A ) and B ) Representative images of stress granule proficient ( siControl ) and deficient ( siG3BP1 / 2 ) MelJuSo cells expressing the nuclear UPS reporter NLS - GFP - CL1 ( A ) and the cytoplasmic UPS reporter NES - GFP - CL1 ( B ) . The cells were left untreated ( - HS ) , exposed to 43 ° C for 30 minutes ( HS ) , or exposed to 43 ° C for 30 minutes and followed by 4 hours recovery ( HS + 4h Rec . ) . Nucleolar accumulation of NLS - GFP - CL1 during the recovery is indicated by arrows in A ) . C ) and D ) Quantifications of the mean cellular GFP fluorescence intensities of A ) and B ) normalized to untreated control cells ( siControl - HS ) . The frequency and distribution of the relative fluorescence intensities per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits . ( n = 3 independent experiments , > 1000 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 , ns : not significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) and B) Representative images of stress granule proficient (siControl) and deficient (siG3BP1/2) MelJuSo cells expressing the nuclear UPS reporter NLS-GFP-CL1 (A) and the cytoplasmic UPS reporter NES-GFP-CL1 (B). The cells were left untreated (- HS), exposed to 43°C for 30 minutes (HS), or exposed to 43°C for 30 minutes and followed by 4 hours recovery (HS + 4h Rec.). Nucleolar accumulation of NLS-GFP-CL1 during the recovery is indicated by arrows in A). C) and D) Quantifications of the mean cellular GFP fluorescence intensities of A) and B) normalized to untreated control cells (siControl - HS). The frequency and distribution of the relative fluorescence intensities per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits. (n=3 independent experiments, >1000 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01, ns: not significant).


E ) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and the nuclear UPS reporter NLS - GFP - CL1 in control ( siControl , left panel ) and G3BP1 / 2 - depleted ( siGRBP1 / 2 , right panel ) MelJuSo cells exposed to a 30 min 43 ° C heat shock , followed by 4 hours at 37 ° C ( Recovery ) . Nucleolar accumulation of NLS - GFP - CL1 is indicated by arrows . F ) Quantification of the nucleolus / nucleoplasm ratios of NLS - GFP - CL1 intensities in images of experiment shown in E ) . The frequency and distribution of the ratio per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits . ( n = 3 independent experiments , 100 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 ) .

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

E) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and the nuclear UPS reporter NLS-GFP-CL1 in control (siControl, left panel) and G3BP1/2-depleted (siGRBP1/2, right panel) MelJuSo cells exposed to a 30 min 43°C heat shock, followed by 4 hours at 37°C (Recovery). Nucleolar accumulation of NLS-GFP-CL1 is indicated by arrows. F) Quantification of the nucleolus/nucleoplasm ratios of NLS-GFP-CL1 intensities in images of experiment shown in E). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits. (n=3 independent experiments, 100 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).


A ) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin - labelled proteins in parental and G3BP1 / 2 knock - out U2OS cells . Cells were subjected to 43 ° C heat shock . B ) Quantification of the nucleus / cytoplasm ratios of puromycin intensities in images from A ) . The frequency and distribution of the ratio per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits ( n = 3 independent experiments , > 50 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin-labelled proteins in parental and G3BP1/2 knock-out U2OS cells. Cells were subjected to 43°C heat shock. B) Quantification of the nucleus/cytoplasm ratios of puromycin intensities in images from A). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).


E ) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin - labelled proteins in control ( siControl ) and Hsp70 - depleted ( siHsp70 ) U2OS cells . Cells were subjected to 43 ° C heat shock F ) Quantification of the numbers of puromycin positive TIA dots per cell in images from E ) . The numbers of positive foci per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits ( n = 3 independent experiments , > 50 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 ) .

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

E) Representative confocal images of immunofluorescent staining of the stress granule marker TIA1 and puromycin-labelled proteins in control (siControl) and Hsp70-depleted (siHsp70) U2OS cells. Cells were subjected to 43°C heat shock F) Quantification of the numbers of puromycin positive TIA dots per cell in images from E). The numbers of positive foci per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).


G ) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and puromycin - labelled proteins in control ( siControl ) and Hsp70 - depleted ( siHsp70 ) G3BP1 / 2 knock - out U2OS cells . Cells were subjected to 43 ° C heat shock . H ) Quantification of the nucleolus / nucleoplasm ratio of puromycin intensities in images from G ) . The frequency and distribution of the ratio per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits ( n = 3 independent experiments , > 50 cells analyzed per condition , Mann - Whitney test , * * P < 0 . 01 ) .

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

G) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and puromycin-labelled proteins in control (siControl) and Hsp70-depleted (siHsp70) G3BP1/2 knock-out U2OS cells. Cells were subjected to 43°C heat shock. H) Quantification of the nucleolus/nucleoplasm ratio of puromycin intensities in images from G). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).


B ) Representative confocal images of immunofluorescent staining of HSF1 in parental and G3BP1 / 2 knock - out U2OS cells directly after heat shock ( + HS ) and 2 hours after 2 hours recovery ( HS + 2h Recovery ) . C ) Quantification of the number of HSF1 foci per cell in images from B ) , shown as box plot with median and 5 - 95 percentiles ( n = 3 independent experiments , > 300 cells analyzed per condition , Kruskal - Wallis test , * * P < 0 . 01 ) .

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

B) Representative confocal images of immunofluorescent staining of HSF1 in parental and G3BP1/2 knock-out U2OS cells directly after heat shock (+ HS) and 2 hours after 2 hours recovery (HS + 2h Recovery). C) Quantification of the number of HSF1 foci per cell in images from B), shown as box plot with median and 5-95 percentiles (n=3 independent experiments, >300 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).


D ) Western blot analysis of parental and G3BP1 / 2 knock - out U2OS cells that were left untreated ( - HS ) , heat shocked ( HS ) , and followed for 1 , 2 , and 4 hours after heat shock ( Rec ) . The blots were probed with antibodies against HSF1 and GAPDH ( * indicated phosphorylated HSF1 , and * * indicates unphosphorylated HSF1 ) .

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

D) Western blot analysis of parental and G3BP1/2 knock-out U2OS cells that were left untreated (- HS), heat shocked (HS), and followed for 1, 2, and 4 hours after heat shock (Rec). The blots were probed with antibodies against HSF1 and GAPDH (* indicated phosphorylated HSF1, and ** indicates unphosphorylated HSF1).


A ) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and SUMO2 / 3 in parental and G3BP1 / 2 knock - out U2OS cells . Cells were subjected to a heat shock followed by 2 hours recovery . B ) Quantification of the nucleolus / nucleoplasm ratio of SUMO2 / 3 intensities in images from A ) . The frequency and distribution of the ratio per cell are shown as violin plots . The solid lines in each distribution represent the median , and dash lines represent the upper and lower interquartile range limits ( n = 3 independent experiments , > 50 cells analyzed per condition , Mann - Whitney test , * * P < 0 . 01 ) .

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

A) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and SUMO2/3 in parental and G3BP1/2 knock-out U2OS cells. Cells were subjected to a heat shock followed by 2 hours recovery. B) Quantification of the nucleolus/nucleoplasm ratio of SUMO2/3 intensities in images from A). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).


C ) Representative confocal images of immunofluorescent staining of PML bodies ( PML ) and SUMO2 / 3 in parental and G3BP1 / 2 knock - out U2OS cells . Cells were heat shocked followed by 2 hours recovery . Arrows indicate dots positive for PML and SUMO2 / 3 staining . D ) Quantification of SUMO2 / 3 positive PML puncta in images from C ) . Data represent the mean ± SD ( n = 3 independent experiments , > 50 cells analyzed per condition , Mann - Whitney test , * * P < 0 . 01 ) .

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

C) Representative confocal images of immunofluorescent staining of PML bodies (PML) and SUMO2/3 in parental and G3BP1/2 knock-out U2OS cells. Cells were heat shocked followed by 2 hours recovery. Arrows indicate dots positive for PML and SUMO2/3 staining. D) Quantification of SUMO2/3 positive PML puncta in images from C). Data represent the mean ± SD (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).


E ) Analysis of SUMO2 / 3 conjugates in parental and G3BP1 / 2 knock - out U2OS cells that were left untreated ( - HS ) , exposed to a heat shock ( + HS ) , and followed for 2 hours ( 2h Rec . ) with or without 100 nM proteasome inhibitor epoxomicin ( EPX ) .

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

E) Analysis of SUMO2/3 conjugates in parental and G3BP1/2 knock-out U2OS cells that were left untreated (- HS), exposed to a heat shock (+ HS), and followed for 2 hours (2h Rec.) with or without 100 nM proteasome inhibitor epoxomicin (EPX).


A ) Representative confocal images of immunofluorescent staining of SUMO2 / 3 and TDP43 in parental and G3BP1 / 2 knock - out U2OS cells . Cells were exposed to a heat shocked followed by 2 hours recovery . Arrows indicate dots positive for TDP - 43 and SUMO2 / 3 staining . B ) Quantification of the percentage of SUMO2 / 3 positive TDP43 puncta in images from experiment shown in A ) . . Data represent the mean ± SD ( n = 3 independent experiments , > 50 cells analyzed per condition , Mann - Whitney test , * * P < 0 . 01 ) .

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

A) Representative confocal images of immunofluorescent staining of SUMO2/3 and TDP43 in parental and G3BP1/2 knock-out U2OS cells. Cells were exposed to a heat shocked followed by 2 hours recovery. Arrows indicate dots positive for TDP-43 and SUMO2/3 staining. B) Quantification of the percentage of SUMO2/3 positive TDP43 puncta in images from experiment shown in A)..Data represent the mean ± SD (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).


D ) Analysis of detergent SDS and NP40 soluble fractions of TDP43 in parental and G3BP1 / 2 knock - out U2OS cells that were left untreated ( - HS ) , exposed to 43 ° C ( + HS ) followed by 2 hours recovery ( 2h Rec . ) in absence or presence of 100 nM proteasome inhibitor epoxomicin ( EPX ) . E ) Quantification of the TDP43 band densities in the SDS - soluble fraction . Data represent the mean ± SD ( n = 3 independent experiments , Student ' s unpaired t - test , * P < 0 . 05 ) .

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

D) Analysis of detergent SDS and NP40 soluble fractions of TDP43 in parental and G3BP1/2 knock-out U2OS cells that were left untreated (-HS), exposed to 43°C (+HS) followed by 2 hours recovery (2h Rec.) in absence or presence of 100 nM proteasome inhibitor epoxomicin (EPX). E) Quantification of the TDP43 band densities in the SDS-soluble fraction. Data represent the mean ± SD (n=3 independent experiments, Student's unpaired t-test, * P<0.05).


A ) Analysis of SUMO2 / 3 conjugates in control ( siControl ) or RNF4 - depleted ( siRNF4 ) G3BP1 / 2 knock - out U2OS cells . Cells were left untreated ( - HS ) , exposed to 43 ° C ( + HS ) , followed by 2 hours recovery ( HS + 2h Rec . ) in the absence or presence of 100 nM proteasome inhibitor epoxomicin ( EPX ) . B ) Quantification of the total SUMO2 / 3 band densities . Data represent the mean ± SD ( n = 3 independent experiments , * P < 0 . 05 ) .

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

A) Analysis of SUMO2/3 conjugates in control (siControl) or RNF4-depleted (siRNF4) G3BP1/2 knock-out U2OS cells. Cells were left untreated (-HS), exposed to 43°C (+ HS), followed by 2 hours recovery (HS + 2h Rec.) in the absence or presence of 100 nM proteasome inhibitor epoxomicin (EPX). B) Quantification of the total SUMO2/3 band densities. Data represent the mean ± SD (n=3 independent experiments, *P<0.05).


E ) Representative confocal images of control ( siControl ) , RNF4 - depleted ( siRNF4 ) , G3BP1 / 2 - depleted ( siG3BP1 / 2 ) and G3BP1 / 2 + RNF4 - depleted ( siG3BP1 / 2 + siRNF4 ) MelJuSo cells expressing the nuclear UPS reporter NLS - GFP - CL1 . Cells were exposed to heat shock followed by 4 hours recovery in the absence or presence of the proteasome inhibitor epoxomicin ( EPX ) . The nucleolar marker nucleolin was stained by immunofluorescence .

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

E) Representative confocal images of control (siControl), RNF4-depleted (siRNF4), G3BP1/2-depleted (siG3BP1/2) and G3BP1/2+RNF4-depleted (siG3BP1/2 + siRNF4) MelJuSo cells expressing the nuclear UPS reporter NLS-GFP-CL1. Cells were exposed to heat shock followed by 4 hours recovery in the absence or presence of the proteasome inhibitor epoxomicin (EPX). The nucleolar marker nucleolin was stained by immunofluorescence.


A ) Representative confocal images of immunofluorescent staining of FLAG - tagged ubiquitin ( FLAG - Ub ) and mutant ataxin1 ( ATX1 ) in parental and G3BP1 / 2 knock - out U2OS cells . Cells were subjected to heat shock followed by 4 hours recovery . B ) Frequency distribution of ataxin - 1 inclusions per cell from three independent experiments .

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

A) Representative confocal images of immunofluorescent staining of FLAG-tagged ubiquitin (FLAG-Ub) and mutant ataxin1 (ATX1) in parental and G3BP1/2 knock-out U2OS cells. Cells were subjected to heat shock followed by 4 hours recovery. B) Frequency distribution of ataxin-1 inclusions per cell from three independent experiments.


A WT and Ccnc - / - MEF cultures were treated with 0 . 4 mM H2O2 as indicated , extracts prepared and immunoprecipitated with antibody recognizing the active conformation of Bax . The immunoprecipitates were subjected to Western blot analysis and probed for total Bax or cyclin C . Whole cell extracts ( WCE ) were subjected to Western blot analysis as indicated to control for protein concentrations in the extracts . Molecular weight markers ( kDa ) are indicated on the left .

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

A WT and Ccnc-/- MEF cultures were treated with 0.4 mM H2O2 as indicated, extracts prepared and immunoprecipitated with antibody recognizing the active conformation of Bax. The immunoprecipitates were subjected to Western blot analysis and probed for total Bax or cyclin C. Whole cell extracts (WCE) were subjected to Western blot analysis as indicated to control for protein concentrations in the extracts. Molecular weight markers (kDa) are indicated on the left.


B Cyclin C and Bax directly interact . Recombinant Bax and SUMO - His6 - cyclin C were either incubated together or separately at room temperature then passed over Co + + resin . The elutions were subjected to Western blot analysis probing for Bax and cyclin C ( lanes 4 - 6 ) . Input controls for each sample ( 1 / 10 ) are included in lanes 1 - 3 . Molecular weight markers ( kDa ) are indicated on the left .

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

B Cyclin C and Bax directly interact. Recombinant Bax and SUMO-His6-cyclin C were either incubated together or separately at room temperature then passed over Co++ resin. The elutions were subjected to Western blot analysis probing for Bax and cyclin C (lanes 4-6). Input controls for each sample (1/10) are included in lanes 1-3. Molecular weight markers (kDa) are indicated on the left.


C Mitochondrial relocalization of cyclin C is required for Bax activation . The experiment described in ( A ) was repeated except that H2O2 treated cells were treated with 1 µM pifithrin - µ ( PFT ) for 24 h as indicated . Molecular weight markers ( kDa ) are indicated on the left .

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

C Mitochondrial relocalization of cyclin C is required for Bax activation. The experiment described in (A) was repeated except that H2O2 treated cells were treated with 1 µM pifithrin-µ (PFT) for 24 h as indicated. Molecular weight markers (kDa) are indicated on the left.


D Cyclin C is required for efficient Bax - Bcl - XL disassociation . Extracts prepared from MEF cells with the indicated genotypes following treatment with H2O2 ( 0 . 4 mM , 4 h ) were immunoprecipitated with active conformation Bax antibodies . The immunoprecipitates were subjected to Western blot analysis probing with Bcl - XL antibodies . Western blot analysis of WCE was performed as input concentration controls . Molecular weight markers ( kDa ) are indicated on the left .

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

D Cyclin C is required for efficient Bax-Bcl-XL disassociation. Extracts prepared from MEF cells with the indicated genotypes following treatment with H2O2 (0.4 mM, 4 h) were immunoprecipitated with active conformation Bax antibodies. The immunoprecipitates were subjected to Western blot analysis probing with Bcl-XL antibodies. Western blot analysis of WCE was performed as input concentration controls. Molecular weight markers (kDa) are indicated on the left.


E The mitochondrial fission complex is required for cyclin C - dependent Bax activation . Extracts were prepared MEF cultures with the indicated genotypes treated with H2O2 ( 0 . 4 mM , 4 h ) . The extracts were treated as in A . Molecular weight markers ( kDa ) are indicated on the left .

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

E The mitochondrial fission complex is required for cyclin C-dependent Bax activation. Extracts were prepared MEF cultures with the indicated genotypes treated with H2O2 (0.4 mM, 4 h). The extracts were treated as in A. Molecular weight markers (kDa) are indicated on the left.


F Drp1 is required for cyclin C - Bax interaction . Extracts were prepared from wild type or Drp1 - / - MEF cultures treated or not with H2O2 and analyzed for the presence of Bax and cyclin C as described in A . Molecular weight markers ( kDa ) are indicated on the left .

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

F Drp1 is required for cyclin C-Bax interaction. Extracts were prepared from wild type or Drp1-/- MEF cultures treated or not with H2O2 and analyzed for the presence of Bax and cyclin C as described in A. Molecular weight markers (kDa) are indicated on the left.


A Wild type MEF cells were fixed before ( control ) and following S - HAD treatment ( 10 µM , 4 h ) then nuclei ( DAPI ) , cyclin C ( indirect immunofluorescence ) and mitochondria ( MitoTracker Red ) were visualized by fluorescence microscopy . Merge panels present DAPI staining ( blue ) only . Zoom panels ( 4X ) are indicated by the boxes in the merge panels . Green arrows indicate cyclin C and mitochondrial co - localization . Bar indicates 10 µM .

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

A Wild type MEF cells were fixed before (control) and following S-HAD treatment (10 µM, 4 h) then nuclei (DAPI), cyclin C (indirect immunofluorescence) and mitochondria (MitoTracker Red) were visualized by fluorescence microscopy. Merge panels present DAPI staining (blue) only. Zoom panels (4X) are indicated by the boxes in the merge panels. Green arrows indicate cyclin C and mitochondrial co-localization. Bar indicates 10 µM.


B Timecourse experiment with wild type and Ccnc - / - MEF cells treated with S - HAD ( 10 µM ) for the times indicated ( n = 3 ) . Error bars indicate SD . Asterisks indicate p values < 0 . 05 from pretreatment control ( Student ' s T - Test ) .

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

B Timecourse experiment with wild type and Ccnc-/- MEF cells treated with S-HAD (10 µM) for the times indicated (n=3). Error bars indicate SD. Asterisks indicate p values <0.05 from pretreatment control (Student's T-Test).


MEF cells were fixed before ( control ) and following S - HAD treatment ( 10 µM , 4 h ) then nuclei ( DAPI ) , cyclin C ( indirect immunofluorescence ) and mitochondria ( MitoTracker Red ) were visualized by fluorescence microscopy . Merge panels present DAPI staining ( blue ) only . S - HAD peptide activity requires cyclin C . The experiment described in A was repeated with Ccnc - / - MEF cultures .

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

MEF cells were fixed before (control) and following S-HAD treatment (10 µM, 4 h) then nuclei (DAPI), cyclin C (indirect immunofluorescence) and mitochondria (MitoTracker Red) were visualized by fluorescence microscopy. Merge panels present DAPI staining (blue) only. S-HAD peptide activity requires cyclin C. The experiment described in A was repeated with Ccnc-/- MEF cultures.


D S - HAD treatment increases Bax mitochondrial localization . Western blot analysis of the indicated proteins in nuclear and mitochondrial fractions before and following S - HAD treatment ( 10 µM , 4 h ) . Atp5A and fibrillarin levels controlled for mitochondrial and nuclear loadings , respectively . Molecular weight markers ( kDa ) are indicated on the left .

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

D S-HAD treatment increases Bax mitochondrial localization. Western blot analysis of the indicated proteins in nuclear and mitochondrial fractions before and following S-HAD treatment (10 µM, 4 h). Atp5A and fibrillarin levels controlled for mitochondrial and nuclear loadings, respectively. Molecular weight markers (kDa) are indicated on the left.


E Bax activation was monitored in wild - type MEF cultures treated with S - HAD ( 10 µM ) for the times indicated . The anti - oxidant N - acetyl cysteine ( NAC ) was added ( 1 mM ) 2 h prior to S - HAD treatment . Active Bax immunoprecipitates were subjected to Western blot analysis for Bax and cyclin C as indicated . Bax , cyclin C and ß - actin levels were monitored in extract preparations as input controls . Molecular weight markers ( kDa ) are indicated on the left .

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

E Bax activation was monitored in wild-type MEF cultures treated with S-HAD (10 µM) for the times indicated. The anti-oxidant N-acetyl cysteine (NAC) was added (1 mM) 2 h prior to S-HAD treatment. Active Bax immunoprecipitates were subjected to Western blot analysis for Bax and cyclin C as indicated. Bax, cyclin C and ß-actin levels were monitored in extract preparations as input controls. Molecular weight markers (kDa) are indicated on the left.


A Caspase activation was measured by fluorescent cell analysis in wild - type MEF cells treated with S - HAD ( 10 µM , 24 h ) or cisplatin ( CP , 30 µM , 16 h ) . n = 3 . Error bars indicate SD with * and * * indicate p < 0 . 05 and p < 0 . 01 , respectively using Student ' s T test .

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

A Caspase activation was measured by fluorescent cell analysis in wild-type MEF cells treated with S-HAD (10 µM, 24 h) or cisplatin (CP, 30 µM, 16 h). n = 3. Error bars indicate SD with * and ** indicate p <0.05 and p<0.01, respectively using Student's T test.


B PARP cleavage was not induced by S - HAD treatment . PARP cleavage was monitored by Western blot analysis in Hela cells treated with S - HAD or cisplatin ( CP ) as described in ( A ) . The blot was stripped and reprobed with ß - actin antibodies as a loading control . Molecular weight markers ( kDa ) are indicated on the left .

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

B PARP cleavage was not induced by S-HAD treatment. PARP cleavage was monitored by Western blot analysis in Hela cells treated with S-HAD or cisplatin (CP) as described in (A). The blot was stripped and reprobed with ß-actin antibodies as a loading control. Molecular weight markers (kDa) are indicated on the left.


C Wild type and Ccnc - / - MEF cultures were treated with S - HAD and cisplatin ( CP ) as just described . Cells were imaged and examined for a " rounding up " phenotype diagnostic for death .

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

C Wild type and Ccnc-/- MEF cultures were treated with S-HAD and cisplatin (CP) as just described. Cells were imaged and examined for a "rounding up" phenotype diagnostic for death.


D Cyclin C is required for normal Bax oligomerization in response to cisplatin . Whole cell extracts prepared from DSP crosslinked wild type and CCNC - / - MEF cells exposed to cisplatin ( 30 µM , 16 h ) were probed for total Bax . Bax multimers are indicated . Molecular weight markers ( kDa ) are indicated on the left .

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

D Cyclin C is required for normal Bax oligomerization in response to cisplatin. Whole cell extracts prepared from DSP crosslinked wild type and CCNC-/- MEF cells exposed to cisplatin (30 µM, 16 h) were probed for total Bax. Bax multimers are indicated. Molecular weight markers (kDa) are indicated on the left.


E S - HAD does not induce Bax oligomerization . Experiments described in D were repeated with cultures treated with S - HAD ( 10 µM , 24 h ) . Bax multimers are indicated . Molecular weight markers ( kDa ) are indicated on the left .

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

E S-HAD does not induce Bax oligomerization. Experiments described in D were repeated with cultures treated with S-HAD (10 µM, 24 h). Bax multimers are indicated. Molecular weight markers (kDa) are indicated on the left.


F S - HAD treatment sensitizes Hela cells to cisplatin . Hela cultures were treated with cisplatin ( 30 µM ) , S - HAD ( 10 µM ) and N - acetyl cysteine ( NAC , 1 mM ) as indicated . Fluorescent cell analysis was used to quantitate the apoptotic ( Annexin V positive ) and necrotic ( PI positive ) population percentages , respectively . n = 3 independent cultures . Error bars indicate SD . Asterisk indicates p < 0 . 05 ( Student ' s T - test ) .

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

F S-HAD treatment sensitizes Hela cells to cisplatin. Hela cultures were treated with cisplatin (30 µM), S-HAD (10 µM) and N-acetyl cysteine (NAC, 1 mM) as indicated. Fluorescent cell analysis was used to quantitate the apoptotic (Annexin V positive) and necrotic (PI positive) population percentages, respectively. n = 3 independent cultures. Error bars indicate SD. Asterisk indicates p <0.05 (Student's T-test).


A S - HAD treatment induces mitochondrial ROS . Wild type and Ccnc - / - MEF cultures were treated with S - HAD ( 10 µM , 24 h ) , rotenone or vehicle control as indicated . Mitochondrial ROS was measured by MitoSox staining ( 4 µM , 15 min ) and quantitative confocal imaging . Error bars indicate SD . n = 3 , asterisk indicates p < 0 . 05 ( Student ' s T - test ) , n . s . , not significantly different .

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

A S-HAD treatment induces mitochondrial ROS. Wild type and Ccnc-/- MEF cultures were treated with S-HAD (10 µM, 24 h), rotenone or vehicle control as indicated. Mitochondrial ROS was measured by MitoSox staining (4 µM, 15 min) and quantitative confocal imaging. Error bars indicate SD. n=3, asterisk indicates p<0.05 (Student's T-test), n.s., not significantly different.


B Quenching mitochondrial ROS reduces cyclin C - Bax interaction . Wild type and Ccnc - / - MEF cultures were treated with S - HAD ( 10 µM , 16 h ) and 100 nM mitochondrial - specific reactive oxygen scavenger MitoTempol . Western blot analysis was conducted as described in Fig . 1 . Molecular weight markers ( kDa ) are indicated on the left .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quenching mitochondrial ROS reduces cyclin C-Bax interaction. Wild type and Ccnc-/- MEF cultures were treated with S-HAD (10 µM, 16 h) and 100 nM mitochondrial-specific reactive oxygen scavenger MitoTempol. Western blot analysis was conducted as described in Fig. 1. Molecular weight markers (kDa) are indicated on the left.


C Insulin treatment stimulates S - HAD induced Bax activation . Wild type and Ccnc - / - MEF cultures were grown with or without insulin ( 10 µg / ml ) then treated with S - HAD ( 10 µM , 2 h ) . Mitochondrial and cytosolic fractions were prepared and probed for the indicated proteins . Atp5A and ß - actin levels control for mitochondrial and cytosolic loading , respectively . The two blots were prepared , processed and developed identically . Molecular weight markers ( kDa ) are indicated on the left .

O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Insulin treatment stimulates S-HAD induced Bax activation. Wild type and Ccnc-/- MEF cultures were grown with or without insulin (10 µg/ml) then treated with S-HAD (10 µM, 2 h). Mitochondrial and cytosolic fractions were prepared and probed for the indicated proteins. Atp5A and ß-actin levels control for mitochondrial and cytosolic loading, respectively. The two blots were prepared, processed and developed identically. Molecular weight markers (kDa) are indicated on the left.


A , B 1 × 105 B16F10 melanoma cells were intravenously injected into C57BL / 6 wild - type ( WT ) or Asm - deficient ( Asm − / − ) mice , and the number of lung metastases was determined after 14 days . The photographs ( A ) show two representative results . The graph ( B ) shows the mean ± SD number of pulmonary metastases in 15 WT and 14 Asm - deficient animals .

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

A, B 1 × 105 B16F10 melanoma cells were intravenously injected into C57BL/6 wild-type (WT) or Asm-deficient (Asm−/−) mice, and the number of lung metastases was determined after 14 days. The photographs (A) show two representative results. The graph (B) shows the mean ± SD number of pulmonary metastases in 15 WT and 14 Asm-deficient animals.


C , D B16F10 melanoma grow as fast or slightly faster in Asm - deficient mice than in wild - type mice after subcutaneous injection at the flank ( C ) or transcutaneous direct intrapulmonary injection ( D ) , excluding a general growth defect of the tumor in Asm - deficient mice . The size of tumors was determined 14 days after local injection at the flank or 10 days after injection into the lung .

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

C, D B16F10 melanoma grow as fast or slightly faster in Asm-deficient mice than in wild-type mice after subcutaneous injection at the flank (C) or transcutaneous direct intrapulmonary injection (D), excluding a general growth defect of the tumor in Asm-deficient mice. The size of tumors was determined 14 days after local injection at the flank or 10 days after injection into the lung.


E The number of lung metastases 25 days after intravenous injection does not differ from the number observed after 14 days in ( B ) .

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

E The number of lung metastases 25 days after intravenous injection does not differ from the number observed after 14 days in (B).


F 5 × 108platelets isolated from wild - type ( WT ) or Asm - deficient ( Asm − / − ) mice were injected intravenously into Asm - deficient or wild - type mice , respectively . After 120 min 1 × 105 , B16F10tumor cells were intravenously injected . The number of lungmetastases was determined after 14 days . Displayed are the mean ± SD of the number of pulmonary metastases , n = 3 in WT platelets in WT mice , n = 5 in WT platelets in Asm − / − mice , and n = 4 in Asm − / − platelets in Asm − / − mice .

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

F 5 × 108platelets isolated from wild-type (WT) or Asm-deficient (Asm−/−) mice were injected intravenously into Asm-deficient or wild-type mice, respectively. After 120 min 1 × 105, B16F10tumor cells were intravenously injected. The number of lungmetastases was determined after 14 days. Displayed are the mean ± SD of the number of pulmonary metastases, n = 3 in WT platelets in WT mice, n = 5 in WT platelets in Asm−/−mice, and n = 4 in Asm−/−platelets in Asm−/−mice.


A Platelets were isolated from wild - type ( WT ) or Asm - deficient ( Asm − / − ) mice by density - gradient centrifugation , and 1 × 107 platelets were incubated with 1 × 105 B16F10 cells . The samples were lysed , and Asm activity was determined . In unstimulated samples ( time point of co - incubation 0 s ) , tumor cells and platelets were admixed after lysis .

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

A Platelets were isolated from wild-type (WT) or Asm-deficient (Asm−/−) mice by density-gradient centrifugation, and 1 × 107 platelets were incubated with 1 × 105 B16F10 cells. The samples were lysed, and Asm activity was determined. In unstimulated samples (time point of co-incubation 0 s), tumor cells and platelets were admixed after lysis.


B The formation of ceramide was determined by a diacylglycerol ( DAG ) kinase assay ( upper panel ) and mass spectrometry ( lower panel ) . In unstimulated samples ( time point of co - incubation 0 s ) , tumor cells and platelets were admixed after lysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The formation of ceramide was determined by a diacylglycerol (DAG) kinase assay (upper panel) and mass spectrometry (lower panel). In unstimulated samples (time point of co-incubation 0 s), tumor cells and platelets were admixed after lysis.


C , D For determination of the secretion of Asm by platelets into the supernatants ( C ) , tumor cells and wild - type ( WT ) or Asm - deficient ( Asm − / − ) platelets were co - incubated for 20 s , the samples were pelleted , the supernatants were removed and acidified , and the Asm activity was measured in the presence or absence of Zn2 + . For measurement of surface Asm and ceramide , samples were co - incubated as indicated ( C , D ) and pelleted ; the supernatants were discarded , incubated with anti - Asm antibodies , washed , and lysed ; and Asm immunocomplexes were immobilized and subjected to immunocomplex enzyme assays . Surface ceramide was measured by incubation of intact cells with DAG kinase in the presence of [ 32P ] γATP followed by extraction and measurement of [ 32P ] - ceramide . Omission of one cell type indicated by " − " here and thereafter .

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

C, D For determination of the secretion of Asm by platelets into the supernatants (C), tumor cells and wild-type (WT) or Asm-deficient (Asm−/−) platelets were co-incubated for 20 s, the samples were pelleted, the supernatants were removed and acidified, and the Asm activity was measured in the presence or absence of Zn2+. For measurement of surface Asm and ceramide, samples were co-incubated as indicated (C, D) and pelleted; the supernatants were discarded, incubated with anti-Asm antibodies, washed, and lysed; and Asm immunocomplexes were immobilized and subjected to immunocomplex enzyme assays. Surface ceramide was measured by incubation of intact cells with DAG kinase in the presence of [32P]γATP followed by extraction and measurement of [32P]-ceramide. Omission of one cell type indicated by "−" here and thereafter.


E Treatment of B16F10tumor cells for 2 min with 1 U / ml ASM or 10 μM C16ceramide restores in vivo metastasis in Asm - deficient mice . After treatment , the cells were injected intravenously into Asm - deficient ( Asm − / − ) mice . Controls were left untreated prior to injection . The number of metastases was determined 14 days after tumor cell injection .

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

E Treatment of B16F10tumor cells for 2 min with 1 U/ml ASM or 10 μM C16ceramide restores in vivo metastasis in Asm-deficient mice. After treatment, the cells were injected intravenously into Asm-deficient (Asm−/−) mice. Controls were left untreated prior to injection. The number of metastases was determined 14 days after tumor cell injection.


Incubation of human melanoma ( HM ) cells with human platelets results in the release of Zn2 + - dependent ASM into the supernatant , Zn2 + - dependent activity of ASM on cell surfaces , and the formation of ceramide . The assay buffer contained 100 μM Zn2 + .

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

Incubation of human melanoma (HM) cells with human platelets results in the release of Zn2+-dependent ASM into the supernatant, Zn2+-dependent activity of ASM on cell surfaces, and the formation of ceramide. The assay buffer contained 100 μM Zn2+.


Addition of human or mouse recombinant ASM to human melanoma or B16F10 cells , respectively , results in binding of ASM to the tumorcell surfaces as determined by flow cytometry . Cytochalasin B was added to control for internalization of added ASM .

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

Addition of human or mouse recombinant ASM to human melanoma or B16F10 cells, respectively, results in binding of ASM to the tumorcell surfaces as determined by flow cytometry. Cytochalasin B was added to control for internalization of added ASM.


Suppression of Asm in B16F10 tumor cells using siRNA technology reduces Asm activity in B16F10 cells ( right panel ) , but does not alter release or surface activity of the Asm after co - incubation with wild - type platelets ( left panels ) .

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

Suppression of Asm in B16F10 tumor cells using siRNA technology reduces Asm activity in B16F10 cells (right panel), but does not alter release or surface activity of the Asm after co-incubation with wild-type platelets (left panels).


Platelets were isolated from wild - type ( WT ) or Asm - deficient ( Asm − / − ) mice by density - gradient centrifugation , and 1 × 107platelets were incubated with 1 × 105B16F10 cells or left untreated . Co - incubation of washed platelets with B16F10 melanoma cells resulted in upregulation of GPIIb / IIIa and CD62P on both wild - type and Asm - deficient platelets in FACS analyses . Platelets were gated in FCS / SSC .

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

Platelets were isolated from wild-type (WT) or Asm-deficient (Asm−/−) mice by density-gradient centrifugation, and 1 × 107platelets were incubated with 1 × 105B16F10 cells or left untreated. Co-incubation of washed platelets with B16F10 melanoma cells resulted in upregulation of GPIIb/IIIa and CD62P on both wild-type and Asm-deficient platelets in FACS analyses. Platelets were gated in FCS/SSC.


Aggregation properties of platelets after co - incubation with collagen or ADP are independent of Asm as determined by aggregometry measurements .

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

Aggregation properties of platelets after co-incubation with collagen or ADP are independent of Asm as determined by aggregometry measurements.


Incubation of tumor cells with platelets resulted in marked change of platelet shape and degranulation as determined by FACS analysis using forward versus side scatter analysis indicating global activation of platelets .

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

Incubation of tumor cells with platelets resulted in marked change of platelet shape and degranulation as determined by FACS analysis using forward versus side scatter analysis indicating global activation of platelets.


4 × 104B16F10tumor cells were incubated with 2 × 107 wild - type ( WT ) , Asm - deficient ( Asm − / − ) platelets ( Plts ) or a 45 : 55 of WT : Asm − / − platelet mixture in the presence or absence of 50 ng / ml PGE1 . Controls were stimulated with Mn2 + . Tumor cells were then incubated for 60 s on fibronectin - coated cover slips , washed , and fixed , and adhesion of the tumor cells was determined . The graph displays the mean ± SD of tumor cells adhering to fibronectin - coated cover slips , n = 7 for WT - platelets , n = 9 for Asm - deficient - platelets , n = 4 for 45 : 55 WT : Asm − / − platelets , and all others n = 3 . Statistical significance was determined by analysis of variance ( ANOVA ) followed by Tukey ' s multiple comparisons test . P - values are indicated .

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

4 × 104B16F10tumor cells were incubated with 2 × 107 wild-type (WT), Asm-deficient (Asm−/−) platelets (Plts) or a 45:55 of WT:Asm−/−platelet mixture in the presence or absence of 50 ng/ml PGE1. Controls were stimulated with Mn2+. Tumor cells were then incubated for 60 s on fibronectin-coated cover slips, washed, and fixed, and adhesion of the tumor cells was determined. The graph displays the mean ± SD of tumor cells adhering to fibronectin-coated cover slips, n = 7 for WT-platelets, n = 9 for Asm-deficient-platelets, n = 4 for 45:55 WT:Asm−/−platelets, and all others n = 3. Statistical significance was determined by analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. P-values are indicated.


A - C 1 × 105 B16F10 cells were incubated with ( A ) 1 U / ml purified acid sphingomyelinase ( ASM ) or ( B ) 5 × 107 wild - type ( WT ) , Asm - deficient ( Asm − / − ) platelets ( Plts ) , or a mixture of 45 : 55 wild - type : Asm - deficient platelets . Cells were fixed with 2 % paraformaldehyde for 15 min and stained with fluorescein isothiocyanate ( FITC ) - coupled anti - α5 integrin , Cy3 - labeled anti - β1 integrin , and Cy5 - labeled anti - ceramide antibodies . Samples were analyzed by confocal microscopy . Shown are representative examples from four independent experiments ( A , B ) or the quantitative analysis of cells positive for ceramide / β1 integrin clusters from at least 100 cells / sample ( C ) . Given is the mean ± SD , n = 4 , ANOVA followed by Tukey ' s multiple comparisons test . P - values are indicated .

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

A-C 1 × 105 B16F10 cells were incubated with (A) 1 U/ml purified acid sphingomyelinase (ASM) or (B) 5 × 107 wild-type (WT), Asm-deficient (Asm−/−) platelets (Plts), or a mixture of 45:55 wild-type:Asm-deficient platelets. Cells were fixed with 2% paraformaldehyde for 15 min and stained with fluorescein isothiocyanate (FITC)-coupled anti-α5 integrin, Cy3-labeled anti-β1 integrin, and Cy5-labeled anti-ceramide antibodies. Samples were analyzed by confocal microscopy. Shown are representative examples from four independent experiments (A, B) or the quantitative analysis of cells positive for ceramide/β1 integrin clusters from at least 100 cells/sample (C). Given is the mean ± SD, n = 4, ANOVA followed by Tukey's multiple comparisons test. P-values are indicated.


D ASM - induced B16F10 tumorcell adhesion to fibronectin - coated cover slips is abrogated by the inhibition of integrins with RGD peptides . Shown is the mean ± SD of the number of cells adherent to the cover slip . Statistical significance was determined by ANOVA followed by Tukey ' s multiple comparisons test . P - values are indicated .

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

D ASM-induced B16F10 tumorcell adhesion to fibronectin-coated cover slips is abrogated by the inhibition of integrins with RGD peptides. Shown is the mean ± SD of the number of cells adherent to the cover slip. Statistical significance was determined by ANOVA followed by Tukey's multiple comparisons test. P-values are indicated.


E Intravenous injection of CFSE - labeled B16F10 cells into wild - type mice results in formation of ceramide - enriched domains that contain β1 integrin clusters on tumor cells in vivo , while injection of tumor cells into Asm - deficient mice does not result in ceramide and integrin clustering . Please note that the cell in the lower panel still binds a platelet . Shown are representative results from four independent experiments each .

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

E Intravenous injection of CFSE-labeled B16F10 cells into wild-type mice results in formation of ceramide-enriched domains that contain β1 integrin clusters on tumor cells in vivo, while injection of tumor cells into Asm-deficient mice does not result in ceramide and integrin clustering. Please note that the cell in the lower panel still binds a platelet. Shown are representative results from four independent experiments each.


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 .

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

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 .

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

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 ) .

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

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 .

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

D 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O 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 .

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

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

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

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


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

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

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


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

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


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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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