( C ) A single living cell expressing FTCDwt - HA - MAO was tracked during mitosis . Mitotic cells were collected by flushing from the HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO , and cultured in the absence or presence of DOX . The cells were stained with MitoTracker at the 22 hr time point and confocal images of 30 - 40 cells were randomly taken at the 23 hr time point . From the 23 hr to 27 hr time point , cells were tracked in the bright field every 30 mins . At the 27 hr time point , confocal images were taken of the 21 - 38 cells that were successfully tracked . Panels a - j display representative images . Asterisks indicate the tracked cells . Scale bar = 10 μm . ( D ) The results of quantification of ( C ) . The experiment was repeated five times and each line shows the average percentage of an independent experiment . The percentages of cells that underwent mitosis are as follows : 41 . 5 % in the DOX ( - ) group and 54 . 7 % in the DOX ( + ) group .

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

(C) A single living cell expressing FTCDwt-HA-MAO was tracked during mitosis. Mitotic cells were collected by flushing from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO, and cultured in the absence or presence of DOX. The cells were stained with MitoTracker at the 22 hr time point and confocal images of 30-40 cells were randomly taken at the 23 hr time point. From the 23 hr to 27 hr time point, cells were tracked in the bright field every 30 mins. At the 27 hr time point, confocal images were taken of the 21-38 cells that were successfully tracked. Panels a-j display representative images. Asterisks indicate the tracked cells. Scale bar = 10 μm. (D) The results of quantification of (C). The experiment was repeated five times and each line shows the average percentage of an independent experiment. The percentages of cells that underwent mitosis are as follows: 41.5 % in the DOX(-) group and 54.7 % in the DOX(+) group.


( E ) FTCD located in mitochondria causes mitochondria aggregation at the end of mitosis . Mitotic cells were collected from the HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO and cultured in the absence or presence of DOX as in ( C ) . Cells were stained with MitoTracker at the 23hr time point and observed by confocal microscopy from the 24 hr to 26 hr time point . Panels a - t display representative images . Scale bar = 10 μm .

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

(E) FTCD located in mitochondria causes mitochondria aggregation at the end of mitosis. Mitotic cells were collected from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO and cultured in the absence or presence of DOX as in (C). Cells were stained with MitoTracker at the 23hr time point and observed by confocal microscopy from the 24 hr to 26 hr time point. Panels a-t display representative images. Scale bar = 10 μm.


( F ) The results of quantification of ( E ) . Cells were classified by their cell cycle phases into the following three categories : prometaphase / metaphase , anaphase / telophase and cytokinesis . Results are shown as the mean ± SD of three sets of independent experiments , with 10 - 14 cells counted in each group in each independent experiment . Asterisks indicate a significant difference at P < 0 . 01 compared with the DOX ( + ) group and with the prometaphase / metaphase category ( Bonferroni method ) .

O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

(F) The results of quantification of (E). Cells were classified by their cell cycle phases into the following three categories: prometaphase/metaphase, anaphase/telophase and cytokinesis. Results are shown as the mean±SD of three sets of independent experiments, with 10-14 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with the DOX(+) group and with the prometaphase/metaphase category (Bonferroni method).


A , Heat map of all reported cancer blood metabolites ( each dark blue mark denotes that the metabolite was reported to be increased or decreased ) in various cancer types , illustrating that most individual studies report only a small subset of all previously measured metabolites . It is typically not described whether the metabolites that were not reported ( white " empty " space ) were not measured , or measured but not reported . Labelings : X - axis ( top ) : ribbon colour code , denoting the cancer type ( right ; indicated by red arrow ) ; X - axis ( bottom ) : cohorts , arranged from 1 to 71 ; Y - axis ( right ) : all 1 , 206 metabolites reported in at least one of the studies ; Y - axis ( left ) : ribbon colour code , denoting the metabolite class ( amino acids , carbohydrates , etc ; " other " refers to all other metabolites than the listed classes ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O

A, Heat map of all reported cancer blood metabolites (each dark blue mark denotes that the metabolite was reported to be increased or decreased) in various cancer types, illustrating that most individual studies report only a small subset of all previously measured metabolites. It is typically not described whether the metabolites that were not reported (white "empty" space) were not measured, or measured but not reported. Labelings: X-axis (top): ribbon colour code, denoting the cancer type (right; indicated by red arrow); X-axis (bottom): cohorts, arranged from 1 to 71; Y-axis (right): all 1,206 metabolites reported in at least one of the studies; Y-axis (left): ribbon colour code, denoting the metabolite class (amino acids, carbohydrates, etc; "other" refers to all other metabolites than the listed classes).


B , Vote counting of cancer blood metabolites ( reported in at least six cohorts ) showed consistently deregulated metabolites . Blue bars : decreased metabolites ; red bars : increased metabolites . * ( asterisk ) in front of the name of the metabolite indicates a statistical trend ( p < 0 . 1 ) ; red arrowheads denote metabolites mentioned in the main text .

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B, Vote counting of cancer blood metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrowheads denote metabolites mentioned in the main text.


C , D , Volcano plot of bloodmetabolites ( C ) and tissuemetabolites ( D ) , reported in at least six cancer studies , with the vote counting score on the X - axis and the - log10 adjusted p - value on the Y - axis . Cyan indicates deregulated metabolites that show a trend ( p < 0 . 1 ; for blood , corresponding to metabolites marked with * in panel 2B ; for tissue , corresponding to metabolites marked with * in Figure 3 ) or statistical significance ( p < 0 . 05 ; above black dashed horizontal line ) ; red indicates metabolites with a p - value > 0 . 1 . A subset of metabolites is annotated ( see Table EV4 ( blood ) and EV5 ( tissue ) for full annotation ) .

O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O

C,D, Volcano plot of bloodmetabolites (C) and tissuemetabolites (D), reported in at least six cancer studies, with the vote counting score on the X-axis and the -log10 adjusted p-value on the Y-axis. Cyan indicates deregulated metabolites that show a trend (p < 0.1; for blood, corresponding to metabolites marked with * in panel 2B; for tissue, corresponding to metabolites marked with * in Figure 3) or statistical significance (p < 0.05; above black dashed horizontal line); red indicates metabolites with a p-value > 0.1. A subset of metabolites is annotated (see Table EV4 (blood) and EV5 (tissue) for full annotation).


Vote counting of cancer tissue metabolites ( reported in at least six cohorts ) showed consistently deregulated metabolites . Blue bars : decreased metabolites ; red bars : increased metabolites . * ( asterisk ) in front of the name of the metabolite indicates a statistical trend ( p < 0 . 1 ) ; red arrows denote key metabolites mentioned in the main text ( see Table EV5 for full annotation ) .

O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

Vote counting of cancer tissue metabolites (reported in at least six cohorts) showed consistently deregulated metabolites. Blue bars: decreased metabolites; red bars: increased metabolites. * (asterisk) in front of the name of the metabolite indicates a statistical trend (p < 0.1); red arrows denote key metabolites mentioned in the main text (see Table EV5 for full annotation).


Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence of H676 cells treated with the indicated compounds for 24h at 1 μM . Scale bars , 5μm . Data are representative of n = 3 biological replicates . ( C ) Western blot images ( top ) and TRF1 protein levels ( bottom ) of h676 cells treated with the indicated compounds for 24h at 1 μM . Data are representative of n = 3 ( PLKi , HSP90i and RTKi ) and n = 4 ( AuroraI , mTOR , CDKi , Docetaxel , Gemcitabine , ERKi , MEKi ) biological replicates .

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Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence of H676 cells treated with the indicated compounds for 24h at 1 μM. Scale bars, 5μm. Data are representative of n=3 biological replicates. (C) Western blot images (top) and TRF1 protein levels (bottom) of h676 cells treated with the indicated compounds for 24h at 1 μM. Data are representative of n=3 (PLKi, HSP90i and RTKi) and n=4 (AuroraI, mTOR, CDKi, Docetaxel, Gemcitabine, ERKi, MEKi) biological replicates.


Representative images ( left ) and percentage ( right ) of cells presenting 1 or more γH2AX and RAP1 colocalizing foci ( TIFs ) upon treatment of CHA9 - 3 lung cancer cells with the indicated compounds . White arrowheads point to colocalization of γH2AX and RAP1 . Scale bars , 5μm . Data are representative of n = 6 ( DMSO ) and n = 3 ( mTORi , PI3Ki , RTKi , MEKi , ERKi , HSPO90i , CDKi , Docetaxel ) biological

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Representative images (left) and percentage (right) of cells presenting 1 or more γH2AX and RAP1 colocalizing foci (TIFs) upon treatment of CHA9-3 lung cancer cells with the indicated compounds. White arrowheads point to colocalization of γH2AX and RAP1. Scale bars, 5μm. Data are representative of n=6 (DMSO) and n=3 (mTORi, PI3Ki, RTKi, MEKi, ERKi, HSPO90i, CDKi, Docetaxel) biological replicates


Representative images ( left ) and percentage ( right ) of γH2AX - positive cells per field in DMSO or compound treated patient - derived h676 GSCs . Scale bars , 50μm . Data are representative of 6 ( DMSO ) and 3 ( mTORi , Docetaxel , ERKi , MEKi , RTKi , HSP90i , Gemcitabine , CDKi ) biological

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Representative images (left) and percentage (right) of γH2AX-positive cells per field in DMSO or compound treated patient-derived h676 GSCs. Scale bars, 50μm. Data are representative of 6 (DMSO) and 3 (mTORi, Docetaxel, ERKi, MEKi, RTKi, HSP90i, Gemcitabine, CDKi) biological replicates


Quantification of multitelomeric signals ( MTS ) in h676 GSC metaphases upon treatment with the indicated compounds . Representative images of the qFISH in the metaphases ( left ) . Multitelomeric signals are indicated by arrowheads . Scale bars , 1μm . Data are representative of n = 31 ( DMSO ) , n = 18 ( mTORi ) , n = 11 ( MEKi ) and n = 24 ( RTKi ) biological replicates . Data are represented as mean ± SEM . Significant differences using unpaired t ‐ test are indicated by * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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Quantification of multitelomeric signals (MTS) in h676 GSC metaphases upon treatment with the indicated compounds. Representative images of the qFISH in the metaphases (left). Multitelomeric signals are indicated by arrowheads. Scale bars, 1μm. Data are representative of n=31 (DMSO), n=18 (mTORi), n=11 (MEKi) and n=24 (RTKi) biological replicates. Data are represented as mean ± SEM. Significant differences using unpaired t‐test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001.


Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations . Data are representative of n = 2 biological replicates . Data are represented as mean ± SEM normalized to DMSO .

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Dose response curves of h543 and h676 GSCs treated with the indicated compounds at several concentrations. Data are representative of n=2 biological replicates. Data are represented as mean ± SEM normalized to DMSO.


1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of mouse ERK2 kinase in the presence of 5μCi [ γ - 32 - P ] ATP . The mixture was resolve by SDS - PAGE followed by autoradiography .

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

1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse ERK2 kinase in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.


1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of human b - Raf kinase ( WT or V600E ) in the presence of 5μCi [ γ - 32 - P ] ATP . The mixture was resolve by SDS - PAGE followed by autoradiography .

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

1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human b-Raf kinase (WT or V600E) in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.


1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of mouse MEK1 kinase in the presence of 5μCi [ γ - 32 - P ] ATP . The mixture was resolve by SDS - PAGE followed by autoradiography .

1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of mouse MEK1 kinase in the presence of 5μCi [γ-32-P]ATP. The mixture was resolve by SDS-PAGE followed by autoradiography.


1 μM of GST - TRF1 and 0 . 2 μM of mouse ERK2 kinase were incubated in the presence of ERK and MEK inhibitors .

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1 μM of GST-TRF1 and 0.2 μM of mouse ERK2 kinase were incubated in the presence of ERK and MEK inhibitors.


2 μM of GST - TRF1 and 0 . 1 μM of human b - Raf kinase were incubated in the presence of the bRaf inhibitors dabrafenib and vemurafenib .

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2 μM of GST-TRF1 and 0.1 μM of human b-Raf kinase were incubated in the presence of the bRaf inhibitors dabrafenib and vemurafenib.


1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of human mTOR

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O B-GENEPROD

1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human mTOR kinase


1 or 2 μM of GST or GST - TRF1 were incubated with the indicated concentrations of human mTOR kinase in the presence of the mTOR inhibitors rapamycin and

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

1 or 2 μM of GST or GST-TRF1 were incubated with the indicated concentrations of human mTOR kinase in the presence of the mTOR inhibitors rapamycin and Ku0063794


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Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 or TRF1 plus ERK2


Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus bRAFWT or bRAFV600E


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Phosphopeptide peak intensity normalized to total TRF1 signal in samples containing only TRF1 plus mTOR


Representative image ( down ) and quantification ( up ) of in vitro phosphorylation assays with the indicated GST - TRF1 wild - type or mutated forms in the presence of mouse ERK2 kinase .

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Representative image (down) and quantification (up) of in vitro phosphorylation assays with the indicated GST-TRF1 wild-type or mutated forms in the presence of mouse ERK2 kinase.


Western blot images of Trf1lox / lox MEFS with or without overexpression of eGFP - Trf1 WT or mutant alleles followed by Cre recombinase transduction .

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Western blot images of Trf1lox/lox MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed by Cre recombinase transduction.


Quantification of eGFP - TRF1 inhibition in Trf1Δ / Δ MEFs transduced with eGFP - Trf1 WT or mutant alleles as indicated . Data are representative of n = 15 biological

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Quantification of eGFP-TRF1 inhibition in Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=15 biological replicates


Growth curves of Trf1Δ / Δ MEFs transduced with eGFP - Trf1 WT or mutant alleles as indicated . Data are representative of n = 5 biological

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Growth curves of Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=5 biological replicates


Western blot images of Trf1lox / lox MEFS with or without overexpression of eGFP - Trf1 WT or mutant alleles followed by Cre recombinase transduction . Quantification of eGFP - TRF1 inhibition in Trf1Δ / Δ MEFs transduced with eGFP - Trf1 WT or mutant alleles as indicated . Data are representative of n = 5 biological

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Western blot images of Trf1lox/lox MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed by Cre recombinase transduction. Quantification of eGFP-TRF1 inhibition in Trf1Δ/Δ MEFs transduced with eGFP-Trf1 WT or mutant alleles as indicated. Data are representative of n=5 biological replicates


Western blot images of p53 - / - MEFS with or without overexpression of eGFP - Trf1 WT or mutant alleles followed treatment with ERKi . Data are representative of n = 2 biological

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Western blot images of p53-/- MEFS with or without overexpression of eGFP-Trf1 WT or mutant alleles followed treatment with ERKi. Data are representative of n=2 biological replicates


Representative images ( above ) and percentage ( bottom ) of Telomeric and 53BP1 colocalizing foci ( TIFs ) per cells of p53 - / - MEFS with or without overexpression of eGFP - Trf1 WT and the indicated mutants upon treatment with the ERKi . White arrowheads : colocalization of telomeric and 53BP1 . Scale bars , 10μm .

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Representative images (above) and percentage (bottom) of Telomeric and 53BP1 colocalizing foci (TIFs) per cells of p53-/- MEFS with or without overexpression of eGFP-Trf1 WT and the indicated mutants upon treatment with the ERKi. White arrowheads: colocalization of telomeric and 53BP1. Scale bars, 10μm.


Western blot image ( above ) and quantification ( bottom ) of TRF1 protein levels upon genetic depletion of ERK1 / 2 in p53 - / - MEF line . Data are representative of n = 3 independent experiments .

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Western blot image (above) and quantification (bottom) of TRF1 protein levels upon genetic depletion of ERK1/2 in p53-/- MEF line. Data are representative of n=3 independent experiments.


Representative images ( above ) and quantification ( bottom ) of TRF1 telomeric foci in ERK1 / 2 RNA interfered p53 - / - MEFs . Scale bars , 5μm . Data are representative of n = 2 independent experiments .

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Representative images (above) and quantification (bottom) of TRF1 telomeric foci in ERK1/2 RNA interfered p53-/- MEFs. Scale bars, 5μm. Data are representative of n=2 independent experiments.


Western blot image of eGFP - tagged and endogenous TRF1 protein levels upon genetic depletion of ERK1 / 2 in p53 - / - MEF line .

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Western blot image of eGFP-tagged and endogenous TRF1 protein levels upon genetic depletion of ERK1/2 in p53-/- MEF line.


Representative images ( above ) and percentage ( bottom ) of telomeric and 53BP1 colocalizing foci ( TIFs ) per cell in p53 - / - MEF with or without overexpression of eGFP - Trf1 WT and indicated mutants upon genetic depletion of ERK1 / 2 . White arrowheads : colocalization of telomeric and 53BP1 . Scale bars , 10μm .

O B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O B-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O O O O O O

Representative images (above) and percentage (bottom) of telomeric and 53BP1 colocalizing foci (TIFs) per cell in p53-/- MEF with or without overexpression of eGFP-Trf1 WT and indicated mutants upon genetic depletion of ERK1/2. White arrowheads: colocalization of telomeric and 53BP1.Scale bars, 10μm.


Representative images ( left ) and quantification ( right ) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination . Scale bars , 100μm . Data are representative of n = 6 biological replicates .

O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images (left) and quantification (right) of number of spheres formed by h676 GSCs 7 days after treatment with the indicated compounds as single agents or in combination. Scale bars, 100μm. Data are representative of n=6 biological replicates.


Western blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of n = 3 ( RTKi in G ( DMSO in

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

Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of n=3 (RTKi in G (DMSO in G


Western blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of ( DMSO in and ERKi in H )

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

Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in and ERKi in H)


Western blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of ( DMSO in ( PI3Ki

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

Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO in (PI3Ki in


Western blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of (

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O

Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of (DMSO


Western blot images ( left ) and TRF1 protein levels ( right ) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination . Data are representative of Docetaxel (

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

Western blot images (left) and TRF1 protein levels (right) measured in h676 GSCs 24h after treatment with the indicated compounds as single agents or in combination. Data are representative of Docetaxel (DMSO


Longitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in A : vehicle n = 16 , PI3Ki n = 8 , ERKi n = 16 , Combination n = 8 Pvalues represent the mean of all the time

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

Longitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in A: vehicle n=16, PI3Ki n=8, ERKi n=16, Combination n=8 Pvalues represent the mean of all the time points


Longitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in B : Vehicle n = 16 , PI3Ki n = 16 , MEKi n = 8 , Combination n = 8 Pvalues represent the mean of all the time

Longitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in B: Vehicle n=16, PI3Ki n=16, MEKi n=8, Combination n=8 Pvalues represent the mean of all the time points


Longitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in C : Vehicle n = 8 , PI3Ki n = 16 , Docetaxel n = 16 , Combination n = 8 Pvalues represent the mean of all the time

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

Longitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in C: Vehicle n=8, PI3Ki n=16, Docetaxel n=16, Combination n=8 Pvalues represent the mean of all the time points


Longitudinal tumor growth follow - up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination . n represents number of tumors , in D : Vehicle n = 16 , PI3K n = 16 , Gemcitabine n = 4 , Combination n = 4 . Pvalues represent the mean of all the time

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

Longitudinal tumor growth follow-up in mice injected with h676 GSCs and treated with the indicated compounds in single agents or combination. n represents number of tumors, in D: Vehicle n=16, PI3K n=16, Gemcitabine n=4, Combination n=4. Pvalues represent the mean of all the time points


Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors : in E : vehicle n = 15 , PI3Ki n = 10 , ERKi n = 8 , Combination n =

O B-EXP_ASSAY O O O O O O O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O

Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors: in E: vehicle n=15, PI3Ki n=10, ERKi n=8, Combination n=2


Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors in F : Vehicle n = 15 , PI3Ki n = 10 , MEKi n = 8 , Combination n =

O B-EXP_ASSAY O O O O O O O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O

Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors in F: Vehicle n=15, PI3Ki n=10, MEKi n=8, Combination n=6


Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors ; in G : Vehicle n = 15 , PI3Ki n = 10 , Docetaxel n = 8 , Combination n =

O B-EXP_ASSAY O O O O O O O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O

Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors ; in G: Vehicle n=15, PI3Ki n=10, Docetaxel n=8, Combination n=4


Representative images ( top ) and quantification ( bottom ) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination . Scale bars , 10 μm . Data are represented as mean ± SEM . n represents number of tumors ; in H : Vehicle n = 15 , PI3K n = 10 , Gemcitabine n = 8 , Combination n = 6 .

O B-EXP_ASSAY O O O O O O O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O

Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence in tumors treated with the indicated compounds as single agents or in combination. Scale bars, 10 μm. Data are represented as mean ± SEM. n represents number of tumors ; in H: Vehicle n=15, PI3K n=10, Gemcitabine n=8, Combination n=6.


B ) Analysis of puromycin incorporation in MelJuSo cells that were left untreated ( - HS ) , exposed to 43 ° C for 30 minutes ( + HS ) , and followed for 4 hours after heat shock ( HS + 4h Rec . ) . The blots were probed with antibodies against puromycin , and GAPDH as loading control .

O O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O

B) Analysis of puromycin incorporation in MelJuSo cells that were left untreated (- HS), exposed to 43°C for 30 minutes (+ HS), and followed for 4 hours after heat shock (HS + 4h Rec.). The blots were probed with antibodies against puromycin, and GAPDH as loading control.


C ) Representative images of MelJuSo cells expressing Ub - YFP , which were either left untreated ( - HS ) , exposed to 43 ° C for 30 minutes ( + HS ) , and followed for 4 hours after heat shock ( HS + 4h Rec . ) .

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Representative images of MelJuSo cells expressing Ub-YFP, which were either left untreated (- HS), exposed to 43 °C for 30 minutes (+ HS), and followed for 4 hours after heat shock (HS + 4h Rec.).


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 B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-CELL_LINE O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-CELL_LINE 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-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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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 B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) 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 B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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 B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

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 B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY 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 B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

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-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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-GENEPROD O O O B-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD 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 B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O 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 .

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

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

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-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

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

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

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

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

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

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