( C , F ) Cells were grown to mid - log phase either in rich medium or shifted for 8 hours to starvation medium . Zygotes and tetrads , which correspond to differentiated cells , were counted under a light microscope . Cells of the following genotypes were analyzed : WT isogenic controls , gcn5Η , tsc1Η , gcn5Η tsc1Η , rhb1 - DA4 , gcn5Η rhb1 - DA4 , gad8Η , and gcn5Η gad8Η . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 4 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . White arrowheads indicate zygotes . Scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,F) Cells were grown to mid-log phase either in rich medium or shifted for 8 hours to starvation medium. Zygotes and tetrads, which correspond to differentiated cells, were counted under a light microscope. Cells of the following genotypes were analyzed: WT isogenic controls, gcn5Η, tsc1Η, gcn5Η tsc1Η, rhb1-DA4, gcn5Η rhb1-DA4, gad8Η, and gcn5Η gad8Η. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 4 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. White arrowheads indicate zygotes. Scale bar, 10 μm.


SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability . ( A , B , D , E ) Expression of ste11 + ( A , D ) using quantitative RT - PCR of RNA extracted from cells grown either in nutrient rich medium ( dark gray ) or shifted for 4 hours to starvation medium ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , tsc1Η , gcn5Η tsc1Η , tsc2Η , gcn5Η tsc2Η , rhb1 - DA4 - a constitutively active ( CA ) rhb1 mutant [ 34 ] , gcn5Η rhb1 - DA4 , tor2 - L1310P - a CA tor2 mutant [ 33 ] , gcn5Η tor2 - L1310P , tor1Η , gcn5Η tor1Η , gad8Η , and gcn5Η gad8Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 3 ( D , E ) independent experiments , overlaid with individual data points and standard error ( SE ) bars . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 3 for D , E ) .

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

SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of ste11+ (A,D) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 3 (D,E) independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 3 for D,E).


SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability . ( A , B , D , E ) Expression of mei2 + ( B , E ) using quantitative RT - PCR of RNA extracted from cells grown either in nutrient rich medium ( dark gray ) or shifted for 4 hours to starvation medium ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , tsc1Η , gcn5Η tsc1Η , tsc2Η , gcn5Η tsc2Η , rhb1 - DA4 - a constitutively active ( CA ) rhb1 mutant [ 34 ] , gcn5Η rhb1 - DA4 , tor2 - L1310P - a CA tor2 mutant [ 33 ] , gcn5Η tor2 - L1310P , tor1Η , gcn5Η tor1Η , gad8Η , and gcn5Η gad8Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 3 ( D , E ) independent experiments , overlaid with individual data points and standard error ( SE ) bars . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 3 for D , E ) .

SAGA is epistatic to the TORC1 and TORC2 pathways in the regulation of differentiation in response to nutrient availability. (A,B,D,E) Expression of mei2+ (B,E) using quantitative RT-PCR of RNA extracted from cells grown either in nutrient rich medium (dark gray) or shifted for 4 hours to starvation medium (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, tsc1Η, gcn5Η tsc1Η, tsc2Η, gcn5Η tsc2Η, rhb1-DA4 - a constitutively active (CA) rhb1 mutant [34], gcn5Η rhb1-DA4, tor2-L1310P - a CA tor2 mutant [33], gcn5Η tor2-L1310P, tor1Η, gcn5Η tor1Η, gad8Η, and gcn5Η gad8Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 3 (D,E) independent experiments, overlaid with individual data points and standard error (SE) bars. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 3 for D,E).


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


SAGA is phosphorylated in response to nutrient starvation . ( A , B ) 4 - 20 % gradient SDS - polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium ( R ) or starved for 45 minutes ( S ) . SAGA was purified using endogenously TAP - tagged Ada1 . A fraction of each eluate was loaded and either stained with silver , to detect all proteins ( A ) , . A strain without any TAP tag is shown as a negative control for the purification . The graph on the left of the gel in B shows the fluorescence intensity of the phospho - specific stain , which was quantified along the left lane in blue ( rich ) and the middle lane in red ( starved ) , using ImageJ . The area of one peak , which corresponds to the bands marked with arrowheads and was coined p55 , is 1 . 8 - fold larger in SAGA purified from starved cells , as compared to rich conditions . Below is an anti - HA immuno - blot ( IB ) of each eluate , to reveal the amount of Ada1 - HA bait recovered . Shown are gels that are representative of 3 independent experiments . A . U . , arbitrary units .

B-MEASURED_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 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 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 O O O O O O O O O O O O O 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

SAGA is phosphorylated in response to nutrient starvation. (A,B) 4-20% gradient SDS-polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium (R) or starved for 45 minutes (S). SAGA was purified using endogenously TAP-tagged Ada1. A fraction of each eluate was loaded and either stained with silver, to detect all proteins (A),. A strain without any TAP tag is shown as a negative control for the purification. The graph on the left of the gel in B shows the fluorescence intensity of the phospho-specific stain, which was quantified along the left lane in blue (rich) and the middle lane in red (starved), using ImageJ. The area of one peak, which corresponds to the bands marked with arrowheads and was coined p55, is 1.8-fold larger in SAGA purified from starved cells, as compared to rich conditions. Below is an anti-HA immuno-blot (IB) of each eluate, to reveal the amount of Ada1-HA bait recovered. Shown are gels that are representative of 3 independent experiments. A.U., arbitrary units.


SAGA is phosphorylated in response to nutrient starvation . ( A , B ) 4 - 20 % gradient SDS - polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium ( R ) or starved for 45 minutes ( S ) . SAGA was purified using endogenously TAP - tagged Ada1 . A fraction of each eluate was loaded and either stained with Pro - Q ® Diamond , which stains phosphorylated proteins ( B ) . A strain without any TAP tag is shown as a negative control for the purification . The graph on the left of the gel in B shows the fluorescence intensity of the phospho - specific stain , which was quantified along the left lane in blue ( rich ) and the middle lane in red ( starved ) , using ImageJ . The area of one peak , which corresponds to the bands marked with arrowheads and was coined p55 , is 1 . 8 - fold larger in SAGA purified from starved cells , as compared to rich conditions . Below is an anti - HA immuno - blot ( IB ) of each eluate , to reveal the amount of Ada1 - HA bait recovered . Shown are gels that are representative of 3 independent experiments . A . U . , arbitrary units .

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

SAGA is phosphorylated in response to nutrient starvation. (A,B) 4-20% gradient SDS-polyacrylamide gel electrophoresis analysis of SAGA purified from cells grown either in rich medium (R) or starved for 45 minutes (S). SAGA was purified using endogenously TAP-tagged Ada1. A fraction of each eluate was loaded and either stained with Pro-Q® Diamond, which stains phosphorylated proteins (B). A strain without any TAP tag is shown as a negative control for the purification. The graph on the left of the gel in B shows the fluorescence intensity of the phospho-specific stain, which was quantified along the left lane in blue (rich) and the middle lane in red (starved), using ImageJ. The area of one peak, which corresponds to the bands marked with arrowheads and was coined p55, is 1.8-fold larger in SAGA purified from starved cells, as compared to rich conditions. Below is an anti-HA immuno-blot (IB) of each eluate, to reveal the amount of Ada1-HA bait recovered. Shown are gels that are representative of 3 independent experiments. A.U., arbitrary units.


( D ) FLAG - tagged Taf12 was purified from cells grown in rich medium ( R ) or shifted for 45 minutes to starvation medium ( S ) . Anti - FLAG immuno - precipitations ( IP ) were incubated with and without λ - phosphatase or its inhibitor and immuno - blotted ( IB ) , together with 1 % of whole cell extracts ( WCE ) , using an anti - FLAG antibody .

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

(D) FLAG-tagged Taf12 was purified from cells grown in rich medium (R) or shifted for 45 minutes to starvation medium (S). Anti-FLAG immuno-precipitations (IP) were incubated with and without λ-phosphatase or its inhibitor and immuno-blotted (IB), together with 1% of whole cell extracts (WCE), using an anti-FLAG antibody.


( E ) Phospho - Taf12 ( P - Taf12 ) , total Taf12 , and tubulin levels were quantified from cells grown in rich medium ( R ) or starved for 45 minutes ( S ) . P - Taf12 levels were normalized to those of total Taf12 , while total Taf12 levels were normalized to those of tubulin . Data points were individually plotted on the graph and overlaid with the mean and SE . Signal intensities were quantified from IBs of 7 independent experiments . Statistical significance was determined using a t - test ( n = 7 , unpaired , two - tailed ) .

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

(E) Phospho-Taf12 (P-Taf12), total Taf12, and tubulin levels were quantified from cells grown in rich medium (R) or starved for 45 minutes (S). P-Taf12 levels were normalized to those of total Taf12, while total Taf12 levels were normalized to those of tubulin. Data points were individually plotted on the graph and overlaid with the mean and SE. Signal intensities were quantified from IBs of 7 independent experiments. Statistical significance was determined using a t-test (n = 7, unpaired, two-tailed).


( F ) P - Taf12 and total Taf12 were followed in WT , taf12 - T283A and taf12 - 5A mutants , grown in rich medium ( R ) or starved for 45 minutes ( S ) .

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

(F) P-Taf12 and total Taf12 were followed in WT, taf12-T283A and taf12-5A mutants, grown in rich medium (R) or starved for 45 minutes (S).


( G , H ) SAGA and TFIID were tandem affinity - purified using endogenously TAP - tagged Spt7 ( G ) from strains containing FLAG - tagged Taf12 , grown in rich medium or starved for 45 minutes . TAP - tagged Spt7 and Taf4 were eluted using the TEV protease , releasing a shorter form of each bait ( Spt7 - CBP or Taf4 - CBP ) . Eluates were loaded and immuno - blotted ( IB ) using anti - FLAG or anti - CBP antibodies , together with 1 % of either whole cell extracts ( WCE ) or IgG - sepharose flow - through ( FT ) . Shown are IBs that are representative of 2 independent experiments .

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

(G,H) SAGA and TFIID were tandem affinity-purified using endogenously TAP-tagged Spt7 (G) from strains containing FLAG-tagged Taf12, grown in rich medium or starved for 45 minutes. TAP-tagged Spt7 and Taf4 were eluted using the TEV protease, releasing a shorter form of each bait (Spt7-CBP or Taf4-CBP). Eluates were loaded and immuno-blotted (IB) using anti-FLAG or anti-CBP antibodies, together with 1% of either whole cell extracts (WCE) or IgG-sepharose flow-through (FT). Shown are IBs that are representative of 2 independent experiments.


( G , H ) SAGA and TFIID were tandem affinity - purified using endogenously TAP - tagged Taf4 ( H ) , from strains containing FLAG - tagged Taf12 , grown in rich medium or starved for 45 minutes . TAP - tagged Spt7 and Taf4 were eluted using the TEV protease , releasing a shorter form of each bait ( Spt7 - CBP or Taf4 - CBP ) . Eluates were loaded and immuno - blotted ( IB ) using anti - FLAG or anti - CBP antibodies , together with 1 % of either whole cell extracts ( WCE ) or IgG - sepharose flow - through ( FT ) . Shown are IBs that are representative of 2 independent experiments .

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

(G,H) SAGA and TFIID were tandem affinity-purified using endogenously TAP-tagged Taf4 (H), from strains containing FLAG-tagged Taf12, grown in rich medium or starved for 45 minutes. TAP-tagged Spt7 and Taf4 were eluted using the TEV protease, releasing a shorter form of each bait (Spt7-CBP or Taf4-CBP). Eluates were loaded and immuno-blotted (IB) using anti-FLAG or anti-CBP antibodies, together with 1% of either whole cell extracts (WCE) or IgG-sepharose flow-through (FT). Shown are IBs that are representative of 2 independent experiments.


( I ) P - Taf12 , total Taf12 , and Ser546 - phosphorylated Gad8AKT were followed in WT cells , grown in rich conditions or over a time - course after a shift to starvation medium . Gad8AKT phosphorylation at Ser546 is a proxy of TORC2 activity in S . pombe .

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

(I) P-Taf12, total Taf12, and Ser546-phosphorylated Gad8AKT were followed in WT cells, grown in rich conditions or over a time-course after a shift to starvation medium. Gad8AKT phosphorylation at Ser546 is a proxy of TORC2 activity in S. pombe.


( J ) P - Taf12 and total Taf12 were followed in WT cells , grown in rich conditions or shifted to different starvation media for 30 minutes . These include both the removal of the nitrogen source , ammonium chloride ( NH4Cl ) , and the reduction of the carbon source , glucose , from a concentration of 2 % to 0 . 5 % . Alternatively , cells were either only deprived of NH4Cl or only exposed to reduced glucose levels ( 2 % to 0 . 5 % ) .

O O O O O B-MEASURED_VAR 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 I-CONTROLLED_VAR 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 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

(J) P-Taf12 and total Taf12 were followed in WT cells, grown in rich conditions or shifted to different starvation media for 30 minutes. These include both the removal of the nitrogen source, ammonium chloride (NH4Cl), and the reduction of the carbon source, glucose, from a concentration of 2% to 0.5%. Alternatively, cells were either only deprived of NH4Cl or only exposed to reduced glucose levels (2% to 0.5%).


( K ) P - Taf12 and total Taf12 were followed in WT cells , grown in rich conditions or deprived of NH4Cl for one hour . Then , NH4Cl was added back to the medium for 30 or 60 minutes . Both short and long exposures of the FLAG IBs are shown to detect total Taf12 and P - Taf12 , respectively , within the linear range of the chemi - luminescence signal . Anti - tubulin IBs are shown as controls for loading between samples . Shown are star that are representative of at least 3 independent experiments . The number sign ( # ) symbol identifies antibody heavy chains and the star ( * ) symbol labels an unspecific band detected by the anti - FLAG antibody in S . pombe .

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

(K) P-Taf12 and total Taf12 were followed in WT cells, grown in rich conditions or deprived of NH4Cl for one hour. Then, NH4Cl was added back to the medium for 30 or 60 minutes. Both short and long exposures of the FLAG IBs are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal. Anti-tubulin IBs are shown as controls for loading between samples. Shown are star that are representative of at least 3 independent experiments. The number sign (#) symbol identifies antibody heavy chains and the star (*) symbol labels an unspecific band detected by the anti-FLAG antibody in S. pombe.


( A , B ) Expression of ste11 + ( A ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 4 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , ppa2Η , par1Η , pab1Η , tsc1Η , and pab1Η tsc1Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 ) .

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

(A,B) Expression of ste11+ (A) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), ppa2Η, par1Η, pab1Η, tsc1Η, and pab1Η tsc1Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4).


( A , B ) Expression of mei2 + ( B ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 4 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , ppa2Η , par1Η , pab1Η , tsc1Η , and pab1Η tsc1Η . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 ) .

(A,B) Expression of mei2+ (B) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), ppa2Η, par1Η, pab1Η, tsc1Η, and pab1Η tsc1Η. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4).


( C ) Cells were grown to mid - log phase either in rich medium or starved for 8 hours . Zygotes and tetrads , which correspond to differentiated cells , were counted under a light microscope . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 4 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . White arrowheads indicate zygotes . Scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells were grown to mid-log phase either in rich medium or starved for 8 hours. Zygotes and tetrads, which correspond to differentiated cells, were counted under a light microscope. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 4 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. White arrowheads indicate zygotes. Scale bar, 10 μm.


( D ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT and pab1Η strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - Rpb1 IB is shown as a control for loading . The signal intensities of P - Taf12 and total Taf12 were quantified in each strain and condition . Ratios of P - Taf12 to total Taf12 were calculated from 6 independent experiments and individually plotted in a graph below the IBs , together with the mean and SE . Averaged values from all WT controls grown in rich medium were set at 1 to allow comparisons across culture conditions and strains . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 6 ; * p < 0 . 01 ; # p < 0 . 05 ) . A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P - Taf12 , respectively , within the linear range of the chemi - luminescence signal .

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

(D) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT and pab1Η strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-Rpb1 IB is shown as a control for loading. The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. Ratios of P-Taf12 to total Taf12 were calculated from 6 independent experiments and individually plotted in a graph below the IBs, together with the mean and SE. Averaged values from all WT controls grown in rich medium were set at 1 to allow comparisons across culture conditions and strains. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 6; *p < 0.01; #p < 0.05). A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal.


( E ) FLAG - tagged Taf12 and TAP - tagged Pab1 were affinity purified separately from cells grown in rich conditions ( upper panels ) . TAP - tagged Pab1 was cleaved off from beads using the Tobacco Etch Virus ( TEV ) protease , releasing a shorter form of Pab1 ( CBP - Pab1 ) in the eluate ( E ) . CBP - Pab1 was then mixed with beads containing FLAG - Taf12 IPs and incubated in a phosphatase buffer , with and without 0 . 5 µM microcystin . Each reaction was then analyzed by IB and simultaneously probed with anti - FLAG or anti - CBP antibodies ( lower panel ) .

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

(E) FLAG-tagged Taf12 and TAP-tagged Pab1 were affinity purified separately from cells grown in rich conditions (upper panels). TAP-tagged Pab1 was cleaved off from beads using the Tobacco Etch Virus (TEV) protease, releasing a shorter form of Pab1 (CBP-Pab1) in the eluate (E). CBP-Pab1 was then mixed with beads containing FLAG-Taf12 IPs and incubated in a phosphatase buffer, with and without 0.5 µM microcystin. Each reaction was then analyzed by IB and simultaneously probed with anti-FLAG or anti-CBP antibodies (lower panel).


( F ) Exponentially growing cells were treated for 1 hour with Torin1 , which was added at increasing concentrations , 7 and 21 µM , to rich medium . Dimethylsulphoxide ( DMSO ) was used as the vehicle and added as a negative control .

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

(F) Exponentially growing cells were treated for 1 hour with Torin1, which was added at increasing concentrations, 7 and 21 µM, to rich medium. Dimethylsulphoxide (DMSO) was used as the vehicle and added as a negative control.


( G ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT and tor2 - ts10 strains , grown in rich conditions at 25 ° C or shifted to 30 ° C for 4 and 6 hours . ( F , G ) Anti - tubulin IBs are shown as a control for loading between samples . Shown are IBs that are representative of 3 independent experiments . The star ( * ) symbol labels unspecific bands detected by the anti - FLAG or anti - CBP antibodies in S . pombe .

O O O O O B-MEASURED_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 O O O O

(G) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT and tor2-ts10 strains, grown in rich conditions at 25°C or shifted to 30°C for 4 and 6 hours. (F,G) Anti-tubulin IBs are shown as a control for loading between samples. Shown are IBs that are representative of 3 independent experiments. The star (*) symbol labels unspecific bands detected by the anti-FLAG or anti-CBP antibodies in S. pombe.


( A ) Igo1 phosphorylation ( P - Igo1 ) was followed by anti - MYC IB of protein extracts from WT , ppk18Η , and igo1 - S64A strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . Igo1 migration was analyzed by electrophoresis of both a 12 % SDS - polyacrylamide gel containing the Phos - tag ™ molecule ( right panel ) and a standard 10 % SDS - polyacrylamide gel ( left panel ) . Arrowheads indicate the various phosphorylated isoforms of Igo1 - MYC in each strain and condition . Shown is an IB representative of 2 independent experiments .

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

(A) Igo1 phosphorylation (P-Igo1) was followed by anti-MYC IB of protein extracts from WT, ppk18Η, and igo1-S64A strains, grown in rich conditions (R) or starved for 45 minutes (S). Igo1 migration was analyzed by electrophoresis of both a 12% SDS-polyacrylamide gel containing the Phos-tag™ molecule (right panel) and a standard 10% SDS-polyacrylamide gel (left panel). Arrowheads indicate the various phosphorylated isoforms of Igo1-MYC in each strain and condition. Shown is an IB representative of 2 independent experiments.


( B , C ) Expression of ste11 + ( C ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 4 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , pab1Η , gcn5Η ppk18Η , pab1Η , pab1Η ppk18Η , igo1Η , igo1 - S64A , gcn5Η igo1 - S64A and pab1Η igo1 - S64A . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 ) .

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

(B,C) Expression of ste11+ (C) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, pab1Η, gcn5Η ppk18Η, pab1Η, pab1Η ppk18Η, igo1Η, igo1-S64A, gcn5Η igo1-S64A and pab1Η igo1-S64A. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4).


( B , C ) Expression of mei2 + ( D ) using igo1 of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 4 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , gcn5Η , ppk18Η , gcn5Η ppk18Η , pab1Η , pab1Η ppk18Η , igo1Η , igo1 - S64A , gcn5Η igo1 - S64A and pab1Η igo1 - S64A . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 4 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 4 ) .

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

(B,C) Expression of mei2+ (D) using igo1 of RNA extracted from cells grown either in rich medium (dark gray) or starved for 4 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), gcn5Η, ppk18Η, gcn5Η ppk18Η, pab1Η, pab1Η ppk18Η, igo1Η, igo1-S64A, gcn5Η igo1-S64A and pab1Η igo1-S64A. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 4 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 4).


( D ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT , S . pombeΗ , and igo1Η strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - MYC IB is shown as a control for loading . ( D , E ) The signal intensities of P - Taf12 and total Taf12 were quantified in each strain and condition . Shown below each blot are average measurements of 3 independent experiments ( n = 3 ) . A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P - Taf12 , respectively , within the linear range of the chemi - luminescence signal . The star ( * ) symbol labels an unspecific band detected by the FLAG antibody in S . pombe .

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

(D) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT, S. pombeΗ, and igo1Η strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-MYC IB is shown as a control for loading. (D,E) The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. Shown below each blot are average measurements of 3 independent experiments (n = 3). A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal. The star (*) symbol labels an unspecific band detected by the FLAG antibody in S. pombe.


( E ) P - Taf12 was followed in WT , igoS64A , pab1Η , and pab1Η igoS64A strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - Rpb1 IB is shown as a control for loading . ( D , E ) The signal intensities of P - Taf12 and total Taf12 were quantified in each strain and condition . Shown below each blot are average measurements of 3 independent experiments ( n = 3 ) . A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P - Taf12 , respectively , within the linear range of the chemi - luminescence signal . The star ( * ) symbol labels an unspecific band detected by the S . pombe antibody in S . pombe .

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

(E) P-Taf12 was followed in WT, igoS64A, pab1Η, and pab1Η igoS64A strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-Rpb1 IB is shown as a control for loading. (D,E) The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. Shown below each blot are average measurements of 3 independent experiments (n = 3). A short and a long exposure of the FLAG IB are shown to detect total Taf12 and P-Taf12, respectively, within the linear range of the chemi-luminescence signal. The star (*) symbol labels an unspecific band detected by the S. pombe antibody in S. pombe.


( A ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT , tor1Η , and gad8Η strains , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - tubulin IB is shown as a control for loading . The signal intensities of P - Taf12 and total Taf12 were quantified in each strain and condition . P - Taf12 to Taf2 ratios were calculated from 3 independent experiments and individually plotted in a graph below the IBs , together with the mean and SE . Averaged values from all WT controls grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 3 ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR O O B-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 O O O O O O O O O O O O O O O O O O O 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) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT, tor1Η, and gad8Η strains, grown in rich conditions (R) or starved for 45 minutes (S). An anti-tubulin IB is shown as a control for loading. The signal intensities of P-Taf12 and total Taf12 were quantified in each strain and condition. P-Taf12 to Taf2 ratios were calculated from 3 independent experiments and individually plotted in a graph below the IBs, together with the mean and SE. Averaged values from all WT controls grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 3).


( B ) P - Taf12 was followed by anti - FLAG IB of protein extracts from sty1Η cells ( left panel ) or ssp2Η cells ( right panel ) , grown in rich conditions ( R ) or starved for 45 minutes ( S ) . An anti - tubulin IB is shown as a control for loading . The star ( * ) symbols label an unspecific band detected by the anti - FLAG antibody in S . pombe . Shown are IBs that are representative of 2 independent experiments .

O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) P-Taf12 was followed by anti-FLAG IB of protein extracts from sty1Η cells (left panel) or ssp2Η cells (right panel), grown in rich conditions (R) or starved for 45 minutes (S). An anti-tubulin IB is shown as a control for loading. The star (*) symbols label an unspecific band detected by the anti-FLAG antibody in S. pombe. Shown are IBs that are representative of 2 independent experiments.


( C ) TAP - tagged Gad8 was immuno - precipitated using IgG - sepharose beads ( IP ) from strains containing FLAG - tagged Taf12 , grown either in rich medium ( left panels ) or starved for 45 minutes ( right panels ) . TAP - tagged Gad8 was eluted from beads using the TEV protease , releasing a shorter form of Gad8 ( CBP - Gad8 ) . Eluates were loaded and immuno - blotted ( IB ) using anti - FLAG or anti - CBP antibodies , together with 1 % of whole cell extracts ( WCE ) , to detect Taf12 co - precipitation with Gad8 . A strain which does not contain any TAP tag is shown as a negative control for the IP . Shown is an IB representative of two independent experiments . ( A , C ) The star ( * ) symbols label unspecific bands detected by the FLAG or the CBP antibodies in S . pombe .

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

(C) TAP-tagged Gad8 was immuno-precipitated using IgG-sepharose beads (IP) from strains containing FLAG-tagged Taf12, grown either in rich medium (left panels) or starved for 45 minutes (right panels). TAP-tagged Gad8 was eluted from beads using the TEV protease, releasing a shorter form of Gad8 (CBP-Gad8). Eluates were loaded and immuno-blotted (IB) using anti-FLAG or anti-CBP antibodies, together with 1% of whole cell extracts (WCE), to detect Taf12 co-precipitation with Gad8. A strain which does not contain any TAP tag is shown as a negative control for the IP. Shown is an IB representative of two independent experiments. (A,C) The star (*) symbols label unspecific bands detected by the FLAG or the CBP antibodies in S. pombe.


( D ) Endogenous Gad8 - HA was affinity purified from starved cells , in order to activate its kinase activity ( left and middle panels ) , and mixed with recombinant , purified GST - Taf12 , GST - Taf12 - 5A , or GST - Fkh2 proteins ( right panels ) in a kinase assay buffer . Each assay was then analyzed by IB and probed with an anti - phospho - AKT substrate antibody .

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

(D) Endogenous Gad8-HA was affinity purified from starved cells, in order to activate its kinase activity (left and middle panels), and mixed with recombinant, purified GST-Taf12, GST-Taf12-5A, or GST-Fkh2 proteins (right panels) in a kinase assay buffer. Each assay was then analyzed by IB and probed with an anti-phospho-AKT substrate antibody.


( A , B ) Expression of quantitative RT - PCR + ( A ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 2 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) and taf12 - 5A mutants . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 6 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 6 ) .

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

(A,B) Expression of quantitative RT-PCR+ (A) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 2 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT) and taf12-5A mutants. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 6 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 6).


mei2 + ( B ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 2 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) and taf12 - 5A mutants . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 6 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 6 ) .

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 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

mei2+ (B) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 2 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT) and taf12-5A mutants. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 6 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 6).


( C , E ) Zygotes and tetrads , which correspond to differentiated cells , were counted from cultures of homothallic cells grown to mid - log phase in rich medium ( t0 ) and shifted to starvation medium for up to 24 hours ( C ) Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , taf12 - 5A , taf12 - 5DE , tor1 - 1972A , and taf12 - 5A tor1 - 1972A . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 6 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . Statistical significance was determined by 2 - way ANOVA followed by Tukey ' s multiple comparison tests ( n = 6 ) .

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

(C,E) Zygotes and tetrads, which correspond to differentiated cells, were counted from cultures of homothallic cells grown to mid-log phase in rich medium (t0) and shifted to starvation medium for up to 24 hours (C) Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), taf12-5A, taf12-5DE, tor1-1972A, and taf12-5A tor1-1972A. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 6 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. Statistical significance was determined by 2-way ANOVA followed by Tukey's multiple comparison tests (n = 6).


( D ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT and tor1 - 1972A strains , grown in rich medium and shifted to starved conditions for 60 minutes . An anti - Tubulin IB is shown as a control for loading . Shown is an IB which is representative of 2 independent experiments .

O O O O O B-MEASURED_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

(D) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT and tor1-1972A strains, grown in rich medium and shifted to starved conditions for 60 minutes. An anti-Tubulin IB is shown as a control for loading. Shown is an IB which is representative of 2 independent experiments.


( C , E ) taf12 and tetrads , which correspond to differentiated cells , were counted from heterothallic cells mixed and incubated for 2 days on mating medium ( E ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , taf12 - 5A , taf12 - 5DE , tor1 - 1972A , and taf12 - 5A tor1 - 1972A . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 6 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . Statistical significance was determined by 2 - way ANOVA followed by Tukey ' s multiple comparison tests ( n = 6 ) .

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

(C,E) taf12 and tetrads, which correspond to differentiated cells, were counted from heterothallic cells mixed and incubated for 2 days on mating medium (E). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), taf12-5A, taf12-5DE, tor1-1972A, and taf12-5A tor1-1972A. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 6 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. Statistical significance was determined by 2-way ANOVA followed by Tukey's multiple comparison tests (n = 6).


A ) IL - 8 production after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysate in HeLa cells ( Hoechst in blue , IL - 8 in red )

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

A) IL-8 production after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysate in HeLa cells (Hoechst in blue, IL-8 in red)


after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysate in HeLa cell B ) Quantification of IL - 8

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR

after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysate in HeLa cell B) Quantification of IL-8 a


with digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysat C ) TIFA oligomerization at 6 h in TIFA - GFP - expressing HeLa cells

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

with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat C) TIFA oligomerization at 6 h in TIFA-GFP-expressing HeLa cells treate


with digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysat at 6 h in TIFA - GFP - expressing HeLa cells treate D ) Quantification of TIFA

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

with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat at 6 h in TIFA-GFP-expressing HeLa cells treate D) Quantification of TIFA oligomerizatio


with digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysat E ) TIFA oligomerization at 30 min in cells

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

with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat E) TIFA oligomerization at 30 min in cells treate


F ) Kinetics of TIFA - GFP oligomerization upon treatment with digitonin alone or in combination with βHBP or S . flexneri

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

F) Kinetics of TIFA-GFP oligomerization upon treatment with digitonin alone or in combination with βHBP or S. flexneri lysate


B ) TIFA - GFP oligomerization at 30 min in HeLa cells treated with digitonin ( mock ) or digitonin and a lysate from indicated S . flexneri strains ( pHldA / C means plasmid - encoded HldA and HldC )

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

B) TIFA-GFP oligomerization at 30 min in HeLa cells treated with digitonin (mock) or digitonin and a lysate from indicated S. flexneri strains (pHldA/C means plasmid-encoded HldA and HldC)


with digitonin ( mock ) or digitonin and a lysate from indicated S . flexneri strain ( pHldA / C means plasmid - encoded HldA and HldC ) C ) TIFA - GFP oligomerization at 6 h in cells

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

with digitonin (mock) or digitonin and a lysate from indicated S. flexneri strain (pHldA/C means plasmid-encoded HldA and HldC) C) TIFA-GFP oligomerization at 6 h in cells treate


with digitonin ( mock ) or digitonin and a lysate from indicated S . flexneri strains ( pHldA / C means plasmid - encoded HldA and HldC ) D ) IL - 8 production at 6 h in cells were

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

with digitonin (mock) or digitonin and a lysate from indicated S. flexneri strains (pHldA/C means plasmid-encoded HldA and HldC) D) IL-8 production at 6 h in cells were treate


E ) TIFA - GFP oligomerization in cells treated for 30 min with digitonin alone , digitonin and wild - type S . flexneri lysate or digitonin and ADP - L - β - d - heptose at indicated

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

E) TIFA-GFP oligomerization in cells treated for 30 min with digitonin alone, digitonin and wild-type S. flexneri lysate or digitonin and ADP-L-β-d-heptose at indicated concentrations


with digitonin alone , digitonin and wild - type S . flexneri lysate or digitonin and ADP - L - β - d - heptose at indicated concentrations . F ) IL - 8 production after 6 h in cells

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 I-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

with digitonin alone, digitonin and wild-type S. flexneri lysate or digitonin and ADP-L-β-d-heptose at indicated concentrations. F) IL-8 production after 6 h in cells treate


A ) Representative images of the formation of TIFA oligomers in HeLa cells infected for 30 min with dsRed - expressing wt or indicated mutants of S . flexneri ( MOI 50 ) . pHldA / C means plasmid - encoded HldA and HldC . Fluorescence intensity was adjusted between strains to optimize visualization of bacteria . Scale bar , 20

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

A) Representative images of the formation of TIFA oligomers in HeLa cells infected for 30 min with dsRed-expressing wt or indicated mutants of S. flexneri (MOI 50). pHldA/C means plasmid-encoded HldA and HldC. Fluorescence intensity was adjusted between strains to optimize visualization of bacteria. Scale bar, 20 μm


B ) Infectivity of indicated S . flexneri

B) Infectivity of indicated S. flexneri strains


C ) TIFA - GFP oligomerization after 30 min of infection with selected mutants of the LPS biosynthesis pathway ( MOI 10 )

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

C) TIFA-GFP oligomerization after 30 min of infection with selected mutants of the LPS biosynthesis pathway (MOI 10)


of the LPS biosynthesis pathway ( MOI 10 ) . D ) TIFA - GFP oligomerization after 4 h of infection with the S . flexneri

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

of the LPS biosynthesis pathway (MOI 10). D) TIFA-GFP oligomerization after 4 h of infection with the S. flexneri mutant


E ) IL - 8 production 4 h post infection ( MOI 5 )

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

E) IL-8 production 4 h post infection (MOI 5)


A ) ADP - heptose - induced TIFA - GFP oligomerization depends on ALPK1 . After siRNA transfection , HeLa cells were transfected with empty pCMV or pCMV - ALPK1 as indicated . They were then infected with wt S . flexneri ( MOI 10 ) or treated with digitonin and ADP - heptose ( 10 - 5 M ) for 30 min . As control , cells were left untreated ( mock ) or treated with

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

A) ADP-heptose-induced TIFA-GFP oligomerization depends on ALPK1. After siRNA transfection, HeLa cells were transfected with empty pCMV or pCMV-ALPK1 as indicated. They were then infected with wt S. flexneri (MOI 10) or treated with digitonin and ADP-heptose (10-5 M) for 30 min. As control, cells were left untreated (mock) or treated with digitonin


ADP - heptose - induced cytokine secretion is ALPK1 - dependent . HeLa cells were transfected with control or ALPK1 - targeting siRNAs and infected with wt S . flexneri ( MOI 2 ) or treated with ADP - heptose ( 10 - 5 M ) . As control , cells were left untreated ( mock ) or treated with digitonin . Cytokines were measured in cell culture supernatants after 8

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-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ADP-heptose-induced cytokine secretion is ALPK1-dependent. HeLa cells were transfected with control or ALPK1-targeting siRNAs and infected with wt S. flexneri (MOI 2) or treated with ADP-heptose (10-5 M). As control, cells were left untreated (mock) or treated with digitonin. Cytokines were measured in cell culture supernatants after 8 h


Representative flow cytometry data showing surface phenotypes of DCs sorted from spleens of wild type ( WT ) or Nlrc3 - / - mice and treated with LPS ( 100 ng / ml ) for 48 hours .

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

Representative flow cytometry data showing surface phenotypes of DCs sorted from spleens of wild type (WT) or Nlrc3-/- mice and treated with LPS (100 ng/ml) for 48 hours.


ELISA of cytokines in culture supernatants of DCs treated


The intracellular production of IFN - γ and IL - 17 by CD4 + T cells among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs

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

The intracellular production of IFN-γ and IL-17 by CD4+ T cells among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated


cytokines in culture supernatants among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs

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

cytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated


thymidine incorporation proliferation assay among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs

thymidine incorporation proliferation assay among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated


CFSE proliferation assay among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs

CFSE proliferation assay among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated


WT and Nlrc3 - / - mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Mean clinical scores of EAE in immunized WT ( n = 10 ) and Nlrc3 - / - mice ( n = 10 ) .

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

WT and Nlrc3-/- mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized WT (n= 10) and Nlrc3-/- mice (n= 10).


Representative flow cytometry data showing intracellular production of IFN - γ and IL - 17A by CD4 + T cells from the spinal cord and brain of WT or Nlrc3 - / - mice 26 d after EAE induction after restimulation with MOG ( 35 - 55 ) peptide . Pooled data are presented in the right panel .

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

Representative flow cytometry data showing intracellular production of IFN-γ and IL-17A by CD4+ T cells from the spinal cord and brain of WT or Nlrc3-/- mice 26 d after EAE induction after restimulation with MOG(35-55) peptide. Pooled data are presented in the right panel.


Representative flow cytometry data showing surface phenotypes of DCs from spleens of WT or Nlrc3 - / - mice 26 d after EAE induction .

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

Representative flow cytometry data showing surface phenotypes of DCs from spleens of WT or Nlrc3-/- mice 26 d after EAE induction.


Expression of Nlrc3 , Il12 , Il6 , Il23 and Il27 mRNA in DCs sorted from WT and Nlrc3 - / - mice 26 d after EAE induction , presented relative to that of Gapdh .

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

Expression of Nlrc3, Il12, Il6, Il23 and Il27 mRNA in DCs sorted from WT and Nlrc3-/- mice 26 d after EAE induction, presented relative to that of Gapdh.


DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Mean clinical scores of EAE in immunized DC ( WT ) ( n = 5 ) and DC ( NLRC3 - KO ) mice ( n = 5 ) .

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

DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized DC(WT) (n= 5) and DC(NLRC3-KO) mice (n= 5).


DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Representative flow cytometry data showing intracellular production of IFN - γ and IL - 17A by CD4 + T cells in the spinal cord and brain from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction after restimulation with MOG ( 35 - 55 ) peptide .

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

DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Representative flow cytometry data showing intracellular production of IFN-γ and IL-17A by CD4+ T cells in the spinal cord and brain from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction after restimulation with MOG(35-55) peptide.


DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Recall response to MOG ( 35 - 55 ) by splenocytes isolated from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction .

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

DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Recall response to MOG(35-55) by splenocytes isolated from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.


DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Expression of Il12 , Il6 , Il23 and Il27 mRNA in DCs sorted from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction , presented relative to that of Gapdh .

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

DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Expression of Il12, Il6, Il23 and Il27 mRNA in DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction, presented relative to that of Gapdh.


DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . CFSE proliferation assay of naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs sorted from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction .

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

DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. CFSE proliferation assay of naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.


DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . cytokine secretion of naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs sorted from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction .

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

DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. cytokine secretion of naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.


DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated with LPS ( 100 ng / ml ) for specified time . DC lysates were probed for phosphorylated p65 ( p - p65 ) , total p65 , p - AKT , AKT , p - p38 , p38 , p - ERK , ERK , p - JNK , JNK and GAPDH .

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

DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated with LPS (100 ng/ml) for specified time. DC lysates were probed for phosphorylated p65 (p-p65), total p65, p-AKT, AKT, p-p38, p38, p-ERK, ERK, p-JNK, JNK and GAPDH.


DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated for 48 hours with LPS ( 100 ng / ml ) in the presence or absence of the p38 inhibitor SB203580 ( 10 μM or indicated concentrations ) . Concentrations of IL - 12 , IL - 6 , IL - 23 and IL - 27 in supernatants were detected by ELISA .

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

DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). Concentrations of IL-12, IL-6, IL-23 and IL-27 in supernatants were detected by ELISA.


DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated for 48 hours with LPS ( 100 ng / ml ) in the presence or absence of the p38 inhibitor SB203580 ( 10 μM or indicated concentrations ) . Cytokines in culture supernatants among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs . NC : negative control .

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 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

DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). Cytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs. NC: negative control.


DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated for 48 hours with LPS ( 100 ng / ml ) in the presence or absence of the p38 inhibitor SB203580 ( 10 μM or indicated concentrations ) . CFSE proliferation assay ( D ) among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs . NC : negative control .

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 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

DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs. NC: negative control.


Activity phosphorylation of p38 were detected in DCs in the spleens from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction . Pooled data are presented in the right panel .

O O O B-MEASURED_VAR 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

Activity phosphorylation of p38 were detected in DCs in the spleens from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction. Pooled data are presented in the right panel.


DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Mean clinical scores of EAE in immunized DC ( WT ) ( n = 5 ) and DC ( NLRC3 - KO ) mice ( n = 5 ) .

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

DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized DC(WT) (n= 5) and DC(NLRC3-KO) mice (n= 5).


DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Frequencies of CD4 + T cells that express IFN - γ and IL - 17A in the spinal cord and brain from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction after restimulation with MOG ( 35 - 55 ) peptide . Pooled data are presented in the right panel .

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

DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Frequencies of CD4+ T cells that express IFN-γ and IL-17A in the spinal cord and brain from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction after restimulation with MOG(35-55) peptide. Pooled data are presented in the right panel.


DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Expression of Il12 , Il6 and Il23 mRNA in DCs sorted from spleens of DC ( WT ) , DC ( NLRC3 - KO ) , DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice 26 d after EAE induction , presented relative to that of Gapdh .

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

DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Expression of Il12, Il6 and Il23 mRNA in DCs sorted from spleens of DC(WT), DC(NLRC3-KO), DC(p38-KO) and DC(p38+NLRC3-KO) mice 26 d after EAE induction, presented relative to that of Gapdh.


DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . DC lysates were probed for p - p38 , p38 , NLRC3 and GAPDH . Densitometry quantification of band intensity were presented in the right panel .

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DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. DC lysates were probed for p-p38, p38, NLRC3 and GAPDH. Densitometry quantification of band intensity were presented in the right panel.


DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . Enzyme - linked immunosorbent assay of cytokines in culture supernatants of DCs treated with LPS for 48 hours .

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DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. Enzyme-linked immunosorbent assay of cytokines in culture supernatants of DCs treated with LPS for 48 hours.


DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . Cytokines in culture supernatants among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs treated with LPS for 48 hours .

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DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. Cytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated with LPS for 48 hours.


DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . CFSE proliferation assay ( D ) among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs treated with LPS for 48 hours .

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DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated with LPS for 48 hours.


EAE was induced by immunization of naive B6 mice with MOG ( 35 - 55 ) , and the mice were randomly divided into five groups . BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 ( LV - NLRC3 ) or only GFP ( LV - Ctrl ) were administered i . v . 4 times , once every 4 days , starting at day 10 after EAE induction . Mean clinical scores of EAE ( n = 5 mice per group ) . Arrows indicate DC vaccine administration .

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

EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. Mean clinical scores of EAE (n= 5 mice per group ). Arrows indicate DC vaccine administration.


EAE was induced by immunization of naive B6 mice with MOG ( 35 - 55 ) , and the mice were randomly divided into five groups . BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 ( LV - NLRC3 ) or only GFP ( LV - Ctrl ) were administered i . v . 4 times , once every 4 days , starting at day 10 after EAE induction . Recall proliferative to MOG ( 35 - 55 ) in splenocytes taken from DCs - treated mice 26 days after EAE induction .

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

EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. Recall proliferative to MOG (35-55) in splenocytes taken from DCs-treated mice 26 days after EAE induction.


EAE was induced by immunization of naive B6 mice with MOG ( 35 - 55 ) , and the mice were randomly divided into five groups . BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 ( LV - NLRC3 ) or only GFP ( LV - Ctrl ) were administered i . v . 4 times , once every 4 days , starting at day 10 after EAE induction . cytokine response ( D ) to MOG ( 35 - 55 ) in splenocytes taken from DCs - treated mice 26 days after EAE induction .

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

EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. cytokine response (D) to MOG (35-55) in splenocytes taken from DCs-treated mice 26 days after EAE induction.


Starvation triggers accumulation of nucleosides , bases and S7P . Wild ‐ type S . cerevisiae cells growing in minimal media were switched to minimal media containing no carbon , no nitrogen or no phosphate . After the indicated duration of starvation , the metabolome was quantified by LC ‐ MS . For media composition , see Supplementary Table S2 . For the list of absolute concentration of metabolties , see Supplementary Table S3 . Data are shown in heat map format , with each line reflecting the dynamics of a particular compound in a particular culture condition . Metabolite levels of biological duplicates were averaged , normalized to cells growing steadily in glucose ( time zero ) , and the resulting fold changes log2 transformed .

O O O 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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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

Starvation triggers accumulation of nucleosides, bases and S7P. Wild‐type S. cerevisiae cells growing in minimal media were switched to minimal media containing no carbon, no nitrogen or no phosphate. After the indicated duration of starvation, the metabolome was quantified by LC‐MS. For media composition, see Supplementary Table S2. For the list of absolute concentration of metabolties, see Supplementary Table S3. Data are shown in heat map format, with each line reflecting the dynamics of a particular compound in a particular culture condition. Metabolite levels of biological duplicates were averaged, normalized to cells growing steadily in glucose (time zero), and the resulting fold changes log2 transformed.


( A ) Experimental design for demonstrating metabolite production via macromolecule degradation . Yeast cells were grown on 2 % unlabeled glucose , and then switched to U ‐ 13C ‐ glucose for 70 min , which completely labels free metabolite but only partially labels macromolecules . Thereafter , glucose was removed and the metabolome analyzed by LC ‐ MS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Experimental design for demonstrating metabolite production via macromolecule degradation. Yeast cells were grown on 2% unlabeled glucose, and then switched to U‐13C‐glucose for 70 min, which completely labels free metabolite but only partially labels macromolecules. Thereafter, glucose was removed and the metabolome analyzed by LC‐MS.


( B ) Fraction of unlabeled nucleosides , nucleic bases and PPP intermediates as a function of starvation time , in wild ‐ type and autophagy deficient ( atg7 deletion ) yeast . The x axis represents minutes after carbon starvation , and the y axis represents fraction of unlabeled metabolites ( mean ± range of N = 2 biological replicates ) .

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(B) Fraction of unlabeled nucleosides, nucleic bases and PPP intermediates as a function of starvation time, in wild‐type and autophagy deficient (atg7 deletion) yeast. The x axis represents minutes after carbon starvation, and the y axis represents fraction of unlabeled metabolites (mean±range of N=2 biological replicates).


( C ) Ratio of metabolite levels in atg7 strain versus wild ‐ type strain in carbon starvation .

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(C) Ratio of metabolite levels in atg7 strain versus wild‐type strain in carbon starvation.


( D ) Ratio of metabolite levels in bcy1 strain and snf1 strain versus wild ‐ type strain in carbon starvation .

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(D) Ratio of metabolite levels in bcy1 strain and snf1 strain versus wild‐type strain in carbon starvation.


( E ) Ratio of metabolite levels in rapamycin treatment versus nitrogen starvation for wild ‐ type yeast . In ( C ) to ( E ) , all reported values are log2 transformed ratios ; data are mean of duplicate samples at each time point .

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(E) Ratio of metabolite levels in rapamycin treatment versus nitrogen starvation for wild‐type yeast. In (C) to (E), all reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.


( A ) Ratio of metabolite levels in sdt1 and phm8 strains versus wild ‐ type strain in carbon starvation . All reported values are log2 transformed ratios ; data are mean of duplicate samples at each time point .

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(A) Ratio of metabolite levels in sdt1 and phm8 strains versus wild‐type strain in carbon starvation. All reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.


( B ) Summary of transcripts data of PHM8 , SDT1 and other enzymes in the pathway ( Gasch et al , 2000 ; Bradley et al , 2009 ; Klosinska et al , 2011 ) . All values are log2 transformed fold changes .

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(B) Summary of transcripts data of PHM8, SDT1 and other enzymes in the pathway (Gasch et al, 2000; Bradley et al, 2009; Klosinska et al, 2011). All values are log2 transformed fold changes.


( C ) Screening of Phm8 ' s phosphatase activity against 90 phosphorylated compounds . Phosphatase activity was measured in the presence of 0 . 5 mM substrate and 5 mM Mg2 + ( pH = 7 . 0 , 30 ° C ) . Compounds with specific activity higher than 0 . 1 μmol / mg / min are shown . The x axis represents specific phosphatase activity ( μmol of phosphate produced per minute per mg of enzyme , mean ± range of N = 2 replicates ) .

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

(C) Screening of Phm8's phosphatase activity against 90 phosphorylated compounds. Phosphatase activity was measured in the presence of 0.5 mM substrate and 5 mM Mg2+ (pH=7.0, 30°C). Compounds with specific activity higher than 0.1 μmol/mg/min are shown. The x axis represents specific phosphatase activity (μmol of phosphate produced per minute per mg of enzyme, mean±range of N=2 replicates).


( D ) Top table : absolute intracellular concentration of nucleotide monophosphates in carbon starvation . Bottom plots : Phosphatase activity of Phm8 and Sdt1 as a function of CMP concentration . The x axis represents CMP concentration and the y axis represents specific phosphatase activity ( μmol of phosphate produced per minute per mg of enzyme , mean ± range of N = 2 replicates ) .

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

(D) Top table: absolute intracellular concentration of nucleotide monophosphates in carbon starvation. Bottom plots: Phosphatase activity of Phm8 and Sdt1 as a function of CMP concentration. The x axis represents CMP concentration and the y axis represents specific phosphatase activity (μmol of phosphate produced per minute per mg of enzyme, mean±range of N=2 replicates).


Confirmation that Pnp1 is the physiological purine nucleoside phosphorylase , Urh1 is the pyrimidine hydrolase , and Prm15 ( Pgm3 ) is the phosphoribomutase . Ratio of metabolite levels in pnp1 / urh1 , pnp1 , urh1 and prm15 ( pgm3 ) strains versus wild ‐ type strain during carbon starvation . Data are shown in heat map format , with each line reflecting the dynamics of the ratio of the metabolite levels in a particular strain versus wild ‐ type strain . All reported values are log2 transformed ratios ; data are mean of duplicate samples at each time point .

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

Confirmation that Pnp1 is the physiological purine nucleoside phosphorylase, Urh1 is the pyrimidine hydrolase, and Prm15 (Pgm3) is the phosphoribomutase. Ratio of metabolite levels in pnp1/urh1, pnp1, urh1 and prm15 (pgm3) strains versus wild‐type strain during carbon starvation. Data are shown in heat map format, with each line reflecting the dynamics of the ratio of the metabolite levels in a particular strain versus wild‐type strain. All reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.


( B ‐ E ) DHAP , SBP and S7P levels in wild type , tkl1 / tkl2 ( B ) , tal1 / nqm1 ( C ) and pfk1 ( E ) strains upon carbon starvation , and wild ‐ type strain upon abruptly switching from glucose to no carbon ( WT ) versus to dihydroxyacetone ( WT + DHA ) as the sole carbon source ( D ) . The x axis represents hours after carbon starvation , and the logarithmic y axis represents absolute intracellular concentration ( mean ± range of N = 2 biological replicates ) .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O 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‐E) DHAP, SBP and S7P levels in wild type, tkl1/tkl2 (B), tal1/nqm1 (C) and pfk1 (E) strains upon carbon starvation, and wild‐type strain upon abruptly switching from glucose to no carbon (WT) versus to dihydroxyacetone (WT+DHA) as the sole carbon source (D). The x axis represents hours after carbon starvation, and the logarithmic y axis represents absolute intracellular concentration (mean±range of N=2 biological replicates).


( A ) Level of hallmark metabolites and energy charge in wild type , phm8 and pnp1 / urh1 strains in starvation . The x axis represents hours after starvation , and the logarithmic y axis represents energy charge ( [ ATP ] + 0 . 5 [ ADP ] / ( [ ATP ] + [ ADP ] + [ AMP ] ) or absolute intracellular concentration ( mean ± range of N = 2 biological replicates ) .

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

(A) Level of hallmark metabolites and energy charge in wild type, phm8 and pnp1/urh1 strains in starvation. The x axis represents hours after starvation, and the logarithmic y axis represents energy charge ([ATP]+0.5[ADP]/([ATP]+[ADP]+[AMP]) or absolute intracellular concentration (mean±range of N=2 biological replicates).


( B and C ) Growth and viability of wild type , phm8 and pnp1 / urh1 strains in starvation .

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(B and C) Growth and viability of wild type, phm8 and pnp1/urh1 strains in starvation.


( D ) Growth of wild type , phm8 and pnp1 / urh1 strains in the switch from glucose to glycerol + ethanol . The x axis represents hours after starvation and the logarithmic y axis represents optical density ( A600 ) in ( B and D ) and percentage of live cells calculated by dividing the number of colonies formed after starvation by the number of colonies formed before starvation in ( C ) ( mean ± range of N = 2 biological replicates ) .

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(D) Growth of wild type, phm8 and pnp1/urh1 strains in the switch from glucose to glycerol+ethanol. The x axis represents hours after starvation and the logarithmic y axis represents optical density (A600) in (B and D) and percentage of live cells calculated by dividing the number of colonies formed after starvation by the number of colonies formed before starvation in (C) (mean±range of N=2 biological replicates).


( C and D ) DHAP , S7P , G6P , and glutathione levels and NADPH / NADP + ratio in wild type , phm8 and pnp1 / urh1 strains in the experiment shown in ( A ) The x axis represents minutes after oxidative stress , and the y axis represents absolute intracellular concentration or ratio of intracellular concentration ( mean ± range of N = 2 biological replicates ) .

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(C and D) DHAP, S7P, G6P, and glutathione levels and NADPH/NADP+ ratio in wild type, phm8 and pnp1/urh1 strains in the experiment shown in (A) The x axis represents minutes after oxidative stress, and the y axis represents absolute intracellular concentration or ratio of intracellular concentration (mean±range of N=2 biological replicates).


( E ) Viability of wild type , phm8 and pnp1 / urh1 strains in oxidative stress . The y axis represents percentage of survived cells calculated by dividing the number of colonies formed after oxidative stress by the number of colonies formed under the same starvation condition but without H2O2 treatment ( mean ± range of N = 2 biological replicates ) .

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(E) Viability of wild type, phm8 and pnp1/urh1 strains in oxidative stress. The y axis represents percentage of survived cells calculated by dividing the number of colonies formed after oxidative stress by the number of colonies formed under the same starvation condition but without H2O2 treatment (mean±range of N=2 biological replicates).