Figure 4Lysates from NMuMG control cells ( Cas9 - expressing cells ) and Ugcg KO cells were subjected to sucrose density gradient ultracentrifugation . The expression levels of flotillin - 1 , β1 - integrin , EEA1 , and TβRI in the sucrose gradient fractions were analyzed by immunoblotting . Fractions 3 , 4 , and 5 contained lipid rafts , whereas fractions 10 - 12 corresponded to the non - lipid raft fractions . Quantification of TβRI percentages in lipid raft and non - lipid raft fractions from Ugcg KO NMuMG and control cells . Isolation of lipid rafts from other cellular components in A549 - VIM - RFP cells treated with the UGCG inhibitor eliglustat ( 2 μM ) for 6 days using sucrose density gradient ultracentrifugation and measurement of flotillin - 1 , β1 - integrin , EEA1 , and TβRI levels in the sucrose gradient fractions using immunoblot analysis . Fractions 3 - 6 contained lipid rafts , whereas fractions 10 - 12 corresponded to the non - lipid raft fractions . Quantification of TβRI percentages in lipid raft and non - lipid raft fractions from A549 - VIM - RFP cells treated with eliglustat ( 2 μM ) for 6 days . Ubiquitination of TβRI was detected by immunoprecipitation ( IP ) of Myc - tagged caTβRI from HA - Ub - transfected HEK293T cells with or without eliglustat ( 2 μM ) treatment for 6 days . All groups were treated with MG132 ( 5 μM ) for 6 h . Immunoblot analysis of TβRI expression levels in control and eliglustat ( 2 μM ) - treated A549 - VIM - RFP cells treated with 50 μg / mL CHX for the indicated times . Vinculin : loading control . Quantification of TβRI expression levels with normalization to the t = 0 controls .

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Figure 4Lysates from NMuMG control cells (Cas9-expressing cells) and Ugcg KO cells were subjected to sucrose density gradient ultracentrifugation. The expression levels of flotillin-1, β1-integrin, EEA1, and TβRI in the sucrose gradient fractions were analyzed by immunoblotting. Fractions 3, 4, and 5 contained lipid rafts, whereas fractions 10-12 corresponded to the non-lipid raft fractions. Quantification of TβRI percentages in lipid raft and non-lipid raft fractions from Ugcg KO NMuMG and control cells. Isolation of lipid rafts from other cellular components in A549-VIM-RFP cells treated with the UGCG inhibitor eliglustat (2 μM) for 6 days using sucrose density gradient ultracentrifugation and measurement of flotillin-1, β1-integrin, EEA1, and TβRI levels in the sucrose gradient fractions using immunoblot analysis. Fractions 3-6 contained lipid rafts, whereas fractions 10-12 corresponded to the non-lipid raft fractions. Quantification of TβRI percentages in lipid raft and non-lipid raft fractions from A549-VIM-RFP cells treated with eliglustat (2 μM) for 6 days.Ubiquitination of TβRI was detected by immunoprecipitation (IP) of Myc-tagged caTβRI from HA-Ub-transfected HEK293T cells with or without eliglustat (2 μM) treatment for 6 days. All groups were treated with MG132 (5 μM) for 6 h.Immunoblot analysis of TβRI expression levels in control and eliglustat (2 μM)-treated A549-VIM-RFP cells treated with 50 μg/mL CHX for the indicated times. Vinculin: loading control.   Quantification of TβRI expression levels with normalization to the t=0 controls.


Figure 5Immunoblot analysis of p - SMAD2 , t - SMAD2 , and ST3GAL5 in A549 - VIM - RFP cells with siRNA - mediated ST3GAL5 knockdown or transfection of nontargeting siRNA and treated with vehicle control or TGF - β for 1 h . Vinculin : loading control . Quantification of the p - SMAD2 level in A549 - VIM - RFP cells transfected with nontargeting or ST3GAL5 siRNA and treated with TGF - β as shown in ( B ) . Expression levels of ST3GAL5 , the epithelial marker E - cadherin , and mesenchymal markers , including N - cadherin , vimentin , and SNAIL , in siRNA ST3GAL5 - depleted and nontargeting siRNA - transfected A549 - VIM - RFP cells treated with vehicle control or TGF - β for 48 h . Vinculin : loading control . Immunoblot analysis of p - SMAD2 and t - SMAD2 in A549 - VIM - RFP cells transduced with empty vector ( pLV - EV ) or the ST3GAL5 expression construct and stimulated with vehicle control or TGF - β for 1 h . GAPDH : loading control . Quantification of the p - SMAD2 level in A549 - VIM - RFP cells transduced with pLV - EV or the ST3GAL5 overexpression construct and treated with TGF - β , as shown in ( F ) . 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 .

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Figure 5Immunoblot analysis of p-SMAD2, t-SMAD2, and ST3GAL5 in A549-VIM-RFP cells with siRNA-mediated ST3GAL5 knockdown or transfection of nontargeting siRNA and treated with vehicle control or TGF-β for 1 h. Vinculin: loading control.   Quantification of the p-SMAD2 level in A549-VIM-RFP cells transfected with nontargeting or ST3GAL5 siRNA and treated with TGF-β as shown in (B).  Expression levels of ST3GAL5, the epithelial marker E-cadherin, and mesenchymal markers, including N-cadherin, vimentin, and SNAIL, in siRNA ST3GAL5-depleted and nontargeting siRNA-transfected A549-VIM-RFP cells treated with vehicle control or TGF-β for 48 h. Vinculin: loading control.Immunoblot analysis of p-SMAD2 and t-SMAD2 in A549-VIM-RFP cells transduced with empty vector (pLV-EV) or the ST3GAL5 expression construct and stimulated with vehicle control or TGF-β for 1 h. GAPDH: loading control. Quantification of the p-SMAD2 level in A549-VIM-RFP cells transduced with pLV-EV or the ST3GAL5 overexpression construct and treated with TGF-β, as shown in (F).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.


Figure 6HEK293T 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 cells with or without ST3GAL5 overexpression . All groups were treated with MG132 ( 5 μM ) for 6 h . 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 . Quantification of TβRI expression in pLV - EV control - and ST3GAL5 - expressing A549 - VIM - RFP cells . The results were normalized to the t = 0 controls .

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Figure 6HEK293T 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 cells with or without ST3GAL5 overexpression. All groups were treated with MG132 (5 μM) for 6 h.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.Quantification of TβRI expression in pLV-EV control- and ST3GAL5-expressing A549-VIM-RFP cells. The results were normalized to the t=0 controls.


Figure 7In 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 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 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 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 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 .

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Figure 7In 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 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 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 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 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.


Fig . 1 . 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 . 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 ) 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 .

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Fig. 1.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. 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) 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.


Fig . 4 . 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 and EK1P against SARS - CoV - 2 S - mediated cell - cell fusion . 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 , and the data are expressed as means ± SD ( error bar ) .

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Fig. 4.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 and EK1P against SARS-CoV-2 S-mediated cell-cell fusion.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, and the data are expressed as means ± SD (error bar).


Fig . 5 . 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 cell - cell fusion mediated by the S proteins of MERS - CoV ( b ) 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 cell - cell fusion mediated by the S proteins of HCoV - 229E ( d ) 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 cell - cell fusion mediated by the S proteins of WIV1 ( f ) 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 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 ) 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 ) Inhibitory activity of EK1C4 in PsV infection assays against HCoV - 229E ( l ) Inhibitory activity of EK1C4 in PsV infection assays against , NL63 ( m ) Inhibitory activity of EK1C4 in PsV infection assays against WIV1 ( n ) Inhibitory activity of EK1C4 in PsV infection assays against Rs3367 ( o ) .

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Fig. 5.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 cell-cell fusion mediated by the S proteins of MERS-CoV (b)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 cell-cell fusion mediated by the S proteins of HCoV-229E (d)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 cell-cell fusion mediated by the S proteins of WIV1 (f)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 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)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)Inhibitory activity of EK1C4 in PsV infection assays against HCoV-229E (l)Inhibitory activity of EK1C4 in PsV infection assays against , NL63 (m)Inhibitory activity of EK1C4 in PsV infection assays against WIV1 (n)Inhibitory activity of EK1C4 in PsV infection assays against Rs3367 (o).


Fig . 6 . 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 . 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 .

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Fig. 6.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. 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.


Figure 1A , 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 .

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Figure 1A, 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.


Figure 3 , 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 , 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 ) 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3, 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, 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) 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).


Figure 4A , ( 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 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A, (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 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 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 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).


Figure 1Double immunolabeling of PDS5A ( green ) and SYCP3 ( red ) in mouse spread spermatocytes in leptoteneDouble 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 ) 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 ) 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 ) 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 ) 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 ) 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 ) 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 ) and kinetochores ( ACA , red ) in a metaphase II spermatocyte . Data information : DAPI staining of the chromatin ( blue ) is shown for some spermatocytes .

O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Double immunolabeling of PDS5A (green) and SYCP3 (red) in mouse spread spermatocytes in leptoteneDouble 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) 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) 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) 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) 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) 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) 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) and kinetochores (ACA, red) in a metaphase II spermatocyte. Data information: DAPI staining of the chromatin (blue) is shown for some spermatocytes.


Figure 2Double immunolabeling of PDS5B ( green ) and SYCP3 ( red ) in spread spermatocytes at the indicated stages . 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 . 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 ) 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 ) 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 ) 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 ) and SYCP3 ( red ) on diplotene spread nuclei . Data information : Sex chromosomes ( X , Y ) and 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Double immunolabeling of PDS5B (green) and SYCP3 (red) in spread spermatocytes at the indicated stages.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.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) 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) 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) 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) and SYCP3 (red) on diplotene spread nuclei. Data information: Sex chromosomes (X, Y) and 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.


Figure 3A - 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 ) and kinetochores ( ACA , red ) , and staining of the chromatin ( DAPI ) in a metaphase II spermatocyte .

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Figure 3A-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) and kinetochores (ACA, red), and staining of the chromatin (DAPI) in a metaphase II spermatocyte.


Figure 4Double 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 ) and SYCP3 ( red ) in Pds5AB cKO spread spermatocytes at the indicated stages . 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 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 ) .

O B-PANEL_START O 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-PANEL_START O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Double 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) and SYCP3 (red) in Pds5AB cKO spread spermatocytes at the indicated stages.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 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).


Figure 5A - 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 .

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Figure 5A-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.


Figure 6Double 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 ) 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 ) 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 ) 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 ) and the cohesin cofactors WAPL ( green , in Pds5AB cKO spread spermatocytes at the indicated stages . Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Double 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) 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) 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) 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) and the cohesin cofactors WAPL (green, in Pds5AB cKO spread spermatocytes at the indicated stages. Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Figure 7Double immunolabeling of SYCP3 ( red ) and either γH2AX ( pseudocolored in blue in Pds5AB cKO spread spermatocytes at the indicated stages . 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 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 white ) and SYCP3 ( red ) in Pds5AB cKO spread spermatocytes . Data information : Sex bivalents ( XY ) are indicated . Scale bar , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Double immunolabeling of SYCP3 (red) and either γH2AX (pseudocolored in blue in Pds5AB cKO spread spermatocytes at the indicated stages.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 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 white) and SYCP3 (red) in Pds5AB cKO spread spermatocytes. Data information: Sex bivalents (XY) are indicated. Scale bar, 10 μm.


Figure 8Double 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 ) 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 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 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 ) 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Double 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) 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 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 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) 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 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 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.


Figure 9A - 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 and pachytene - like bivalents presenting regular ( left ) or altered ( i - iv ) telomere FISH signals . Double immunolabeling of SYCP3 ( red ) and RAP1 ( blue ) and telomere FISH ( PNA - Tel , green ) in Pds5AB cKO spread spermatocytesDouble 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 ) 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 ) 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9A-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 and pachytene-like bivalents presenting regular (left) or altered (i-iv) telomere FISH signals.Double immunolabeling of SYCP3 (red) and RAP1 (blue) and telomere FISH (PNA-Tel, green) in Pds5AB cKO spread spermatocytesDouble 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) 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) 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.


Figure 10Double 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 ) 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 ) and telomere - less ( T ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 10Double 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) 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) and telomere-less (T).


Figure 1HCT116 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1HCT116 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 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 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 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 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.


Figure 2U87 , 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 . , 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 ) 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2U87, 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., 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) 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 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 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.


Figure 3 , 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 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 , 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 cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of Noxa , BAK and Vinculin . 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3, 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 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, 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 cell protein lysates were collected and analyzed by capillary electrophoresis for the expression of Noxa, BAK and Vinculin.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.


Figure 4U87 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 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 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 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 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 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 . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4U87 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 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 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 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 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 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.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 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 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.


Figure 5U87 cells were treated as in A . Shown are the levels of TCA - cycle metabolites . 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 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 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 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 acidU87 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5U87 cells were treated as in A. Shown are the levels of TCA-cycle metabolites.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 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 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 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 acidU87 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 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 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.


Figure 6U87 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 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 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6U87 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 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 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 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 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 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 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.


Figure 71x106 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 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 the experiment in 7A were quantified for apoptotic bodies , Sections from the experiment in 7A were quantified for , number of TUNEL positive cellsSections from the experiment in 7A were quantified for number of mitosis per multiple high - power fields .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O

Figure 71x106 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 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 the experiment in 7A were quantified for apoptotic bodies, Sections from the experiment in 7A were quantified for , number of TUNEL positive cellsSections from the experiment in 7A were quantified for number of mitosis per multiple high-power fields.