Immunoblot of epithelial marker E - cadherin mesenchymal markers , including N - cadherin , vimentin , SNAIL , in A549 - VIM - RFP transduced with pLV - EV control or ST3GAL5 expression construct treated with vehicle control or TGF - β for 48 h . GAPDH : loading control .

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Immunoblot analysis of the epithelial marker E-cadherin and mesenchymal markers, including N-cadherin, vimentin, and SNAIL, in A549-VIM-RFP cells transduced with the pLV-EV control or ST3GAL5 expression construct and treated with vehicle control or TGF-β for 48 h. GAPDH: loading control.


HEK293T transfected with Myc - tagged caTβRI , ( HA - Ub , nontargeting siRNA or ST3GAL5 siRNA were collected for IP with an anti - Myc antibody immunoblot . groups were treated with MG132 ( 5 μM ) for 6 h .

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HEK293T cells transfected with Myc-tagged caTβRI, (HA-Ub, and nontargeting siRNA or ST3GAL5 siRNA were collected for IP with an anti-Myc antibody and immunoblot analysis. All groups were treated with MG132 (5 μM) for 6 h.


Ubiquitination of TβRI was detected by IP of Myc - tagged caTβRI from HA - Ub - transfected HEK293T with or without ST3GAL5 overexpression . groups were treated with MG132 ( 5 μM ) for 6 h .

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Ubiquitination of TβRI was detected by IP of Myc-tagged caTβRI from HA-Ub-transfected HEK293T cells with or without ST3GAL5 overexpression. All groups were treated with MG132 (5 μM) for 6 h.


Western blot of TβRI expression in A549 - VIM - RFP transduced with pLV - EV control or ST3GAL5 expression construct treated with CHX for indicated times . Vinculin : loading control .

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Western blot analysis of TβRI expression in A549-VIM-RFP cells transduced with the pLV-EV control or ST3GAL5 expression construct and treated with CHX for the indicated times. Vinculin: loading control.


of TβRI expression in pLV - EV control - ST3GAL5 - expressing A549 - VIM - RFP . results were normalized to t = 0 controls .

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Quantification of TβRI expression in pLV-EV control- and ST3GAL5-expressing A549-VIM-RFP cells. The results were normalized to the t=0 controls.


In vivo zebrafish extravasation experiments with mCherry - labeled A549 with or without ectopic expression of ST3GAL5 . Representative with magnified regions ( outlined with dotted squares ) of extravasated were acquired 4 days after injection by confocal microscopy . number of extravasated clusters was quantified in 30 injected with A549 transduced with pLV - EV control or ST3GAL5 expression construct .

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In vivo zebrafish extravasation experiments with mCherry-labeled A549 cells with or without ectopic expression of ST3GAL5. Representative images with magnified regions (outlined with dotted squares) of extravasated cells were acquired 4 days after injection by confocal microscopy. The number of extravasated cell clusters was quantified in 30 embryos injected with A549 cells transduced with the pLV-EV control or ST3GAL5 expression construct.


Kaplan - Meier survival curves showing first progression survival of lung cancer in a publicly available lung cancer dataset according to ST3GAL5 expression ( n = 982 ) .

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Kaplan-Meier survival curves showing the first progression survival of lung cancer patients in a publicly available lung cancer dataset according to ST3GAL5 expression (n=982).


Box plots of ST3GAL5 gene expression in lung cancer tissues normal tissues in Bhattacharjee Lung database . central bands indicate medium expression values of ST3GAL5 , boxes indicate expression level ranges of ST3GAL5 . Data are presented as means ± SDs from indicated number of tissues .

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Box plots of ST3GAL5 gene expression levels in lung cancer tissues and normal tissues in the Bhattacharjee Lung database. The central bands indicate the medium expression values of ST3GAL5, boxes indicate the expression level ranges of ST3GAL5. Data are presented as the means ± SDs from the indicated number of tissues.


Representative of ST3GAL5 immunohistochemistry in a human lung tissue microarray including normal cancer tissues . Wide field magnified ( outlined with dotted squares ) are shown .

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Representative images of ST3GAL5 immunohistochemistry in a human lung tissue microarray including normal and cancer tissues. Wide field and magnified images (outlined with dotted squares) are shown.


Scatter plot showing expression of ST3GAL5 in normal cancerous lung tissues . Each point represents H - score of a single tissue sample with ST3GAL5 ranging from completely absent ( H - score 0 ) to very strong ( H - score 300 ) . H - scores are presented as means ± SDs ; normal tissues , n = 48 ; cancer tissues , n = 97 ; * * * * , P < 0 . 0001 on unpaired Student ' s t .

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Scatter plot showing the expression of ST3GAL5 in normal and cancerous lung tissues. Each point represents the H-score of a single tissue sample with ST3GAL5 staining ranging from completely absent (H-score 0) to very strong (H-score 300). H-scores are presented as the means ± SDs; normal tissues, n=48; cancer tissues, n=97; ****, P < 0.0001 based on unpaired Student's t test.


emerging timeline for highly pathogenic proposed Disease X . b . Schematic representation of SARS - CoV - 2 S . Its S1 subunit contains NTD ( 14 - 305 aa ) , RBD ( 319 - 541 aa ) , RBM ( 437 - 508 aa ) . Its S2 subunit contains FP ( 788 - 806 aa ) , HR1 ( 912 - 984 aa ) , HR2 ( 1163 - 1213 aa ) , TM ( 1214 - 1237 aa ) CP ( 1238 - 1273 aa ) . c . formation of syncytium in Huh - 7 24 h after SARS - CoV - 2 infection , with scale bar of 200 μm .

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The emerging timeline for highly pathogenic viruses and the proposed Disease X. b. Schematic representation of SARS-CoV-2 S protein. Its S1 subunit contains NTD (14-305 aa), RBD (319-541 aa), and RBM (437-508 aa). Its S2 subunit contains FP (788-806 aa), HR1 (912-984 aa), HR2 (1163-1213 aa), TM (1214-1237 aa) and CP (1238-1273 aa). c. The formation of syncytium in Huh-7 cells 24 h after SARS-CoV-2 infection, with scale bar of 200 μm.


d . of SARS - CoV - SARS - CoV - 2 S - mediated - fusion on 293T / ACE2 at 2 h ( left ) 24 h ( right ) .

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d. Images of SARS-CoV- and SARS-CoV-2 S-mediated cell-cell fusion on 293T/ACE2 cells at 2 h (left) and 24 h (right).


e . SARS - CoV ( I - II ) SARS - CoV - 2 ( III - IV ) S - mediated syncytium formation on 293T / ACE2 at 48 h . f . SARS - CoV ( I - II ) SARS - CoV - 2 ( III - IV ) S - mediated syncytium formation on Huh - 7 at 48 h . Scale bar equals 400 μm in d - f .

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e. SARS-CoV (I-II) and SARS-CoV-2 (III-IV) S-mediated syncytium formation on 293T/ACE2 cells at 48 h. f. SARS-CoV (I-II) and SARS-CoV-2 (III-IV) S-mediated syncytium formation on Huh-7 cells at 48 h. Scale bar equals 400 μm in d-f.


b . SARS - CoV - 2 S - mediated - fusion in presence of EK1 - scramble ( I ) , EK1 ( II ) , EK1C ( III ) , EK1P ( IV ) at 2 . 5 μM ( scale bar : 400 μm ) .

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b. SARS-CoV-2 S protein-mediated cell-cell fusion in the presence of EK1-scramble (I), EK1 (II), EK1C (III), and EK1P (IV) at 2.5 μM (scale bar: 400 μm).


c . Inhibitory activity of EK1 - scramble , EK1 , EK1C EK1P against SARS - CoV - 2 S - mediated - fusion .

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c. Inhibitory activity of EK1-scramble, EK1, EK1C and EK1P against SARS-CoV-2 S-mediated cell-cell fusion.


e . Inhibitory activity of EK1 - lipopeptides on SARS - CoV - 2 S - mediated - fusion .

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e. Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 S-mediated cell-cell fusion.


f . Inhibitory activity of EK1 - lipopeptides on SARS - CoV - 2 PsV infection . Experiments were repeated twice , data are expressed as means ± SD ( error bar ) .

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f. Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 PsV infection. Experiments were repeated twice, and the data are expressed as means ± SD (error bar).


HCoVs . a to h . Inhibitory activity of EK1C4 in - fusion mediated by S of SARS - CoV ( a )

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HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of SARS-CoV (a)


HCoVs . a to h . Inhibitory activity of EK1C4 in - fusion mediated by S of MERS - CoV ( b )

HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of MERS-CoV (b)


HCoVs . a to h . Inhibitory activity of EK1C4 in - fusion mediated by S of HCoV - OC43 ( c )

HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of HCoV-OC43 (c)


HCoVs . a to h . Inhibitory activity of EK1C4 in - fusion mediated by S of HCoV - 229E ( d )

HCoVs.a to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of HCoV-229E (d)


to h . Inhibitory activity of EK1C4 in - fusion mediated by S of HCoV - NL63 ( e )

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to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of HCoV-NL63 (e)


to h . Inhibitory activity of EK1C4 in - fusion mediated by S of WIV1 ( f )

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to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of WIV1 (f)


to h . Inhibitory activity of EK1C4 in - fusion mediated by S of Rs3367 ( g )

to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of Rs3367 (g)


to h . Inhibitory activity of EK1C4 in - fusion mediated by S of SHC014 ( h ) .

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to h. Inhibitory activity of EK1C4 in cell-cell fusion mediated by the S proteins of SHC014 (h).


i to o . Inhibitory activity of EK1C4 in PsV infection assays against SARS - CoV ( i )

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i to o. Inhibitory activity of EK1C4 in PsV infection assays against SARS-CoV (i)


to o . Inhibitory activity of EK1C4 in PsV infection assays against MERS - CoV ( j )

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to o. Inhibitory activity of EK1C4 in PsV infection assays against MERS-CoV (j)


Inhibitory activity of EK1C4 in PsV infection assays against HCoV - OC43 ( k )

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Inhibitory activity of EK1C4 in PsV infection assays against HCoV-OC43(k)


Inhibitory activity of EK1C4 in PsV infection assays against HCoV - 229E ( l )

Inhibitory activity of EK1C4 in PsV infection assays against HCoV-229E (l)


Inhibitory activity of EK1C4 in PsV infection assays against , NL63 ( m )

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Inhibitory activity of EK1C4 in PsV infection assays against , NL63 (m)


Inhibitory activity of EK1C4 in PsV infection assays against WIV1 ( n )

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Inhibitory activity of EK1C4 in PsV infection assays against WIV1 (n)


Inhibitory activity of EK1C4 in PsV infection assays against Rs3367 ( o ) .

Inhibitory activity of EK1C4 in PsV infection assays against Rs3367 (o).


Inhibitory activity of EK1 on live HCoV replication for SARS - CoV - 2 ( a ) , MERS - CoV ( b ) , HCoV - OC43 ( c ) , HCoV - 229E ( d ) , HCoV - NL63 ( e ) .

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Inhibitory activity of EK1 on live HCoV replication for SARS-CoV-2 (a), MERS-CoV (b), HCoV-OC43 (c), HCoV-229E (d), and HCoV-NL63 (e).


In vivo prophylactic efficacy of EK1C4 against HCoV - OC43 infection in mice . change ( f ) survival curves ( g ) of mice challenged with HCoV - OC43 . h - i . In vivo therapeutic efficacy of EK1C4 against HCoV - OC43 infection in mice . change ( h ) survival curves ( i ) of mice challenged with HCoV - OC43 . Experiments were repeated twice , data are expressed as means ± SD .

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In vivo prophylactic efficacy of EK1C4 against HCoV-OC43 infection in mice. Body weight change (f) and survival curves (g) of mice challenged with HCoV-OC43. h-i. In vivo therapeutic efficacy of EK1C4 against HCoV-OC43 infection in mice. Body weight change (h) and survival curves (i) of mice challenged with HCoV-OC43. Experiments were repeated twice, and the data are expressed as means ± SD.


A , Representative immunofluorescence of normal , , residual 3D organoids grown from mammary of a preclinical mouse model of breast cancer ( Methods ) . Similar of normal residual organoids shown on left with polarity markers ITGA6 ( red ) , ZO - 1 ( green ) , GM - 130 ( magenta ) ; DAPI ( blue ) . Right : human MYC oncoprotein ( green ) is expressed only in , CDH1 ( red ) . Scale bar : 25 μm . Data information : Number of replicates corresponds to mice used to derive organoids .

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A, Representative immunofluorescence staining of normal, tumor, and residual 3D organoids grown from primary mammary cells of a preclinical mouse model of breast cancer (Methods). Similar morphology of normal and residual organoids shown on the left with polarity markers ITGA6 (red), ZO-1 (green), GM-130 (magenta); DAPI (blue). Right: human MYC oncoprotein (green) is expressed only in tumor cells, CDH1 (red). Scale bar: 25 μm. Data information: Number of replicates corresponds to individual mice used to derive organoids.


, Selective secreted metabolites with significant change linked to urea cycle components from healthy ( n = 3 ; blue ) regressed ( n = 3 ; green ) mammary . Values represent metabolite abundance as quantified by under curve ( AUC ) of marker fragment ions / transitions for each metabolite . Values are plotted on log2 scale . Statistics were calculated using limma package n R with significance threshold corresponding to a Benjamini - Hochberg adjusted p - value ≤ 0 . 05 ( residual compared to normal ) . ata information : umbers of replicates correspond to mice or humans . ox plots : midline , median ; box , 25 - 75th percentile ; whisker , minimum to maximum . umbers marking comparisons ( gray lines ) show p values ( corresponding statistical are described in panel legends ) .

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, Selective secreted metabolites with significant change linked to urea cycle components from healthy (n=3; blue) and regressed (n=3; green) mammary glands. Values represent metabolite abundance levels as quantified by the area under the curve (AUC) of the marker fragment ions/transitions for each metabolite. Values are plotted on the log2 scale. Statistics were calculated using the limma package n R with the significance threshold corresponding to a Benjamini-Hochberg adjusted p-value ≤ 0.05 (residual compared to normal). ata information: umbers of replicates correspond to individual mice or humans. ox plots: midline, median; box, 25-75th percentile; whisker, minimum to maximum. umbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


C , Left , of expressing ARG1 ( top ) , an enzyme converting arginine to urea ornithine , of ARG1 ( bottom ) in normal from healthy ( n = 5 , 2921 analyzed ; blue ) residual from regressed ( n = 5 , 2241 analyzed ; green ) mammary tissue sections . Statistical differences were calculated with Mann - Whitney U ( Wilcoxon rank - sum ) . Right , Representative of immunofluorescence in normal ( top ) residual ( bottom ) stained for ARG1 ( green ) , CDH1 ( red ) , DAPI ( blue ) . Scale bar : 20 μm . Data information : Numbers of replicates correspond to mice or humans . Box plots : midline , median ; box , 25 - 75th percentile ; whisker , minimum to maximum . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical are described in panel legends ) .

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C, Left, Quantification of cells expressing ARG1 (top), an enzyme converting arginine to urea and ornithine, and intensity of ARG1 (bottom) in normal cells from healthy (n=5, 2921 cells analyzed; blue) and residual cells from regressed (n=5, 2241 cells analyzed; green) mammary gland tissue sections. Statistical differences were calculated with the Mann-Whitney U test (Wilcoxon rank-sum test). Right, Representative images of immunofluorescence staining in normal (top) and residual (bottom) duct stained for ARG1 (green), CDH1 (red), and DAPI (blue). Scale bar: 20 μm. Data information: Numbers of replicates correspond to individual mice or humans. Box plots: midline, median; box, 25-75th percentile; whisker, minimum to maximum. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


D , Nitric oxide synthase ( NOS ) activity , an enzyme involved in arginine metabolism , in healthy ( n = 3 ; blue ) in residual ( n = 3 ; green ) mouse mammary . difference is statistically significant by unpaired two - samples t - . E , Fractional labeling of lactate after cultivation of isolated regressed ( n = 3 ; green ) healthy ( n = 3 ; blue ) mouse mammary in growth medium supplemented with [ U - 13C ] glucose for 8 h ( Methods ) . three - carbon labeled ( 13C ) isotopologue ( M + 3 ) of lactate is depicted shows enrichment in residual of regressed mammary . difference is statistically significant by unpaired two - samples t - . Data information : Numbers of replicates correspond to mice or humans . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical are described in panel legends ) .

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D, Nitric oxide synthase (NOS) activity, an enzyme involved in arginine metabolism, in healthy (n=3; blue) and in residual (n=3; green) mouse mammary glands. The difference is statistically significant by unpaired two-samples t-test. E, Fractional labeling of lactate after cultivation of isolated regressed (n=3; green) and healthy (n=3; blue) mouse mammary glands in cell growth medium supplemented with [U-13C] glucose for 8 h (Methods). The three-carbon labeled (13C) isotopologue (M+3) of lactate is depicted and shows enrichment in the residual cells of the regressed mammary glands. The difference is statistically significant by unpaired two-samples t-test. Data information: Numbers of replicates correspond to individual mice or humans. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


A , ( Top ) Experimental design ( bottom ) of ( Methods ) of normal , , residual after 72 - h treatment with 3 - BP at indicated doses ( n = 5 mice per condition ) . Two - way ANOVA with Tukey ' s HSD multiple comparison testing was utilized to calculate statistical significance . Dox , doxycycline . Data information : Box plots : midline , median ; box , 25 - 75th percentile ; whiskers , minimum to maximum . Number of replicates corresponds to mice used to derive organoids . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical are described in panel legends ) .

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A, (Top) Experimental design and (bottom) quantification of cell death (Methods) of normal, tumor, and residual cells after 72-h treatment with 3-BP at the indicated doses (n=5 mice per condition). Two-way ANOVA with Tukey's HSD multiple comparison testing was utilized to calculate statistical significance. Dox, doxycycline. Data information: Box plots: midline, median; box, 25-75th percentile; whiskers , minimum to maximum. Number of replicates corresponds to individual mice used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


B , Representative bright - field of normal ( left ) , ( middle ) , residual ( right ) organoids , treated with vehicle ( top ) or with 50 μM 3 - BP ( bottom ) . Scale bar , 100 μm .

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B, Representative bright-field images of normal (left), tumor (middle), and residual (right) organoids, treated with vehicle (top) or with 50 μM 3-BP (bottom). Scale bar, 100 μm.


C , ( Top ) Experimental design ( bottom ) - of passaged residual normal after 48 h of treatment with 3 - BP ( n = 5 mice per condition ) . Statistics were calculated with multiple t - . Data information : Box plots : midline , median ; box , 25 - 75th percentile ; whiskers minimum to maximum . Number of replicates corresponds to mice used to derive organoids . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical are described in panel legends ) .

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C, (Top) Experimental design and (bottom) cell-death quantification of passaged residual and normal cells after 48 h of treatment with 3-BP (n=5 mice per condition). Statistics were calculated with multiple t-tests. Data information: Box plots: midline, median; box, 25-75th percentile; whiskers minimum to maximum. Number of replicates corresponds to individual mice used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


D , ( Main ) Extracellular glucose abundance changes upon treatment with 3 - BP in three populations ( n = 4 mice per condition ) . Values represent of glucose abundance in 3 - BP - treated versus untreated . Statistics were calculated using limma package in R with significance threshold corresponding to a Benjamini - Hochberg adjusted p - value ≤ 0 . 05 ( residual compared to normal ) . ( Insert ) Fractional labeling of lactate in untreated 3 - BP treated normal ( n = 2 ) residual ( n = 2 ) following cultivation in growth medium supplemented with [ U - 13C ] glucose . three - carbon labeled ( 13C ) isotopologue ( M + 3 ) of lactate is depicted . Statistics were calculated with unpaired two - samples t - . Data information : , Box plots : midline , median ; box , 25 - 75th percentile minimum to maximum . Number of replicates corresponds to mice used to derive organoids . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical are described in panel legends ) .

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D, (Main) Extracellular glucose abundance changes upon treatment with 3-BP in all three populations (n=4 mice per condition). Values represent the ratio of glucose abundance in 3-BP-treated versus untreated cells. Statistics were calculated using the limma package in R with the significance threshold corresponding to a Benjamini-Hochberg adjusted p-value ≤ 0.05 (residual compared to normal). (Insert) Fractional labeling of lactate in untreated and 3-BP treated normal (n=2) and residual (n=2) cells following cultivation in growth medium supplemented with [U-13C] glucose. The three-carbon labeled (13C) isotopologue (M+3) of lactate is depicted. Statistics were calculated with unpaired two-samples t-tests. Data information: , Box plots: midline, median; box, 25-75th percentile minimum to maximum. Number of replicates corresponds to individual mice used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


Double immunolabeling of PDS5A ( green ) SYCP3 ( red ) in mouse spread spermatocytes in

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Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in leptotene


Double immunolabeling of PDS5A ( green ) SYCP3 ( red ) in mouse spread spermatocytes in zygotene Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

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Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in zygotene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5A ( green ) SYCP3 ( red ) in mouse spread spermatocytes in , early pachytene Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

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Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in , early pachytene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5A ( green ) SYCP3 ( red ) in mouse spread spermatocytes in mid pachytene Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

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Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in mid pachytene Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5A ( green ) SYCP3 ( red ) in mouse spread spermatocytes in diplotene Arrows in ( I - L ) indicate position of some chromocenters . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated . DAPI of ( blue ) is shown for some spermatocytes .

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Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in diplotene Arrows in (I-L) indicate the position of some chromocenters. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. DAPI staining of the chromatin (blue) is shown for some spermatocytes.


Double immunolabeling of PDS5A ( green ) SYCP3 ( red ) in mouse spread spermatocytes in diakinesis Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in diakinesis Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


O , P Double immunolabeling of PDS5A ( green ) kinetochores revealed with an ACA serum ( red ) in a metaphase I spermatocyte . Data information : DAPI of ( blue ) is shown for some spermatocytes .

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O, P Double immunolabeling of PDS5A (green) and kinetochores revealed with an ACA serum (red) in a metaphase I spermatocyte. Data information: DAPI staining of the chromatin (blue) is shown for some spermatocytes.


Q , R Double immunolabeling of PDS5A ( green ) REC8 ( pseudocolored in purple ) in a metaphase I spermatocyte . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated . DAPI of ( blue ) is shown for some spermatocytes .

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Q, R Double immunolabeling of PDS5A (green) and REC8 (pseudocolored in purple) in a metaphase I spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated. DAPI staining of the chromatin (blue) is shown for some spermatocytes.


S , T Double immunolabeling of PDS5A ( green ) kinetochores ( ACA , red ) in a metaphase II spermatocyte . Data information : DAPI of ( blue ) is shown for some spermatocytes .

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S, T Double immunolabeling of PDS5A (green) and kinetochores (ACA, red) in a metaphase II spermatocyte. Data information: DAPI staining of the chromatin (blue) is shown for some spermatocytes.


Double immunolabeling of PDS5B ( green ) SYCP3 ( red ) in spread spermatocytes at indicated stages .

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Double immunolabeling of PDS5B (green) and SYCP3 (red) in spread spermatocytes at the indicated stages.


Double immunolabeling of PDS5B ( green ) SYCP3 ( red ) in spread spermatocytes at indicated stages . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

Double immunolabeling of PDS5B (green) and SYCP3 (red) in spread spermatocytes at the indicated stages. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Immunolabeling of PDS5B ( green ) REC8 ( pseudocolored in blue onto a spread pachytene spermatocyte . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

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Immunolabeling of PDS5B (green) and REC8 (pseudocolored in blue onto a spread pachytene spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Immunolabeling of PDS5B ( green ) SYCP3 ( red ) TRF1 ( pseudocolored in yellow onto a spread pachytene spermatocyte . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

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Immunolabeling of PDS5B (green) and SYCP3 (red) and TRF1 (pseudocolored in yellow onto a spread pachytene spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Immunolabeling of PDS5B ( green ) ACA ( pseudocolored in purple onto a spread pachytene spermatocyte . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

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Immunolabeling of PDS5B (green) and ACA (pseudocolored in purple onto a spread pachytene spermatocyte. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5B ( green ) SYCP3 ( red ) on diplotene spread nuclei . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

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Double immunolabeling of PDS5B (green) and SYCP3 (red) on diplotene spread nuclei. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


Double immunolabeling of PDS5B ( green ) SYCP3 ( red ) on diakinesis spread nuclei . Data information : Sex chromosomes ( X , Y ) bivalents ( XY ) are indicated .

Double immunolabeling of PDS5B (green) and SYCP3 (red) on diakinesis spread nuclei. Data information: Sex chromosomes (X, Y) and bivalents (XY) are indicated.


A - F Double immunolabeling of PDS5B ( green ) SYCP3 ( red ) in a metaphase I spermatocyte ( A - B ) selected bivalents ( C - F ) . sex bivalent ( XY ) is indicated .

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A-F Double immunolabeling of PDS5B (green) and SYCP3 (red) in a metaphase I spermatocyte (A-B) and selected bivalents (C-F). The sex bivalent (XY) is indicated.


G - J Double immunolabeling of PDS5B ( green ) kinetochores ( ACA , red ) , of ( DAPI ) in a metaphase II spermatocyte .

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G-J Double immunolabeling of PDS5B (green) and kinetochores (ACA, red), and staining of the chromatin (DAPI) in a metaphase II spermatocyte.


Double immunolabeling of PDS5A ( green , A - E ) SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at indicated stages .

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Double immunolabeling of PDS5A (green, A-E) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages.


Double immunolabeling of PDS5B ( green , F - J ) SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at indicated stages .

Double immunolabeling of PDS5B (green, F-J) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages.


K - M Preparations from same mice were immunolabeled with testis - specific histone variant H1t ( green ) SYCP3 ( red ) .

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K-M Preparations from the same mice were immunolabeled with the testis-specific histone variant H1t (green) and SYCP3 (red).


N Scatter dot - plot graph of total SC measured in autosomal pairs of 33 early pachytene from three different individuals of each genotype . Bars indicate mean standard error . Statistical significance was assessed using an unpaired two - T - function ( * * * * , p < 0 . 0001 ) .

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N Scatter dot-plot graph of total SC length measured in the autosomal pairs of 33 early pachytene cells from three different individuals of each genotype. Bars indicate mean and standard error. Statistical significance was assessed using an unpaired two-tailed T-test function (****, p < 0.0001).


A - H Double immunolabeling of SYCP1 ( green ) SYCP3 ( red ) in wild - type ( A - D ) Pds5AB cKO ( E - H ) spread spermatocytes at indicated stages . I - L Double immunolabeling of HORMAD2 ( pseudocolored in ) SYCP3 ( red ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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A-H Double immunolabeling of SYCP1 (green) and SYCP3 (red) in wild-type (A-D) and Pds5AB cKO (E-H) spread spermatocytes at the indicated stages. I-L Double immunolabeling of HORMAD2 (pseudocolored in white) and SYCP3 (red). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) cohesin subunits SMC3 ( green , in Pds5AB cKO spread spermatocytes at indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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Double immunolabeling of SYCP3 (red) and the cohesin subunits SMC3 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) cohesin subunits REC8 ( green in Pds5AB cKO spread spermatocytes at indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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Double immunolabeling of SYCP3 (red) and the cohesin subunits REC8 (green in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) cohesin subunits SMC1β ( green , in Pds5AB cKO spread spermatocytes at indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and the cohesin subunits SMC1β (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) cohesin cofactors Sororin ( green , in Pds5AB cKO spread spermatocytes at indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and the cohesin cofactors Sororin (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) cohesin cofactors WAPL ( green , in Pds5AB cKO spread spermatocytes at indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and the cohesin cofactors WAPL (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) either γH2AX ( pseudocolored in blue in Pds5AB cKO spread spermatocytes at indicated stages .

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Double immunolabeling of SYCP3 (red) and either γH2AX (pseudocolored in blue in Pds5AB cKO spread spermatocytes at the indicated stages.


Double immunolabeling of SYCP3 ( red ) RAD51 ( green in Pds5AB cKO spread spermatocytes at indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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Double immunolabeling of SYCP3 (red) and RAD51 (green in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


I Scatter dot - plot graph of of RAD51 foci in in 15 zygotene nuclei randomly selected from three different individuals of each genotype . Bars indicate mean standard error statistical significance was assessed with an unpaired two - T - function ( * * * * , p < 0 . 0001 ) .

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I Scatter dot-plot graph of the quantification of RAD51 foci in in 15 zygotene nuclei randomly selected from three different individuals of each genotype. Bars indicate mean and standard error and statistical significance was assessed with an unpaired two-tailed T-test function (****, p < 0.0001).


J - L Double immunolabeling of MLH1 ( pseudocolored in ) SYCP3 ( red ) in Pds5AB cKO spread spermatocytes . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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J-L Double immunolabeling of MLH1 (pseudocolored in white) and SYCP3 (red) in Pds5AB cKO spread spermatocytes. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) TRF1 ( green , in Pds5AB cKO spread spermatocytes at indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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Double immunolabeling of SYCP3 (red) and TRF1 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) RAP1 ( green , in Pds5AB cKO spread spermatocytes at indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

Double immunolabeling of SYCP3 (red) and RAP1 (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


I Selected zygotene - like pachytene - like bivalents presenting altered TRF1 distribution as ( from left to right ) ( i ) telomere stretches , ( ii ) multiple telomere , ( iii ) distant telomeres ( iv ) telomere - less . Arrowheads in ( I ) indicate position of altered telomeres . J of telomeres with a regular or altered disposition of TRF1 in Pds5AB cKO spermatocytes at indicated stages ( n = 1646 telomeres in zygotene - like spermatocytes 738 in pachytene - like ones ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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I Selected zygotene-like and pachytene-like bivalents presenting altered TRF1 distribution as (from left to right) (i) telomere stretches, (ii) multiple telomere, (iii) distant telomeres and (iv) telomere-less. Arrowheads in (I) indicate the position of altered telomeres. J Quantification of telomeres with a regular or altered disposition of TRF1 in Pds5AB cKO spermatocytes at the indicated stages (n=1646 telomeres in zygotene-like spermatocytes and 738 in pachytene-like ones). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


K , L As in ( I , J ) , respectively , but for RAP1 ( n = 1376 telomeres in zygotene - like spermatocytes 328 in pachytene - like ones ) . Arrowheads in ( K ) indicate position of altered telomeres . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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K, L As in (I, J), respectively, but for RAP1 staining (n=1376 telomeres in zygotene-like spermatocytes and 328 in pachytene-like ones). Arrowheads in (K) indicate the position of altered telomeres. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


M - P Double immunolabeling of SUN1 ( green ) SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at indicated stages . 0 , 1 2 in ( N O ) indicate number of chromosome ends labelled by SUN1 in a given bivalent . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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M-P Double immunolabeling of SUN1 (green) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages. 0, 1 and 2 in (N and O) indicate the number of chromosome ends labelled by SUN1 in a given bivalent. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Q As in ( I ) , but for SUN1 . Arrowheads in ( Q ) indicate position of altered telomeres . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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Q As in (I), but for SUN1 staining. Arrowheads in (Q) indicate the position of altered telomeres. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


R Selected zygotene - like early pachytene - like bivalents from Pds5AB cKO spermatocytes immunolabeled with SYCP3 ( red ) SUN1 ( green ) , their respective of appearance . S of telomeres with a regular or altered disposition of SUN1 ( n = 715 telomeres in zygotene - like spermatocytes 410 in pachytene - like ones ) . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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R Selected zygotene-like and early pachytene-like bivalents from Pds5AB cKO spermatocytes immunolabeled with SYCP3 (red) and SUN1 (green), and their respective percentage of appearance. S Quantification of telomeres with a regular or altered disposition of SUN1 (n=715 telomeres in zygotene-like spermatocytes and 410 in pachytene-like ones). Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


A - C Immunolabeling of SYCP3 ( red ) telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at indicated stages . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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A-C Immunolabeling of SYCP3 (red) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


D 300 % magnification of selected zygotene - like pachytene - like bivalents presenting regular ( left ) or altered ( i - iv ) telomere FISH signals .

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D 300% magnification of selected zygotene-like and pachytene-like bivalents presenting regular (left) or altered (i-iv) telomere FISH signals.


Double immunolabeling of SYCP3 ( red ) RAP1 ( blue ) telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread

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Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes


Double immunolabeling of SYCP3 ( red ) RAP1 ( blue ) telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at indicated stages . Asterisks indicate position of enlarged chromosomes / bivalents shown in a 300 % magnification displaying telomere stretches , multiple telomere , distant telomeres telomere - less . arrowheads indicate telomeres with an altered organization of telomeric into multiple telomere signals with RAP1 signals telomere stretches Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Asterisks indicate the position of the enlarged chromosomes/bivalents shown in a 300% magnification displaying telomere stretches, multiple telomere, distant telomeres and telomere-less. White arrowheads indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals with RAP1 signals and telomere stretches Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) RAP1 ( blue ) telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at indicated stages . Asterisks indicate position of enlarged chromosomes / bivalents shown in a 300 % magnification displaying telomere stretches , multiple telomere , distant telomeres telomere - less . arrowheads indicate telomeres with an altered organization of telomeric into multiple telomere signals without RAP1 signals telomere stretches Pink arrowheads in ( T ) indicate a telomere presenting a regular telomere FISH signal without a RAP1 signal . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

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Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Asterisks indicate the position of the enlarged chromosomes/bivalents shown in a 300% magnification displaying telomere stretches, multiple telomere, distant telomeres and telomere-less. White arrowheads indicate telomeres with an altered organization of telomeric DNA into multiple telomere signals without RAP1 signals telomere stretches Pink arrowheads in (T) indicate a telomere presenting a regular telomere FISH signal without a RAP1 signal. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Double immunolabeling of SYCP3 ( red ) RAP1 ( blue ) telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytes at indicated stages . Asterisks indicate position of enlarged chromosomes / bivalents shown in a 300 % magnification displaying telomere stretches , multiple telomere , distant telomeres telomere - less .

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

Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytes at the indicated stages. Asterisks indicate the position of the enlarged chromosomes/bivalents shown in a 300% magnification displaying telomere stretches, multiple telomere, distant telomeres and telomere-less.


Double immunolabeling of TRF1 ( green ) SYCP3 ( red ) in squashed spermatocytes from wild - type or Pds5AB cKO mice M - P ) . first three columns correspond to z - projections of 15 focal planes throughout Top , Equator Bottom regions of nucleus . projection of 65 focal planes across same spermatocyte nucleus is shown at fourth column ( Z - Projection ) . arrowheads in ( F indicate presence of TRF1 signals non - attached to NE . Scale bar , 10 μm .

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

Double immunolabeling of TRF1 (green) and SYCP3 (red) in squashed spermatocytes from wild-type or Pds5AB cKO mice M-P). The first three columns correspond to z-projections of 15 focal planes throughout the Top, Equator and Bottom regions of the nucleus. The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column (Z-Projection). White arrowheads in (F indicate the presence of TRF1 signals non-attached to the NE. Scale bar, 10 μm.


Double immunolabeling of TRF1 ( green ) SYCP3 ( red ) in squashed spermatocytes from wild - type or Pds5AB cKO mice first three columns correspond to z - projections of 15 focal planes throughout Top , Equator Bottom regions of nucleus . projection of 65 focal planes across same spermatocyte nucleus is shown at fourth column ( Z - Projection ) . arrowheads in N ) indicate presence of TRF1 signals non - attached to NE . Scale bar , 10 μm .

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

Double immunolabeling of TRF1 (green) and SYCP3 (red) in squashed spermatocytes from wild-type or Pds5AB cKO mice The first three columns correspond to z-projections of 15 focal planes throughout the Top, Equator and Bottom regions of the nucleus. The projection of 65 focal planes across the same spermatocyte nucleus is shown at the fourth column (Z-Projection). White arrowheads in N) indicate the presence of TRF1 signals non-attached to the NE. Scale bar, 10 μm.


Q - T Selected bivalents from Pds5AB cKO spermatocytes displaying structural telomere abnormalities as telomere stretches ( Q ) , multiple telomere ( R ) , distant telomere ( S ) telomere - less ( T ) .

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

Q-T Selected bivalents from Pds5AB cKO spermatocytes displaying structural telomere abnormalities as telomere stretches (Q), multiple telomere (R), distant telomere (S) and telomere-less (T).


HCT116 U87 were treated with 20 µM LXR623 for 24h . Transcriptome gene set enrichment was performed . Shown are enrichment plots . NES : normalized enrichment score .

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HCT116 and U87 cells were treated with 20 µM LXR623 for 24h. Transcriptome and gene set enrichment analysis was performed. Shown are enrichment plots. NES: normalized enrichment score.


HCT116 were treated with 20 µM LXR623 for 24h . Transcriptome gene set enrichment was performed . Shown are enrichment plots . NES : normalized enrichment score .

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

HCT116 cells were treated with 20 µM LXR623 for 24h. Transcriptome and gene set enrichment analysis was performed. Shown are enrichment plots. NES: normalized enrichment score.


U87 GBM were treated with 20 µM LXR623 or 50 µM Simvastatin ( Sim ) for 48h . Thereafter , lysates were collected analyzed for total cholesterol . Shown are means SD ( n = 3 ) .

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

U87 GBM cells were treated with 20 µM LXR623 or 50 µM Simvastatin (Sim) for 48h. Thereafter, lysates were collected and analyzed for total cholesterol levels. Shown are means and SD (n = 3).


Shown is a volcano plot of from TCGA ( database ) with high vs . low of ABCA1 . A close - up of most significantly down - regulated genes is presented ( mRNAs related to electron transport chain mitochondrial metabolism ) . x - axis highlights fold changes ( log2 scale ) ( down - regulated genes , negative values ) , whereas y - axis indicates level of statistical significance ( shown in - log10 format ) .

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

Shown is a volcano plot of patients from the TCGA (database) with high vs. low levels of ABCA1. A close-up of the most significantly down-regulated genes is presented (mRNAs related to the electron transport chain and mitochondrial metabolism). The x-axis highlights the fold changes (log2 scale) (down-regulated genes, negative values), whereas the y-axis indicates the level of statistical significance (shown in -log10 format).


GBM12 GBM ( short term - derived xenograft ) , U87 GBM , HCT116 colonic carcinoma A375 melanoma were treated with indicated concentrations of ABT263 , LXR623 or combination for 72h . Thereafter , viability was analyzed statistical was performed .

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GBM12 GBM cells (short term patient-derived xenograft), U87 GBM cells, HCT116 colonic carcinoma cells and A375 melanoma cells were treated with the indicated concentrations of ABT263, LXR623 or the combination for 72h. Thereafter, cellular viability was analyzed and statistical analysis was performed.


U87 , T98G LN229 were treated with 1 µM ABT263 , 20 µM LXR623 or combination of both for 72h . Mewo melanoma HCT116 colon carcinoma were treated with 1 µM ABT263 , 10 µM LXR623 or combination of both for 72h ( MeWo ) or for 48h ( HCT116 ) . Thereafter , were stained with annexin V / propidium iodide analyzed by multi - parametric flow cytometry .

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

U87, T98G and LN229 cells were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 72h. Mewo melanoma and HCT116 colon carcinoma cells were treated with 1 µM ABT263, 10 µM LXR623 or the combination of both for 72h (MeWo) or for 48h (HCT116). Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.


, - like GBM , NCH644 , NCH421k NCH690 were treated with 1 µM ABT263 , 20 µM LXR623 or combination of both for 48h . Thereafter , were stained with annexin V / propidium iodide analyzed by multi - parametric flow cytometry .

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, Stem-like GBM cells, NCH644, NCH421k and NCH690 were treated with 1 µM ABT263, 20 µM LXR623 or the combination of both for 48h. Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.


U87 ( 72h treatment ) HCT116 ( 48h treatment ) were treated with 1 µM ABT263 , 20 µM ( U87 ) or 10 µM ( HCT116 ) LXR623 or combination of both , stained with TMRE analyzed by flow cytometric for dissipation of mitochondrial potential .

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U87 (72h treatment) and HCT116 (48h treatment) were treated with 1 µM ABT263, 20 µM (U87) or 10 µM (HCT116) LXR623 or the combination of both, stained with TMRE and analyzed by flow cytometric analysis for dissipation of mitochondrial membrane potential.


HCT116 colonic carcinoma were treated with ABT263 , LXR623 or combination of both . Whole lysates were collected subjected to capillary electrophoresis for expression / cleavage of PARP , total caspase - 9 ( CP9 ) Vinculin . CF : fragment , FL : full .

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

HCT116 colonic carcinoma cells were treated with ABT263, LXR623 or the combination of both. Whole protein lysates were collected and subjected to capillary electrophoresis for the expression/cleavage of PARP, total caspase-9 (CP9) and Vinculin. CF: cleaved fragment, FL: full length.


HCT116 colonic carcinoma or NCH644 glioblastoma were treated with selective BH3 - mimetics , WEHI - 539 ( Bcl - xL inhibitor ) , ABT199 ( Bcl - 2 inhibitor ) or A1210477 ( Mcl - 1 inhibitor ) in presence or absence of LXR623 for 48h . Thereafter , were labeled with annexin V / propidium iodide analyzed by multi - parametric flow cytometry .

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

HCT116 colonic carcinoma or NCH644 glioblastoma stem cells were treated with selective BH3-mimetics, WEHI-539 (Bcl-xL inhibitor), ABT199 (Bcl-2 inhibitor) or A1210477 (Mcl-1 inhibitor) in the presence or absence of LXR623 for 48h. Thereafter, cells were labeled with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry.


, LN229 , T98G , U87 , U87 - EGFRvIII HCT116 were treated with increasing concentrations of LXR623 for 72h . Thereafter , lysates were collected subjected to standard western blot ( LN229 , T98G U87 ) or capillary electrophoresis ( U87 - EGFR - vIII HCT116 ) for expression of Usp9X , Mcl - 1 , Bcl - 2 , Bcl - xL , Noxa , BIM / or Actin Vinculin . In U87 - EGFRvIII HCT116 , Vinculin was used in lieu of Actin .

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, LN229, T98G, U87, U87-EGFRvIII and HCT116 cells were treated with increasing concentrations of LXR623 for 72h. Thereafter, protein lysates were collected and subjected to standard western blot (LN229, T98G and U87) or capillary electrophoresis (U87-EGFR-vIII and HCT116) for the expression of Usp9X, Mcl-1, Bcl-2, Bcl-xL, Noxa, BIM and/or Actin and Vinculin. In U87-EGFRvIII and HCT116, Vinculin was used in lieu of Actin.


LN229 , T98G , U87 , U87 - EGFRvIII HCT116 were treated with LXR623 as described in A . Thereafter , lysates were collected subjected to capillary electrophoresis for expression of ABCA1 Vinculin .

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LN229, T98G, U87, U87-EGFRvIII and HCT116 cells were treated with LXR623 as described in A. Thereafter, protein lysates were collected and subjected to capillary electrophoresis for the expression of ABCA1 and Vinculin.


LN229 were transfected with siNT , siNoxa 1 , siNoxa 2 or BAK - siRNA for 72h . Thereafter , were treated with combination treatment of 1 µM ABT263 20 µM LXR623 for another 24h . After conclusion of treatment , were harvested , fixed , stained with propidium iodide analyzed by flow cytometry for - fragmentation .

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LN229 were transfected with siNT, siNoxa 1, siNoxa 2 or BAK-siRNA for 72h. Thereafter, cells were treated with the combination treatment of 1 µM ABT263 and 20 µM LXR623 for another 24h. After conclusion of the treatment, cells were harvested, fixed, stained with propidium iodide and analyzed by flow cytometry for DNA - fragmentation.


LN229 were transfected as described in B . Thereafter , whole lysates were collected analyzed by capillary electrophoresis for expression of Noxa , BAK Vinculin .

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LN229 were transfected as described in B. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of Noxa, BAK and Vinculin.


LN229 GBM were treated with 10 µM LXR623 ( LXR ) , 1 µM ABT263 or combination for 48h . Thereafter , lysates were prepared immunoprecipitated with an antibody against BAK . Standard western blotting was performed ( immunoprecipitation corresponding inputs ) with antibody against BAK Mcl - 1 . arrows highlight specific bands , while stars indicate immunoglobulin - chains .

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

LN229 GBM cells were treated with 10 µM LXR623 (LXR), 1 µM ABT263 or the combination for 48h. Thereafter, protein lysates were prepared and immunoprecipitated with an antibody against BAK. Standard western blotting was performed (immunoprecipitation and the corresponding inputs) with antibody against BAK and Mcl-1. The arrows highlight the specific protein bands, while stars indicate the immunoglobulin light-chains.


U87 were treated with 20 µM LXR623 for 7h 24h . Thereafter , were stained with annexin V / propidium iodide analyzed by multi - parametric flow cytometry . lower left quadrant indicates of viable .

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U87 cells were treated with 20 µM LXR623 for 7h and 24h. Thereafter, cells were stained with annexin V/propidium iodide and analyzed by multi-parametric flow cytometry. The lower left quadrant indicates the percentage of viable cells.


U87 were treated with LXR623 as indicated for 7h analyzed for total ATP by a luminescence . To account for dead , assays were normalized to - content ( number ) .

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U87 cells were treated with LXR623 as indicated for 7h and analyzed for total ATP levels by a luminescence assay. To account for dead cells, assays were normalized to DNA-content (cell number).


U87 were treated with LXR623 as indicated for 24h . lysates were prepared subjected to LC / MS for determination of ATP .

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U87 cells were treated with LXR623 as indicated for 24h. Cell lysates were prepared and subjected to LC/MS for the determination of ATP levels.


U87 were treated with increasing concentrations of LXR623 for 7h 24h . Thereafter , whole lysates were collected analyzed by capillary electrophoresis for expression of phosphorylated AMPK ( threonine 172 ) total AMPK .

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U87 cells were treated with increasing concentrations of LXR623 for 7h and 24h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK (threonine 172) and total AMPK.


U87 were treated with increasing concentrations of LXR623 in presence or absence of exogenous ATP for 7h . Thereafter , whole lysates were collected analyzed by capillary electrophoresis for expression of ATF4 .

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U87 cells were treated with increasing concentrations of LXR623 in the presence or absence of exogenous ATP for 7h. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4.


U87 were treated with increasing concentrations of LXR623 for 24h in presence or absence of cholesterol . Thereafter , whole lysates were collected analyzed by capillary electrophoresis for expression of phosphorylated AMPK ( threonine 172 ) total AMPK .

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

U87 cells were treated with increasing concentrations of LXR623 for 24h in the presence or absence of cholesterol. Thereafter, whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of phosphorylated AMPK (threonine 172) and total AMPK.


were treated as in F whole lysates were collected analyzed by capillary electrophoresis for expression of ATF4 Vinculin ( G ) by standard western blot for expression of Noxa Actin .

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Cells were treated as in F and whole cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of ATF4 and Vinculin (G) and by the standard western blot for the expression of Noxa and Actin.


U87 were treated with increasing concentrations of LXR623 . Thereafter , whole lysates were prepared analyzed for classical ER - stress markers , GRP78 , ATF4 Noxa by capillary electrophoresis .

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U87 cells were treated with increasing concentrations of LXR623. Thereafter, whole cell protein lysates were prepared and analyzed for classical ER-stress markers, GRP78, ATF4 and Noxa by capillary electrophoresis.


U87 were transfected with non - targeting siRNA ( n . t . - siRNA ) , ATF3 - siRNA , ATF4 siRNA or combination of both ATF4 ATF3 siRNAs . After 48h , were treated with LXR623 , whole lysates were harvested analyzed by capillary electrophoresis for expression of ATF3 , ATF4 , Noxa Vinculin .

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

U87 cells were transfected with non-targeting siRNA (n.t.-siRNA), ATF3-siRNA, ATF4 siRNA or the combination of both ATF4 and ATF3 siRNAs. After 48h, cells were treated with LXR623, whole cell protein lysates were harvested and analyzed by capillary electrophoresis for the expression of ATF3, ATF4, Noxa and Vinculin.


U87 were treated as in A . Shown are of TCA - cycle metabolites .

U87 cells were treated as in A. Shown are the levels of TCA-cycle metabolites.


U87 were incubated in DMEM ( devoid of phenol red , glucose , pyruvate glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine 1 . 5 % dialyzed FBS in presence or absence of 20 µM LXR623 for 24 hours . were then harvested for LC / MS . fractions of each different isotopologue of each metabolite were calculated ( of entire pool ) . Shown are isotopologues of TCA cycle intermediates labeled by glucose carbons non - labeled isotopologues ( m + 0 ) .

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

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of the TCA cycle intermediates labeled by glucose carbons and non-labeled isotopologues (m+0).


U87 were incubated in DMEM ( devoid of phenol red , glucose , pyruvate glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine 1 . 5 % dialyzed FBS in presence or absence of 20 µM LXR623 for 24 hours . were then harvested for LC / MS . fractions of each different isotopologue of each metabolite were calculated ( of entire pool ) . Shown are isotopologues of non - essential aminoacids ( glutamatic acid aspartic acid ) .

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

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of non-essential aminoacids (glutamatic acid and aspartic acid).


U87 were incubated in DMEM ( devoid of phenol red , glucose , pyruvate glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine 1 . 5 % dialyzed FBS in presence or absence of 20 µM LXR623 for 24 hours . were then harvested for LC / MS . fractions of each different isotopologue of each metabolite were calculated ( of entire pool ) . Shown are isotopologues of non - essential aminoacids ( glutamatic

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

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of non-essential aminoacids (glutamatic acid


U87 were incubated in DMEM ( devoid of phenol red , glucose , pyruvate glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine 1 . 5 % dialyzed FBS in presence or absence of 20 µM LXR623 for 24 hours . were then harvested for LC / MS . fractions of each different isotopologue of each metabolite were calculated ( of entire pool ) . Shown are isotopologues of glutathione nucleotides .

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

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool). Shown are the isotopologues of glutathione and nucleotides.


U87 were incubated in DMEM ( devoid of phenol red , glucose , pyruvate glutamine ) supplemented with 25 mM U - 13C - glucose , 4 mM glutamine 1 . 5 % dialyzed FBS in presence or absence of 20 µM LXR623 for 24 hours . were then harvested for LC / MS . fractions of each different isotopologue of each metabolite were calculated ( of entire pool ) .

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

U87 cells were incubated in DMEM (devoid of phenol red, glucose, pyruvate and glutamine) supplemented with 25 mM U-13C-glucose, 4 mM glutamine and 1.5% dialyzed FBS in the presence or absence of 20 µM LXR623 for 24 hours. Cells were then harvested for LC/MS analysis. The fractions of each different isotopologue of each metabolite were calculated (percentage of the entire pool).


U87 were treated with DMSO or LXR623 20 µM for 24h . Thereafter , were processed for polar metabolite by LC / MS . Shown are of NAD NADH2 .

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

U87 cells were treated with DMSO or LXR623 20 µM for 24h. Thereafter, cells were processed for polar metabolite analysis by LC/MS. Shown are the levels of NAD and NADH2.


U87 were treated with LXR623 for 1h , 3h or 5h subjected to extracellular flux on Seahorse XFp instrument in context of a mitochondrial stress ( A - C ) . From this , coupled respiration was calculated ( D - F ) .

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

U87 cells were treated with LXR623 for 1h, 3h or 5h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay (A-C). From this assay, coupled respiration was calculated (D-F).


U87 were treated with LXR623 , cholesterol or combination of both for 24h subjected to extracellular flux on Seahorse XFp instrument in context of a mitochondrial stress ( G ) . From this , mitochondrial OCR coupled respiration was calculated ( H - I ) .

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

U87 cells were treated with LXR623, cholesterol or the combination of both for 24h and subjected to extracellular flux analysis on the Seahorse XFp instrument in the context of a mitochondrial stress assay (G). From this assay, mitochondrial OCR and coupled respiration was calculated (H-I).


U87 were treated with DMSO or LXR623 20 µM with indicated time point . Thereafter , whole lysates were collected subjected to standard western blot for expression of respiratory complexes ( OXPHOS ) , Noxa , ATF4 . A star indicates an unspecific band .

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

U87 cells were treated with DMSO or LXR623 20 µM with the indicated time point. Thereafter, whole cell protein lysates were collected and subjected to standard western blot for the expression of the respiratory complexes (OXPHOS), Noxa, and ATF4. A star indicates an unspecific band.


U87 were treated with DMSO or LXR623 20 µM in presence or absence of cycloheximide for 7h . Thereafter , whole lysates were collected of components of V were analyzed by standard western blot .

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

U87 cells were treated with DMSO or LXR623 20 µM in the presence or absence of cycloheximide for 7h. Thereafter, whole cell protein lysates were collected and the protein levels of the components of complex V were analyzed by standard western blot.


U87 were treated with increasing of LXR623 in presence or absence of cholesterol . Thereafter , whole lysates were collected analyzed of respiratory complexes ( OXPHOS ) by standard western blot .

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

U87 cells were treated with increasing concentration of LXR623 in the presence or absence of cholesterol. Thereafter, whole cell protein lysates were collected and analyzed the protein levels of the respiratory complexes (OXPHOS) by standard western blot.


U87 transduced with non - targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h . Thereafter , extracellular flux was performed in context of a mitochondrial stress . From this , basal respiration coupled respiration was calculated ( M - N ) .

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

U87 cells transduced with non-targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h. Thereafter, extracellular flux analysis was performed in the context of a mitochondrial stress assay. From this assay, basal respiration and coupled respiration was calculated (M-N).


U87 transduced with non - targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h . were collected analyzed for expression of LXRβ mRNA ( O ) or LXRβ by capillary electrophoresis ( P ) . Shown are means SD ( n = 5 ) . Statistical significance was determined by two - sided student ' s t . .

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

U87 cells transduced with non-targeting or LXRβ shRNA were treated with vehicle or 20 µM LXR623 for 24h. RNA and protein were collected and analyzed for the expression of LXRβ mRNA (O) or LXRβ protein by capillary electrophoresis (P). Shown are means and SD (n = 5). Statistical significance was determined by two-sided student's t.test.


1x106 HCT116 colon carcinoma were implanted subcutaneously . were treated intraperitoneally with vehicle , LXR623 ( 100 - 200 mg / kg ) , ABT263 ( 100 mg / kg ) or both agents ( 3 days per week for 1 . 5 . growth curves show development of for each treatment group . Scatter plots display quantitative representation of among different treatments toward end of experiment . Shown are means SD ( n ≥ 5 ) . * p = 0 . 022 , * * p = 0 . 0063 , * * * * p < 0 . 0001 . Statistical significance was determined by one - way ANOVA . B , E , GBM43 PDXs were implanted subcutaneously . were treated intraperitoneally with vehicle , LXR623 ( 100 mg / kg ) , ABT263 ( 75 mg / kg ) or both agents ( 3 days per week for 1 . 5 ) . growth curves show development of for each treatment group . Scatter plots display quantitative representation of among different treatments toward end of experiment . Shown are means SD ( n ≥ 5 ) . * p = 0 . 022 , * * p = 0 . 0031 . Statistical significance was determined by one - way ANOVA . C , F , A375 BRAF V600E mutated melanomas were implanted subcutaneously . were treated intraperitoneally with vehicle , LXR623 ( 100 mg / kg ) , ABT263 ( 75 mg / kg ) or both agents ( 3 days per week for 1 . 5 ) . growth curves show development of for each treatment group . Scatter plots display quantitative representation of among different treatments toward end of experiment . Shown are means SD ( n ≥ 5 ) . * p = 0 . 0109 ( GW3965 vs Combination ) , * p = 0 . 0166 ( ABT263 vs Combination ) , * * p = 0 . 0047 . Statistical significance was determined by one - way ANOVA .

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

1x106 HCT116 colon carcinoma cells were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100-200 mg/kg), ABT263 (100 mg/kg) or both agents (3 days per week for 1.5 weeks. Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.022, **p=0.0063, ****p<0.0001. Statistical significance was determined by one-way ANOVA. B, E, GBM43 PDXs were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100 mg/kg), ABT263 (75 mg/kg) or both agents (3 days per week for 1.5 weeks). Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.022, **p=0.0031. Statistical significance was determined by one-way ANOVA. C, F, A375 BRAF V600E mutated melanomas were implanted subcutaneously. Animals were treated intraperitoneally with vehicle, LXR623 (100 mg/kg), ABT263 (75 mg/kg) or both agents (3 days per week for 1.5 weeks). Tumor growth curves show the development of tumor size for each treatment group. Scatter plots display the quantitative representation of the tumor size among the different treatments toward the end of the experiment. Shown are means and SD (n ≥ 5). *p=0.0109 (GW3965 vs Combination), *p=0.0166 (ABT263 vs Combination), **p=0.0047. Statistical significance was determined by one-way ANOVA.


Related to HCT116 colonic xenograft model , representative histopathological ( hematoxylin eosin stain ) are shown from each treatment group . Scale bar : 50 µm . Sections from same treatment groups as in G were stained with TUNEL SDHB , repectively . Displayed are representative of . Scale bar 50 µM .

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

Related to the HCT116 colonic xenograft model, representative histopathological images (hematoxylin and eosin stain) are shown from each treatment group. Scale bar: 50 µm. Sections from the same treatment groups as in G were stained with TUNEL and SDHB, repectively. Displayed are representative images of the staining. Scale bar 50 µM.


Sections from experiment in 7A were quantified for apoptotic

Sections from the experiment in 7A were quantified for apoptotic bodies


, Sections from experiment in 7A were quantified for , number of TUNEL positive

, Sections from the experiment in 7A were quantified for , number of TUNEL positive cells


Sections from experiment in 7A were quantified for number of mitosis per multiple high - power fields .

Sections from the experiment in 7A were quantified for number of mitosis per multiple high-power fields.


Total carbonyl - bearing detected by shotgun redox proteomic measurement in three biological replicates each of E . coli D . radiodurans with without irradiation . left axis is number of - unique detected as carbonylated . right axis is number of sites in total detected as carbonylated ( red ) or not oxidized ( black ) in peptides bearing at least one carbonyl . Stripes indicate carbonylated carbonylatable sites detected only in irradiated samples .

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

Total carbonyl-bearing proteins detected by shotgun redox proteomic measurement in three biological replicates each of E. coli and D. radiodurans with and without irradiation. The left axis is the number of sequence-unique proteins detected as carbonylated. The right axis is the number of sites in total detected as carbonylated (red) or not oxidized (black) in peptides bearing at least one carbonyl. Stripes indicate carbonylated proteins and carbonylatable sites detected only in irradiated samples.


Volcano plots for relative abundance changes measured by mass spectrometry in E . coli ( left ) D . radiodurans ( right ) after irradiation using same biological replicates as in Fig 2A . Black - circled points are those with significant changes ( paired , 2 - sided t - p - value < 0 . 05 ) of > 2 - fold or < 0 . 5 - fold . Red points are with at least one carbonylated peptide detected . Fold change p - value cutoffs considered for significance are indicated by dashed lines .

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

Volcano plots for relative protein abundance changes measured by mass spectrometry in E. coli (left) and D. radiodurans (right) after irradiation using the same biological replicates as in Fig 2A. Black-circled points are those proteins with significant changes (paired, 2-sided t-test p-value < 0.05) of >2-fold or <0.5-fold. Red points are proteins with at least one carbonylated peptide detected. Fold change and p-value cutoffs considered for significance are indicated by dashed lines.


Prevalence of RKPT residues prevalence of carbonylated form in experimentally measured peptides combining 3 biological replicates of both conditions for each organism . Ratios are given above each pair of bars . proportions are significantly different between each RKPT their respective carbonylation state by two - z - of two proportions ( p - values < 0 . 01 ; see Materials Methods ) , meaning carbonylated proportions are not determined simply by relative prevalence of RKPT . See also Appendix Fig S1 .

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

Prevalence of individual RKPT residues and prevalence of carbonylated form in experimentally measured peptides combining all 3 biological replicates of both conditions for each organism. Ratios are given above each pair of bars. All proportions are significantly different between each RKPT and their respective carbonylation state by two-tailed z-test of two proportions (p-values < 0.01; see Materials and Methods), meaning carbonylated proportions are not determined simply by relative prevalence of RKPT. See also Appendix Fig S1.


Prevalence of canonical before irradiation of E . coli D . radiodurans , combining 3 biological replicates for each condition . Ratios are given above each pair of bars . proportions are significantly different between species by two - z - of two proportions ( p - values < 0 . 01 ) . See also Fig EV1 EV2 .

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

Prevalence of all canonical amino acids before irradiation of E. coli and D. radiodurans, combining all 3 biological replicates for each condition. Ratios are given above each pair of bars. All proportions are significantly different between species by two-tailed z-test of two proportions (p-values < 0.01). See also Fig EV1 and EV2.


A , B . RT - qPCR of Zfp207 Oct4 in mouse ESCs during ( A ) retinoic acid ( RA ) - induced ( B ) embryoid ( EB ) - mediated differentiation . Nestin was used as a neuronal differentiation marker to monitor RA - mediated differentiation . mRNA are relative to expression at day 0 .

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

A, B. RT-qPCR analysis of Zfp207 and Oct4 in mouse ESCs during (A) retinoic acid (RA)-induced and (B) embryoid body (EB)-mediated differentiation. Nestin was used as a neuronal differentiation marker to monitor RA-mediated differentiation. mRNA levels are relative to the expression at day 0.


J , K . ( J ) AP of shScr Zfp207 - depleted ( sh1 sh2 ) mouse ESCs . Scale bars , 20 µM . ( K ) of fully differentiated ( FD ) , partially differentiated ( PD ) undifferentiated ( UN ) ESC colonies in shScr , sh1 sh2 .

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

J, K. (J) AP staining of shScr and Zfp207-depleted (sh1 and sh2) mouse ESCs. Scale bars, 20 µM. (K) Percentage of fully differentiated (FD), partially differentiated (PD) and undifferentiated (UN) ESC colonies in shScr, sh1 and sh2.


M , N . ( M ) Western blot of ZFP207 , OCT4 , NANOG , SOX2 in shScr , sh1 sh2 ESCs ( N ) RT - qPCR of Oct4 , Nanog , Sox2 in shScr , sh1 sh2 ESCs ; data is relative to shScr .

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

M, N. (M) Western blot of ZFP207, OCT4, NANOG, and SOX2 in shScr, sh1 and sh2 ESCs and (N) RT-qPCR analysis of Oct4, Nanog, and Sox2 in shScr, sh1 and sh2 ESCs; data is relative to shScr.


( O ) Western blot of OCT4 during a 9 - h cycloheximide ( CHX ) time course treatment in shScr , sh1 sh2 ESCs .

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

(O) Western blot of OCT4 during a 9-h cycloheximide (CHX) time course treatment in shScr, sh1 and sh2 ESCs.


( Q ) Western blot of OCT4 during 4 8 h treatment with proteasome inhibitor MG132 .

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

(Q) Western blot of OCT4 during 4 and 8 h treatment with the proteasome inhibitor MG132.


D . RT - qPCR of Zfp207 , pluripotency genes ( Oct4 , Nanog Sox2 ) , mesodermal markers ( Msx1 Brachyury , T ) , endodermal markers ( Foxa2 , Sox17 ) , neural - associated genes ( Pax6 , Sox11 Nestin ) in shScr , sh1 sh2 ESCs along time - course of EB - mediated differentiation . mRNA are relative to expression of shScr at day 0 .

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

D. RT-qPCR of Zfp207, the pluripotency genes (Oct4, Nanog and Sox2), the mesodermal markers (Msx1 and Brachyury, T), the endodermal markers (Foxa2, Sox17), and the neural-associated genes (Pax6, Sox11 and Nestin) in shScr, sh1 and sh2 ESCs along the time-course of EB-mediated differentiation. mRNA levels are relative to the expression of shScr at day 0.


B . Immunostaining of NESTIN ( green ) TUJ1 ( red ) of neural progenitors generated from shScr on day 8 10 of neuroectodermal differentiation . Nuclei were counterstained with DAPI . Scale bar , 20 μM .

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

B. Immunostaining of NESTIN (green) and TUJ1 (red) of neural progenitors generated from shScr on day 8 and 10 of the neuroectodermal differentiation. Nuclei were counterstained with DAPI. Scale bar, 20 μM.


( E ) of signal in shScr , sh1 sh2 at day 5 of neuroectodermal differentiation .

(E) quantification of the signal in shScr, sh1 and sh2 at day 5 of neuroectodermal differentiation.


G . RT - qPCR of Zfp207 neural - associated markers in shScr , sh1 sh2 along course of neural differentiation . mRNA are relative to shScr at day 0 .

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

G. RT-qPCR of Zfp207 and neural-associated markers in shScr, sh1 and sh2 along the course of neural differentiation. mRNA levels are relative to shScr at day 0.


( A ) Volcano plots of common differentially expressed genes in shScr Zfp207 - depleted ( sh1 sh2 ) mouse ESCs . Up - regulated ( Up ) down - regulated ( Down ) genes are indicated in red blue , respectively ( p < 0 . 05 ; > 1 . 5 - fold ) . Zfp207 is depicted in black . Grey dots indicate non - significant ( NS ) < 1 . 5 - fold differential expressed genes .

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

(A) Volcano plots of common differentially expressed genes in shScr and Zfp207-depleted (sh1 and sh2) mouse ESCs. Up-regulated (Up) and down-regulated (Down) genes are indicated in red and blue, respectively (p < 0.05; > 1.5-fold). Zfp207 is depicted in black. Grey dots indicate non-significant (NS) and < 1.5-fold differential expressed genes.


A . of column z - scores of log2 transformed values of genes involved in alternative splicing ( AS ) in shScr , sh1 sh2 mouse ESCs .

O O O O 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. Heatmap of column z-scores of log2 transformed values of genes involved in alternative splicing (AS) in shScr, sh1 and sh2 mouse ESCs.


F . RT - PCR of Zfp207 - regulated AS events . For Mta1 , AS1 ( alternative splicing 1 ) AS2 ( alternative splicing 2 ) . # 1 # 2 indicate distinct biological replicates . * Denotes an isoform that was not taken in consideration for . structure of each isoform is indicated ( not to scale ) . Alternative exons are blue . spliced in ( PSI ) was quantified for each condition .

O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RT-PCR of Zfp207-regulated AS events. For Mta1, AS1 (alternative splicing 1) and AS2 (alternative splicing 2). #1 and #2 indicate distinct biological replicates. *Denotes an isoform that was not taken in consideration for the quantification. The structure of each isoform is indicated (not to scale). Alternative exons are blue. The percent spliced in (PSI) was quantified for each condition.


( E ) FISH of poly ( A ) + distribution in shScr , sh1 sh2 stained with 5 ´ Cys3 - oligo dT ( 50 ) . Nuclei were counterstained with DAPI . Scale bar , 20 µM .

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

(E) FISH of poly(A)+ RNA distribution in shScr, sh1 and sh2 stained with 5´Cys3-oligo dT(50). Nuclei were counterstained with DAPI. Scale bar, 20 µM.


of ( F ) , ( G ) , ( H ) of / poly ( A ) + FISH signals . ( I ) Relative total in sh1 sh2 compared to shScr . Data are presented as mean ± of n ≥ 3 independent biological experiments . * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( shScr vs . sh1 or sh2 ) ; unpaired Student ' s t - .

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

Quantification of (F) nuclear, (G) cytoplasmic, and (H) of nuclear/cytoplasmic poly(A)+ FISH signals. (I) Relative nuclear and cytosolic total RNA levels in sh1 and sh2 compared to shScr. Data are presented as mean ± SEM of n ≥ 3 independent biological experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001 (shScr vs. sh1 or sh2); unpaired Student's t-test.


( a ) Flow cytometry of LPS - stimulated B stained with LysoTracker Red .

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

(a) Flow cytometry of LPS-stimulated B cells stained with LysoTracker Red.


( b ) Autophagic flux of GFP - LC3 splenic B stimulated with LPS then left untreated ( UT ) or treated for 1 h with bafimolycin A1 ( BafA1 ) , assessed by live - microscopy ( left ) automated of spots ( right ) . Scale bar ( left ) , 5 μm .

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

(b) Autophagic flux of GFP-LC3 splenic B cells stimulated with LPS and then left untreated (UT) or treated for 1 h with bafimolycin A1 (BafA1), assessed by live-cell fluorescence microscopy (left) and automated quantification of spots (right). Scale bar (left), 5 μm.