Human melanoma cells were stimulated for 10 min with 1 U / ml purified ASM or 10 μM C16 ceramide ( C16 - Cer ) in the presence or absence of 500 ng ceramidase ( CDase ) or 1 μg / ml neutralizing anti - ceramide antibodies ( anti - Cer ) , and lysed and active β1 integrin was immunoprecipitated using the HUTS - 4 antibody . Octylglucopyranoside ( OGP , final concentration 0 . 01 % ) served to resuspend C16 ceramide . Internalization was excluded by addition of cytochalasin B ( CTB ) as indicated . Samples were separated by SDS - PAGE and blotted with an activation - independent anti - β1 Integrin antibody to detect the amount of immunoprecipitated , that is active β1 integrin . Control immunoprecipitates ( control Ipt ) were performed with an irrelevant isotype control antibody . Activation of β1 integrin in human melanoma cells by incubation with Asm or C16 ceramide was confirmed by FACS analysis upon staining with FITC - labeled HUTS - 4 antibodies .

B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O

Human melanoma cells were stimulated for 10 min with 1 U/ml purified ASM or 10 μM C16 ceramide (C16-Cer) in the presence or absence of 500 ng ceramidase (CDase) or 1 μg/ml neutralizing anti-ceramide antibodies (anti-Cer), and lysed and active β1 integrin was immunoprecipitated using the HUTS-4 antibody. Octylglucopyranoside (OGP, final concentration 0.01%) served to resuspend C16 ceramide. Internalization was excluded by addition of cytochalasin B (CTB) as indicated. Samples were separated by SDS-PAGE and blotted with an activation-independent anti-β1 Integrin antibody to detect the amount of immunoprecipitated, that is active β1 integrin. Control immunoprecipitates (control Ipt) were performed with an irrelevant isotype control antibody. Activation of β1 integrin in human melanoma cells by incubation with Asm or C16 ceramide was confirmed by FACS analysis upon staining with FITC-labeled HUTS-4 antibodies.


Incubation of human melanoma with human platelets or ASM for 5 min triggers co - clustering of ceramide and activated β1 integrin on the surface of the tumor cells . Cells were stained with FITC - coupled anti - active β1 integrin ( HUTS - 4 ) antibodies and Cy3 - coupled anti - ceramide antibodies . Shown are representative stainings from each four independent experiments .

O O B-ORGANISM O O B-ORGANISM B-CELL_TYPE O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

Incubation of human melanoma with human platelets or ASM for 5 min triggers co-clustering of ceramide and activated β1 integrin on the surface of the tumor cells. Cells were stained with FITC-coupled anti-active β1 integrin (HUTS-4) antibodies and Cy3-coupled anti-ceramide antibodies. Shown are representative stainings from each four independent experiments.


B16F10tumor cells or B16F10 transfected with siRNA targeting Asm expression or with scrambled control siRNA were labeled with [ 3H ] thymidine , washed , and treated ( + ) for 10 min with 1 U / ml ASM , 10 μM C16ceramide , 10 μM S1P , 10 μM PDMP , 20 μM sphingosine kinase inhibitor SKI II , and 10 μM myriocin , or left untreated ( − ) . The cells were then injected intravenously into wild - type or Asm - deficient mice as indicated . If indicated , 10 μg of arginine - glycine - aspartic acid ( RGD ) peptide or 10 μg / ml neutralizing anti - β1 integrin antibodies were added to B16F10tumor cells together with ASM for 15 min prior injection . Mice were sacrificed 30 min after tumorcell injection , the lungs carefully flushed via the right heart , and the radioactivity in the lung as a measurement for adherent tumor cells was determined . Shown is the mean ± SD of the counts per minute ( cpm ) in the lung lysates from three independent experiments . Statistical significance was determined by analysis of variance ( ANOVA ) followed by Tukey ' s test for multiple comparison to determine P - values .

B-CELL_LINE O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O O O O B-TISSUE O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B16F10tumor cells or B16F10 transfected with siRNA targeting Asm expression or with scrambled control siRNA were labeled with [3H]thymidine, washed, and treated (+) for 10 min with 1 U/ml ASM, 10 μM C16ceramide, 10 μM S1P, 10 μM PDMP, 20 μM sphingosine kinase inhibitor SKI II, and 10 μM myriocin, or left untreated (−). The cells were then injected intravenously into wild-type or Asm-deficient mice as indicated. If indicated, 10 μg of arginine-glycine-aspartic acid (RGD) peptide or 10 μg/ml neutralizing anti-β1 integrin antibodies were added to B16F10tumor cells together with ASM for 15 min prior injection. Mice were sacrificed 30 min after tumorcell injection, the lungs carefully flushed via the right heart, and the radioactivity in the lung as a measurement for adherent tumor cells was determined. Shown is the mean ± SD of the counts per minute (cpm) in the lung lysates from three independent experiments. Statistical significance was determined by analysis of variance (ANOVA) followed by Tukey's test for multiple comparison to determine P-values.


A Amitriptyline ( 2 mg / kg , Ami ) was intraperitoneally injected into C57BL / 6mice for five times every 12 h . Sixty minutes after the last injection , 1 × 105B16F10tumor cells were intravenously injected . Control experiments confirmed that amitriptyline inhibited Asm activity in the blood by approximately 85 % . Asm heterozygous mice were injected with 1 × 105B16F10tumor cells . The number of lungmetastases was determined after 14 days . Shown is the mean ± SD from six mice each , ANOVA followed by Tukey ' s test for multiple comparison . P - values are indicated .

O B-SMALL_MOLECULE O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O 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-SMALL_MOLECULE O B-GENEPROD O O O B-TISSUE O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O

A Amitriptyline (2 mg/kg, Ami) was intraperitoneally injected into C57BL/6mice for five times every 12 h. Sixty minutes after the last injection, 1 × 105B16F10tumor cells were intravenously injected. Control experiments confirmed that amitriptyline inhibited Asm activity in the blood by approximately 85%. Asm heterozygous mice were injected with 1 × 105B16F10tumor cells. The number of lungmetastases was determined after 14 days. Shown is the mean ± SD from six mice each, ANOVA followed by Tukey's test for multiple comparison. P-values are indicated.


B , C MT / ret cells ( 105 ) suspended in 200 μl Matrigel / PBS ( 1 : 1 ) were injected s . c . in the left and right flanks of 8 - week - old Asm - deficient mice or wild - type mice . Mice were sacrificed at day 20 after injection , and spleens were inspected for the presence of metastases . The representative photographs ( B ) show the results from one of four experiments , and the quantitative analysis is displayed in ( C ) .

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B, C MT/ret cells (105) suspended in 200 μl Matrigel/PBS (1:1) were injected s.c. in the left and right flanks of 8-week-old Asm-deficient mice or wild-type mice. Mice were sacrificed at day 20 after injection, and spleens were inspected for the presence of metastases. The representative photographs (B) show the results from one of four experiments, and the quantitative analysis is displayed in (C).


D Platelets were isolated from wild - type or Asm - deficient mice by density - gradient centrifugation , and 1 × 107 platelets were incubated with 1 × 105 MT / ret melanoma cells . The samples were lysed , and Asm activity in cell lysates was determined . In unstimulated samples ( time point 0 of co - incubation ) , tumorcell and platelet lysates were admixed after lysis .

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D Platelets were isolated from wild-type or Asm-deficient mice by density-gradient centrifugation, and 1 × 107 platelets were incubated with 1 × 105 MT/ret melanoma cells. The samples were lysed, and Asm activity in cell lysates was determined. In unstimulated samples (time point 0 of co-incubation), tumorcell and platelet lysates were admixed after lysis.


E To measure secretion of Asm by platelets , tumor cells and platelets were co - incubated for 30 s , the samples were pelleted , the supernatants were acidified , and the Asm activity was measured . All Asm activity measurements were performed in the presence of 100 μM Zn2 + .

O O O O O B-GENEPROD O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O

E To measure secretion of Asm by platelets, tumor cells and platelets were co-incubated for 30 s, the samples were pelleted, the supernatants were acidified, and the Asm activity was measured. All Asm activity measurements were performed in the presence of 100 μM Zn2+.


F Local tumor growth in wild - type or Asm - deficient mice at the flank was measured at day 20 .

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F Local tumor growth in wild-type or Asm-deficient mice at the flank was measured at day 20.


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 4 ( A , B ) 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 = 4 for A , B )

O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4 (A,B)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 = 4 for A,B)


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 4 ( A , B ) 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 = 4 for A , B )

O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 4 (A,B) 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 = 4 for A,B)


( 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 B-TISSUE O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-TISSUE 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-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-TISSUE O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O 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-TISSUE 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-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O 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-GENEPROD O O B-GENEPROD O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD 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-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O 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-GENEPROD O O B-GENEPROD O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD 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 .

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(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 ) .

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(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 ) .

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(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 .

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(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 .

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(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 .

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(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 % ) .

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(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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM 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-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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(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-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD 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 .

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(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 .

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(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-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM 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-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O 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-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM 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-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O 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-GENEPROD 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-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM 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 B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM 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-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD 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-EXP_ASSAY O B-GENEPROD I-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD 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 B-TISSUE O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_LINE O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD

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 B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE 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 B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O B-GENEPROD

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 B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-ORGANISM I-ORGANISM I-ORGANISM

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-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD O B-GENEPROD 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 B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD 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 B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD 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-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-EXP_ASSAY O O O O B-GENEPROD O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY 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

O O B-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM

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-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM

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-GENEPROD I-GENEPROD I-GENEPROD 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE

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 B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE

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-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-DISEASE O O B-EXP_ASSAY I-EXP_ASSAY O B-DISEASE O O O O O O O O O B-GENEPROD O O O B-ORGANISM 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O B-TISSUE O O O B-GENEPROD O O O B-ORGANISM O O O B-DISEASE O O O O B-SMALL_MOLECULE 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O B-TISSUE O O O B-GENEPROD O O O B-ORGANISM O O O B-DISEASE 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-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O B-GENEPROD O O O B-ORGANISM O O O B-DISEASE O O O O O O O B-GENEPROD 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 ) .

B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-DISEASE O O B-EXP_ASSAY I-EXP_ASSAY O B-DISEASE O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM 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 .

B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-DISEASE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O B-TISSUE O B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O B-DISEASE O O O O B-SMALL_MOLECULE 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 .

B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-DISEASE O O O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O B-DISEASE 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 .

B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-DISEASE O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O B-DISEASE O O O O O O O B-GENEPROD 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 .

B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-DISEASE O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O B-DISEASE 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 .

B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-DISEASE O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O B-DISEASE 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 .

B-CELL_TYPE O O O B-TISSUE O O O B-GENEPROD O O O B-ORGANISM O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 .

B-CELL_TYPE O O O B-TISSUE O O O B-GENEPROD O O O B-ORGANISM O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY 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 .

B-CELL_TYPE O O O B-TISSUE O O O B-GENEPROD O O O B-ORGANISM O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE 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 .

B-CELL_TYPE O O O B-TISSUE O O O B-GENEPROD O O O B-ORGANISM O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE 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 B-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE O O B-TISSUE O B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O B-DISEASE 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 ) .

B-CELL_TYPE O B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-DISEASE O O B-EXP_ASSAY I-EXP_ASSAY O B-DISEASE O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM 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 .

B-CELL_TYPE O B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-DISEASE O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE I-TISSUE O B-TISSUE O B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O B-DISEASE O O O O B-SMALL_MOLECULE 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 .

B-CELL_TYPE O B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-DISEASE O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O B-TISSUE O B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-DISEASE O O O O O O O B-GENEPROD 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 .

B-CELL_TYPE O O O O B-CELL_TYPE O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O

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.