quantification of indicated metabolites in 6 PDXs. Relative quantities of metabolites are expressed in log scale and normalized to tissue quantity used for extract preparation. IDHwt glioblastomas (GBM) in blue, IDHm GBM in orange, IDHm lower grade gliomas (LGG) in red, contralateral control brain (CB) in grey. Sdh7-P: sedoheptulose-7-phosphate.


C ) Energetic charge defined as of ( ATP + 1 / 2ADP ) / ( ATP + ADP + AMP ) was calculated at each pixel to present its distribution throughout .

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C) Energetic charge defined as the ratio of (ATP+1/2ADP)/(ATP+ADP+AMP) was calculated at each pixel to present its distribution throughout the tumor.


( D ) energetic charge was confirmed by LC - MS data performed on separate extracts ( n = 3 / sample / group ) . Indicated statistical significant group differences are on T , pv : p - values .

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(D) The energetic charge ratio was confirmed by the LC-MS data performed on separate tumor extracts (n=3/ sample/ group). Indicated statistical significant group differences are based on T test, pv: p-values.


A ) LC - MS of steady - state level of glycolysis TCA cycle metabolites in three IDH1wt three IDH1m PDXs control brain ( CB ) ( n = 3 / sample / group ) . Metabolites showing statistically significant group differences between IDH1wt IDH1m are indicated ( pvalue : * < 0 . 05 ) . Color code as in Fig . 1 - 2 .

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A) LC-MS quantification of steady-state level of glycolysis and TCA cycle metabolites in three IDH1wt and three IDH1m PDXs and contralateral control brain (CB) (n=3/ sample/ group). Metabolites showing statistically significant group differences between IDH1wt and IDH1m are indicated (pvalue: * <0.05). Color code as in Fig. 1-2.


C ) In vivo flux of 13C - labelled glucose injected 20 minutes prior to sacrifice in one IDH1wt PDX ( P3 ) one IDH1m PDX ( E478 ) ( n = 3 / sample ) . of glucose - derived 13C label is shown in selected metabolites containing 0 to 6 13C atoms ( 13C0 - 13C6 as indicated by color code ) . Note absence of 13C2 label of αKG D2HG in IDHm tumours , while label was detectable in IDHwt PDX in brain of both tumors ( lower panel ) .

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C) In vivo flux analysis of 13C-labelled glucose injected 20 minutes prior to sacrifice in one IDH1wt PDX (P3) and one IDH1m PDX (E478) (n=3/ sample). The percentage of glucose-derived 13C label is shown in selected metabolites containing 0 to 6 13C atoms (13C0 - 13C6 as indicated by color code). Note the absence of 13C2 label of αKG and D2HG in IDHm tumours, while the label was detectable in IDHwt PDX and in contralateral brain of both tumors (lower panel).


D ) Quantities of 13C3 13C2 labeled metabolites from glucose tracer experiment in ( C ) . D2HG in IDH1m PDX shows a detectable 13C2 signature here , but represents a minor fraction of total D2HG pool in this . Hex - P : hexose phosphate , PEP : phosphoenolpyruvate , Pyr : pyruvate , Cit : citrate , Mal : malate , Asp : aspartate , Glu : glutamate , Gln : glutamine , α - KG : α - Ketoglutarate , D2HG : D2 - Hydroxyglutarate . Indicated statistical significant group differences are on T , pv : p - values .

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D) Quantities of 13C3 and 13C2 labeled metabolites from glucose tracer experiment in (C). D2HG in IDH1m PDX shows a detectable 13C2 signature here, but represents a minor fraction of the total cellular D2HG pool in this tumor. Hex-P: hexose phosphate, PEP: phosphoenolpyruvate, Pyr: pyruvate, Cit: citrate, Mal: malate, Asp: aspartate, Glu: glutamate, Gln: glutamine, α-KG: α-Ketoglutarate, D2HG: D2-Hydroxyglutarate. Indicated statistical significant group differences are based on T test, pv: p-values.


A ) MALDI imaging of key metabolites in IDHwt ( P3 ) IDHm ( E478 ) glioma PDX , compared to control brain ( CB ) sections .

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A) MALDI imaging of key metabolites in IDHwt (P3) and IDHm (E478) glioma PDX, compared to control brain (CB) sections.


B ) of metabolite distribution by MSI in 6 PDX with IDH1wt IDH1m status ( n = 3 / group ) in control brain ( CB ) . No significant differences between IDH groups were detected , although significant differences are found between tumors control brain ( glutamate , apsartate ) . Color code as in Fig . 1 , NAA : N - acetylaspartic acid ; NAAG : N - acetylaspartylglutamic acid .

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B) Quantification of metabolite distribution by MSI in 6 PDX with IDH1wt and IDH1m status (n=3 / group) and in contralateral control brain (CB). No significant differences between IDH groups were detected, although significant differences are found between tumors and control brain (glutamate, apsartate). Color code as in Fig. 1, NAA: N-acetylaspartic acid; NAAG: N-acetylaspartylglutamic acid.


A ) NADPH are reduced in IDHm compared to wildtype tumors , resulting in a reduced NADPH / NAD + ( n = 3 / sample ) . p - value from T : * < 0 . 05 . NADPH , NADP : reduced , oxydized nicotinamide adenine dinucleotide phosphate , NADH , NAD : reduced , oxydized nicotinamide adenine dinucleotide .

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A) NADPH levels are reduced in IDHm compared to wildtype tumors, resulting in a reduced NADPH/NAD+ ratio (n=3/ sample). p-value from T test: *<0.05. NADPH, NADP: reduced, oxydized nicotinamide adenine dinucleotide phosphate, NADH, NAD: reduced, oxydized nicotinamide adenine dinucleotide.


B ) MALDI for glutathione ( GSH ) cystathionine on IDHwt ( P3 ) IDHm ( E478 ) PDXs .

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B) MALDI images for glutathione (GSH) and cystathionine on IDHwt (P3) and IDHm (E478) PDXs.


C ) of MSI signals for GSH cystathionine from 6 PDXs ( n = 3 / group ) .

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C) Quantification of MSI signals for GSH and cystathionine from 6 PDXs (n=3 / group).


D ) LC - MS of independent samples for GSH , GSSG ( oxidized GSH ) , GSSG / GSH cystathionine from 6 PDXs ( n = 3 / sample / group ) . Statistical significance between IDH groups calculated by T , pv : p - value .

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D) LC-MS quantification of independent samples for GSH, GSSG (oxidized GSH), GSSG/GSH ratio and cystathionine from 6 PDXs (n=3/ sample / group). Statistical significance between IDH groups calculated by T test, pv: p-value.


A ) Comparative gene expression of key enzymes involved in GSH cysteine synthesis supply in IDHwt ( n = 220 ) , IDHm with 1p / 19q co - deletion ( n = 151 ) or IDHm without 1p / 19q co - deletion ( n = 226 ) on TCGA - seq data from a cohort of 597 . CBS : Cystathionine beta synthase , key enzyme of transsulfuration pathway . GCLC : catalytic subunit of glutamate - cysteine ligase . SLC7A11 : gene encoding xCT , cystine - glutamate antiporter .

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A) Comparative gene expression analysis of key enzymes involved in GSH and cysteine synthesis and supply in IDHwt (n=220), IDHm with 1p/19q co-deletion (n=151) or IDHm without 1p/19q co-deletion (n=226) patients based on TCGA RNA-seq data from a cohort of 597 patients. CBS: Cystathionine beta synthase, key enzyme of the transsulfuration pathway. GCLC: catalytic subunit of glutamate-cysteine ligase. SLC7A11: gene encoding xCT, the cystine-glutamate antiporter.


B ) Kaplan - Meier curves of CBS gene expression versus overall survival in with IDHwt , IDHm with or without 1p / 19q co - deletion . Statistical significance for survival is shown as : pv : p - value , ns = non significant .

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B) Kaplan-Meier curves of CBS gene expression versus overall survival in patients with IDHwt, IDHm with or without 1p/19q co-deletion. Statistical significance for the survival analysis is shown as: pv: p-value, ns= non significant.


B . structural predictions for S . pombe Rbd2 ( blue ) E . coli GlpG ( red ) . structures were predicted using Phyre2 server ( http : / / www . sbg . bio . ic . ac . uk / phyre2 ) . Rbd2 model structure was aligned to GlpG using PyMOL .

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B. Protein structural predictions for S. pombe Rbd2 (blue) and E. coli GlpG (red). Protein structures were predicted using the Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2). Rbd2 model structure was aligned to GlpG using PyMOL.


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E. Cells carrying a plasmid expressing rbd2-6xmCherry and either of two Golgi proteins dsc2-6xGFP(dsc2-6xGFPrbd2-6xmCherry) or Sec72-6xGFP (Sec72-6xGFPrbd2-6xmCherry) were imaged by confocal fluorescence microscopy.


A . Wild - type ( WT ) , rbd2Δ or sre1Δ yeast ( 200 ) containing either empty vector ( EV ) or a plasmid expressing sre1N ( Sre1amino 1 - 440 ) were grown on rich medium in presence or absence of cobalt chloride ( CoCl2 ) .

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A. Wild-type (WT), rbd2Δ or sre1Δ yeast (200 cells) containing either empty vector (EV) or a plasmid expressing sre1N (Sre1amino acids 1-440) were grown on rich medium in the presence or absence of cobalt chloride (CoCl2).


B . Western blot was probed with anti - Sre1 IgG of phosphatase - treated , whole lysates from WT indicated mutants grown for indicated time in absence of oxygen . Dsc5 serves as a loading control was detected by chemiluminescence . P N denote Sre1 precursor forms , respectively .

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B. Western blot was probed with anti-Sre1 IgG of phosphatase-treated, whole cell lysates from WT and the indicated mutants grown for the indicated time in the absence of oxygen. Dsc5 serves as a loading control and was detected by chemiluminescence. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.


C . Indicated yeast strains expressing rbd2 or chromosomal Flag - tagged rbd2 , rbd2 - S130A or rbd2 - H182A were analyzed for Sre1 cleavage . Western blot was performed using anti - Sre1 or anti - FlagIgG of phosphatase - treated , whole lysates from grown for 3 hr in presence or absence of oxygen .

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C. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre1 cleavage. Western blot was performed using anti-Sre1 or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown for 3 hr in the presence or absence of oxygen.


D . Indicated yeast strains expressing rbd2 or chromosomal Flag - tagged rbd2 , rbd2 - S130A or rbd2 - H182A were analyzed for Sre2 cleavage by western blot probed with anti - Sre2 serum or anti - FlagIgG of phosphatase - treated , whole lysates from grown in presence of oxygen .

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D. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre2 cleavage by western blot probed with anti-Sre2 serum or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown in the presence of oxygen.


E . Sre1 cleavage was performed as in ( C ) . G128V , A127D A186T mutant strains were obtained from our previous mutagenesis screening ( Stewart et al , 2012 ) . Dsc5 serves as a loading control .

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E. Sre1 cleavage assay was performed as in (C). G128V, A127D and A186T mutant strains were obtained from our previous mutagenesis screening (Stewart et al, 2012). Dsc5 serves as a loading control.


F . Sre2 cleavage for indicated yeast strains was performed as in ( D ) .

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F. Sre2 cleavage assay for indicated yeast strains was performed as in (D).


G . A model substrate of Sre2 containing 423 - 793 ( Sre2MS ) with an N - terminal 3xFlag epitope tag was expressed on a plasmid under control of a CaMV promoter in sre2Δ , dsc1Δsre2Δ or rbd2Δsre2Δ yeast strains . Whole lysates from indicated yeast strains were analyzed by western blot with anti - Flag antibody . Three independent isolates ( A - C ) are shown for each . Empty vector ( EV ) transformation serves as a negative control .

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G. A model substrate of Sre2 containing amino acids 423-793 (Sre2MS) with an N-terminal 3xFlag epitope tag was expressed on a plasmid under the control of a CaMV promoter in sre2Δ, dsc1Δsre2Δ or rbd2Δsre2Δ yeast strains. Whole cell lysates from indicated yeast strains were analyzed by western blot with anti-Flag antibody. Three independent isolates (A-C) are shown for each strain. Empty vector (EV) transformation serves as a negative control.


A . Wild - type ( WT ) indicated mutant strains were grown for 3 hr at non - permissive ( 36 ° C ) to inactivate Mts3 . Phosphatase - treated lysates were analyzed by western blot with anti - Sre2 serum imaged using chemiluminescence . P N denote Sre2 precursor forms , respectively .

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A. Wild-type (WT) and the indicated mutant strains were grown for 3 hr at the non-permissive temperature (36°C) to inactivate Mts3. Phosphatase-treated cell lysates were analyzed by western blot with anti-Sre2 serum and imaged using chemiluminescence. P and N denote Sre2 precursor and cleaved nuclear forms, respectively.


B . WT , rbd2Δ sre2Δ were treated with bortezomib ( Bz ) for 3 hr , phosphatase - treated , whole lysates were analyzed by western blot with anti - Sre2 serum .

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B. WT, rbd2Δ and sre2Δ cells were treated with bortezomib (Bz) for 3 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum.


C . WT , dsc1Δ rbd2Δ carrying either empty vector ( EV ) or a plasmid expressing 3xFlag - Sre2MS ( MS ) were treated with bortezomib ( Bz ) for 3 hr . Whole lysates were analyzed by western blot with anti - Flag antibody .

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C. WT, dsc1Δ and rbd2Δ cells carrying either empty vector (EV) or a plasmid expressing 3xFlag-Sre2MS (MS) were treated with bortezomib (Bz) for 3 hr. Whole cell lysates were analyzed by western blot with anti-Flag antibody.


D . Whole lysates from indicated strains were analyzed by western blot with anti - Sre2 serum .

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D. Whole cell lysates from the indicated strains were analyzed by western blot with anti-Sre2 serum.


E . Catalytically dead Dsc1 E3 ligase ( dsc1 - C634A ) mutant other indicated yeast strains were analyzed by western blot probed with anti - Sre2 serum imaged using chemiluminescence .

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E. Catalytically dead Dsc1 E3 ligase (dsc1-C634A) mutant and other indicated yeast strains were analyzed by western blot probed with anti-Sre2 serum and imaged using chemiluminescence.


A . HumanHEK293T were transfected with plasmids expressing different 3xHA - tagged rhomboid proteases : S . pomberbd2 , S . pomberbd2 recoded for human expression , humanRHBDL2 , D . melanogaster Rho1 , P . falciparumROM4 . Whole lysates were analyzed by western blot to detect HA - tagged rhomboid [ asterisks indicate loading control bands detected with antibody ab179726 ( abcam ) ] .

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A. HumanHEK293T cells were transfected with plasmids expressing different 3xHA-tagged rhomboid proteases: S. pomberbd2, S. pomberbd2 recoded for human expression, humanRHBDL2, D. melanogaster Rho1, and P. falciparumROM4. Whole cell lysates were analyzed by western blot to detect the HA-tagged rhomboid [asterisks indicate loading control bands detected with antibody ab179726 (abcam)].


B . HumanHEK293T were transfected with plasmids expressing indicated rhomboid substrates either recoded S . pombe rbd2 , D . melanogaster Rho1 , or P . falciparumROM4 . Conditioned media lysates were analyzed by western blot . P C denote precursor products , respectively .

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B. HumanHEK293T cells were transfected with plasmids expressing the indicated rhomboid substrates and either recoded S. pombe rbd2, D. melanogaster Rho1, or P. falciparumROM4. Conditioned media and cell lysates were analyzed by western blot. P and C denote precursor and cleaved products, respectively.


C . HumanHEK293T were transfected with plasmids expressing GFP - TatA - Flag substrate indicated rhomboid proteases . Whole lysates were analyzed by western blot . P , CN , CC denote precursor , N - terminal C - terminal products , respectively .

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C. HumanHEK293T cells were transfected with plasmids expressing the GFP-TatA-Flag substrate and the indicated rhomboid proteases. Whole cell lysates were analyzed by western blot. P, CN, CC denote precursor, N-terminal cleaved and C-terminal cleaved products, respectively.


D . HumanHEK293T were transfected with plasmids expressing indicated GFP - TatA - Flag substrates either wild - type or mutant recoded S . pomberbd2 . Whole lysates were analyzed by western blot .

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D. HumanHEK293T cells were transfected with plasmids expressing the indicated GFP-TatA-Flag substrates and either wild-type or mutant recoded S. pomberbd2. Whole cell lysates were analyzed by western blot.


E . HumanHEK293T were transfected with plasmids expressing indicated GFP - TatA - Flag substrates different rhomboid proteases . Whole lysates were analyzed by western blot .

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E. HumanHEK293T cells were transfected with plasmids expressing the indicated GFP-TatA-Flag substrates and different rhomboid proteases. Whole cell lysates were analyzed by western blot.


F . HumanHEK293T were transfected with plasmids expressing GFP - TatA ( A8L ) - Flag recoded S . pomberbd2 . C - terminal cleavage products were purified using anti - Flag antibodies purified was analyzed by mass spectrometry .

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F. HumanHEK293T cells were transfected with plasmids expressing the GFP-TatA (A8L)-Flag and recoded S. pomberbd2. C-terminal cleavage products were purified using anti-Flag antibodies and purified protein was analyzed by mass spectrometry.


B . Whole lysates from WT , rbd2Δ dsc1Δ carrying a plasmid expressing 3xFlag - Sre2MS WT or indicated mutants were analyzed by western blot with anti - Flag antibody . P N denote Sre2MS precursor forms , respectively .

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B. Whole cell lysates from WT, rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag-Sre2MS WT or indicated mutants were analyzed by western blot with anti-Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively.


D - E . Whole lysates from WT , rbd2Δ dsc1Δ carrying a plasmid expressing 3xFlag - Sre2MS WT or indicated mutants were analyzed by western blot with anti - Flag antibody . P N denote Sre2MS precursor forms , respectively . Asterisks indicate non - specific , loading control bands , which were used to normalize intensities of P N in Figure 5F . Two independent isolates , A B , are shown for each .

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D-E. Whole cell lysates from WT, rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag-Sre2MS WT or indicated mutants were analyzed by western blot with anti-Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively. Asterisks indicate non-specific, loading control bands, which were used to normalize the intensities of P and N in Figure 5F. Two independent isolates, A and B, are shown for each strain.


F . Intensities of P N bands from western blots were quantified using LI - COR software . Relative of P N to corresponding non - specific band ( asterisk ) in each lane was normalized to that of P level in isolate A of WT for each mutagenesis . of N to P or relative P level was presented in bar graphs .

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F. Intensities of individual P and N bands from western blots were quantified using LI-COR software. Relative intensity of P and N to corresponding non-specific band (asterisk) in each lane was normalized to that of P level in isolate A of WT strain for each mutagenesis. The ratio of N to P or relative P level was presented in bar graphs.


G . average of N to P level for different Sre2MS mutants in wild - type ( Appendix Fig S2 ) was plotted . Cleavage was scored as normal if was > 2 .

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G. The average ratio of N to P level for different Sre2MS mutants in wild-type cells (Appendix Fig S2) was plotted. Cleavage was scored as normal if the ratio was >2.


H . For Sre2MS mutants showing defects in cleavage , average fold - increase in P level in dsc1Δ compared to WT from two isolates ( Appendix Fig S2 ) was plotted . Mutants were grouped into three phenotypic classes ( intermediate , dsc1Δ , rbd2 ∆ ) according to observed precursor accumulation . Precursor accumulation was defined as dsc1Δ if < 1 . 5 ( orange ) , rbd2Δ if > 3 ( pink ) , intermediate if in between ( blue ) .

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H. For Sre2MS mutants showing defects in cleavage, the average fold-increase in P level in dsc1Δ cells compared to WT cells from two isolates (Appendix Fig S2) was plotted. Mutants were grouped into three phenotypic classes (intermediate, dsc1Δ, and rbd2∆) according to observed precursor accumulation. Precursor accumulation was defined as dsc1Δ if <1.5 (orange), rbd2Δ if >3 (pink), and intermediate if in between (blue).


B . Recombinant GST - fused Rbd2 C - terminus ( Rbd2200 - 251 ) , Dsc5 UBX ( Dsc5323 - 425 ) , Dsc2 UBA ( Dsc2298 - 372 ) , GST - HA - V5 control were bound to GST magnetic beads incubated with S . pombe cytosol fraction from wild - type . Equivalent amounts of unbound bound fractions were probed for anti - Cdc48 , anti - ubiquitin anti - GSTIgG .

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B. Recombinant proteins GST-fused Rbd2 C-terminus (Rbd2200-251), Dsc5 UBX (Dsc5323-425), Dsc2 UBA (Dsc2298-372), and GST-HA-V5 control were bound to GST magnetic beads and incubated with S. pombe cytosol fraction from wild-type cells. Equivalent amounts of unbound and bound fractions were probed for anti-Cdc48, anti-ubiquitin and anti-GSTIgG.


C . GSTpulldown was performed as in ( B ) using truncated forms of Rbd2 C - terminus ; Rbd2200 - 251 , Rbd2200 - 225 , Rbd2225 - 251 Rbd2200 - 240 . Unbound ( 1x ) bound ( 5x ) fractions were analyzed by western

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C. GSTpulldown assay was performed as in (B) using truncated forms of Rbd2 C-terminus; Rbd2200-251, Rbd2200-225, Rbd2225-251 and Rbd2200-240. Unbound (1x) and bound (5x) fractions were analyzed by western blotting


D . Single residue mutants of conserved glycine residues in Rbd2200 - 251 ( G244R G246R ) were tested for Cdc48 binding as in ( C ) . Western blots ( B - D ) were imaged using chemiluminescence .

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D. Single residue mutants of conserved glycine residues in Rbd2200-251 (G244R and G246R) were tested for Cdc48 binding as in (C). Western blots (B-D) were imaged using chemiluminescence.