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 .

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

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


U87 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 .

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

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 .

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

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


U87 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 ) .

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

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

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


1x106 HCT116 colon carcinoma 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 .

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

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


Related to 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 .

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

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 cells


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

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


C ) MiNA analysis of HeLa cells stained with the membrane potential - independent dye MitoTracker ™ Green ( MTG , 100 nM ) to visualize and determine the mitochondrial area ( = mitochondrial footprint ) . Left : HeLa WT cells grown in 10 mM galactose stained with 7 nM TMRE ( red ) plus 100 nM MTG ( green ) . Co - localization shown in yellow . Right : mitochondrial footprint ( white ) and mitochondrial skeleton ( red ) generated from MiNA analysis of the MTG channel ( green ) . D ) Left : HeLa IF1 - KO cells grown in 10 mM galactose stained with 7 nM TMRE ( red ) plus 100 nM MTG ( green ) . Right : mitochondrial footprint ( white ) and mitochondrial skeleton ( red ) generated from MiNA analysis of the MTG channel ( green ) . E ) Left : HeLa IF1 - H49K cells grown in 10 mM galactose stained with 7 nM TMRE ( red ) plus 100 nM MTG ( green ) . Right : mitochondrial footprint ( white ) and mitochondrial skeleton ( red ) generated from MiNA analysis of the MTG channel ( green ) . Co - localization shown in yellow . Scale bars = 10 µm . F )

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

C) MiNA analysis of HeLa cells stained with the membrane potential-independent dye MitoTracker™Green (MTG, 100 nM) to visualize and determine the mitochondrial area (= mitochondrial footprint). Left: HeLa WT cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Co-localization shown in yellow. Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). D) Left: HeLa IF1-KO cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). E) Left: HeLa IF1-H49K cells grown in 10 mM galactose stained with 7 nM TMRE (red) plus 100 nM MTG (green). Right: mitochondrial footprint (white) and mitochondrial skeleton (red) generated from MiNA analysis of the MTG channel (green). Co-localization shown in yellow. Scale bars = 10 µm. F)


A ) BN - PAGE and subsequent immuno - blotting of mitochondrial extracts from indicated HeLa cell lines showing the assembly of F1FO ATP synthase and binding of IF1 . Monomers ( M ) , dimers ( D ) and oligomers ( O ) were detected . Left panel : Coomassie - stained gel . Right panels : Immunoblotting and detection of ATP synthase and IF1 . WT : wild type HeLa cells ; IF * : cells expressing IF1 - H49K . Stars indicate oligomeric IF1 forms . I : complex I ; III : complex III .

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

A) BN-PAGE and subsequent immuno-blotting of mitochondrial extracts from indicated HeLa cell lines showing the assembly of F1FO ATP synthase and binding of IF1. Monomers (M), dimers (D) and oligomers (O) were detected. Left panel: Coomassie-stained gel. Right panels: Immunoblotting and detection of ATP synthase and IF1. WT: wild type HeLa cells; IF*: cells expressing IF1-H49K. Stars indicate oligomeric IF1 forms. I: complex I; III: complex III.


D ) Assembly of sEcGFP - tagged subunits CoxVIIIa - sEcGFP ( CIV # ) into complex IV , and assembly of subunits γ - sEcGFP ( SUγ # ) and SU e - sEcGFP ( Sue # ) into ATP synthase . Proteins were separated by BN - PAGE and immunoblotted . Lanes # 1 - # 10 were cut into three pieces for further evaluation . Lanes 1 - 2 : complex IV assembly in WT and CoxVIIIa - sEcGFP - expressing cells , detection of CIV with anti - Cox1 . Lanes 3 - 4 : complex V assembly in WT and SUγ - sEcGFP - expressing cells , detection with anti - SU γ . Lanes 8 - 10 : assembly of CIV # into CIV , SU γ - sEcGFP ( SUγ # ) into CV , and SU e - sEcGFP ( SUe # ) into CV . At lane # 6 in the Coomassie - stained gel , the positions of molecular weight markers ( M ) are shown . SC : respiratory supercomplexes ; CIV2 : complex IV dimers ; CIV : complex IV monomer ; s1 : F1 - c8 subcomplexes ; s2 : subcomplexes of CV containing subunits b , e and g He et al . , 2018 ( ) . Blue * * : dimeric F1F0 ATP synthase ; blue * : monomeric F1F0 ATP synthase . Red asterisks : respiratory

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

D) Assembly of sEcGFP-tagged subunits CoxVIIIa-sEcGFP (CIV#) into complex IV, and assembly of subunits γ-sEcGFP (SUγ#) and SU e-sEcGFP (Sue#) into ATP synthase. Proteins were separated by BN-PAGE and immunoblotted. Lanes #1-#10 were cut into three pieces for further evaluation. Lanes 1-2: complex IV assembly in WT and CoxVIIIa-sEcGFP-expressing cells, detection of CIV with anti-Cox1. Lanes 3-4: complex V assembly in WT and SUγ-sEcGFP-expressing cells, detection with anti-SU γ. Lanes 8-10: assembly of CIV# into CIV, SU γ-sEcGFP (SUγ#) into CV, and SU e-sEcGFP (SUe#) into CV. At lane #6 in the Coomassie-stained gel, the positions of molecular weight markers (M) are shown. SC: respiratory supercomplexes; CIV2: complex IV dimers; CIV: complex IV monomer; s1: F1-c8 subcomplexes; s2: subcomplexes of CV containing subunits b, e and g He et al., 2018(). Blue **: dimeric F1F0 ATP synthase; blue*: monomeric F1F0 ATP synthase. Red asterisks: respiratory supercomplexes


A , A ' Trajectory maps of ATP synthase labeled at SU γ ( A ) and SU g ( A ' ) in single mitochondria . Insets : Detailed views of the diffusion of CV - SU γ ( A ) and CV - SU g ( A ' ) . Scale bars : 1 μm overview and 0 . 5 μm insets .

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

A,A' Trajectory maps of ATP synthase labeled at SU γ (A) and SU g (A') in single mitochondria. Insets: Detailed views of the diffusion of CV-SU γ (A) and CV-SU g (A'). Scale bars: 1 μm overview and 0.5 μm insets.


B ) Diffusion coefficients for ATP synthase under control conditions ( 5 . 6 mM glucose ) , and under OXPHOS conditions . The probability density function ( PDF ) and cumulative density function ( CDF ) of - log D is plotted . Number of trajectories analyzed : 8622 ( Glc ) and 10333 ( Gal ) from mitochondria in at least 5 different cells .

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

B) Diffusion coefficients for ATP synthase under control conditions (5.6 mM glucose), and under OXPHOS conditions. The probability density function (PDF) and cumulative density function (CDF) of -log D is plotted. Number of trajectories analyzed: 8622 (Glc) and 10333 (Gal) from mitochondria in at least 5 different cells.


C ) Images showing matrix pH equilibration with extracellular pH . Shown are ratiometric fluorescence images of MPP - sEcGFP before and after addition of the uncouplers and inhibitors FCCP , nigericin and oligomycin ( plus 0 . 002 % w / v Triton X - 100 where indicated ) . False color images ( λ511 / λ464 ) indicate ratio values . D ) Ratiometric imaging of mitochondrial pH after equilibration with extracellular pH values pH = 8 , pH = 7 . 4 and pH = 6 . 5 . Scale bars = 10 µm .

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

C) Images showing matrix pH equilibration with extracellular pH. Shown are ratiometric fluorescence images of MPP-sEcGFP before and after addition of the uncouplers and inhibitors FCCP, nigericin and oligomycin (plus 0.002 % w/v Triton X-100 where indicated). False color images (λ511/λ464) indicate ratio values. D) Ratiometric imaging of mitochondrial pH after equilibration with extracellular pH values pH=8, pH=7.4 and pH=6.5. Scale bars = 10 µm. E


B ) Mitochondrial pH values at SU e , SU γ and in the matrix bulk in hyper - glycolytic cells ( high glucose : HGlc , 25 mM ) . C ) pH at SU e , SU γ and in the matrix in respiring cells ( Gal , 10 mM ) . D ) pH at CoxVIIIa at complex IV in the ICS before and after stimulation of the respiratory chain .

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

B) Mitochondrial pH values at SU e, SU γ and in the matrix bulk in hyper-glycolytic cells (high glucose: HGlc, 25 mM). C) pH at SU e , SU γ and in the matrix in respiring cells (Gal, 10 mM). D) pH at CoxVIIIa at complex IV in the ICS before and after stimulation of the respiratory chain. Da


A ) Mitochondrial pH at CV SU e measured in HeLa WT in glycolytic ( HGlc ) and respiring ( Gal ) conditions . Where indicated , 5 µg / mL oligomycin was added to inhibit CV activity . B ) Mitochondrial pH at CV SU e in IF1 - KO cells . Conditions : glycolytic ( HGlc ) and respiring ( Gal ) with and without oligomycin . C ) Mitochondrial pH at CV SU e in IF1 - OE cells . Conditions : glycolytic ( HGlc ) and respiring ( Gal ) with and without oligomycin . D )

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

A) Mitochondrial pH at CV SU e measured in HeLa WT in glycolytic (HGlc) and respiring (Gal) conditions. Where indicated, 5 µg/mL oligomycin was added to inhibit CV activity. B) Mitochondrial pH at CV SU e in IF1-KO cells. Conditions: glycolytic (HGlc) and respiring (Gal) with and without oligomycin. C) Mitochondrial pH at CV SU e in IF1-OE cells. Conditions: glycolytic (HGlc) and respiring (Gal) with and without oligomycin. D)


F ) Effect of inhibiting the respiratory chain at complex III ( CIII ) with antimycin A ( AA ) on the local pH at SU e in WT cells ( glycolytic conditions ) . G ) Local pH at SU e in IF1 - KO cells after inhibiting CIII . H ) Local pH at SU e in IF1 - OE cells after inhibiting CIII .

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

F) Effect of inhibiting the respiratory chain at complex III (CIII) with antimycin A (AA) on the local pH at SU e in WT cells (glycolytic conditions). G) Local pH at SU e in IF1-KO cells after inhibiting CIII. H) Local pH at SU e in IF1-OE cells after inhibiting CIII. Da


A ) Mitochondrial pH at CV SU e is compared for HeLa WT , IF1 - KO and IF1 - H49K cells under glycolytic and respiring conditions .

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

A) Mitochondrial pH at CV SU e is compared for HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions.


D ) pH at respiratory complex IV at SU CoxVIIIa in the ICS measured in HeLa WT , IF1 - KO and IF1 - H49K cells under glycolytic and respiring conditions .

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

D) pH at respiratory complex IV at SU CoxVIIIa in the ICS measured in HeLa WT, IF1-KO and IF1-H49K cells under glycolytic and respiring conditions.


J ) pH measurements in mitochondria in live cells expressing sEcGFP as the pH sensor at SU γ . The pH was color coded according to respective emission ratio . The pH at SU γ in IF1 - H49K - expressing cells is compared with WT cells . ( Scale bar : 1 µm ) .

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

J) pH measurements in mitochondria in live cells expressing sEcGFP as the pH sensor at SU γ. The pH was color coded according to respective emission ratio. The pH at SU γ in IF1-H49K-expressing cells is compared with WT cells. (Scale bar: 1 µm).


A ) Mitochondrial ATP production rates in HeLa cells the presence and absence of IF1 . Oxygen consumption rates ( OCR ) and extracellular acidification rates ( ECAR ) were determined with an automatic flux analyzer ( Seahorse / Agilent ) . After monitoring basal respiration , oligomycin ( 1 µM ) , and rotenone plus antimycin A ( Rot ; 0 . 5 µM ; AA , 0 . 5 µM ) were added sequentially to determine glycolytic and mitochondrial ATP production rates . ~ 12 technical replicates per measurement , N = 4 independent assays .

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

A) Mitochondrial ATP production rates in HeLa cells the presence and absence of IF1. Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were determined with an automatic flux analyzer (Seahorse/Agilent). After monitoring basal respiration, oligomycin (1 µM), and rotenone plus antimycin A (Rot; 0.5 µM; AA, 0.5 µM) were added sequentially to determine glycolytic and mitochondrial ATP production rates. ~12 technical replicates per measurement, N=4 independent assays.


( A ) MYB knockdown ( MiaPaCa - shMYB and Panc1 - shMYB ) and forced MYB - overexpressing ( BxPC3 - MYB ) PC cells along with their respective control cell lines ( MiaPaCa - Scr , Panc1 - Scr , and BxPC3 - Neo ) were subjected to hypoxia ( 1 % O2 ) treatment for different time intervals ( 0 - 96 h ) . Their growth was measured by viable cell counting using a trypan blue exclusion assay .

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

(A) MYB knockdown (MiaPaCa-shMYB and Panc1-shMYB) and forced MYB-overexpressing (BxPC3-MYB) PC cells along with their respective control cell lines (MiaPaCa-Scr, Panc1-Scr, and BxPC3-Neo) were subjected to hypoxia (1% O2) treatment for different time intervals (0-96 h). Their growth was measured by viable cell counting using a trypan blue exclusion assay.


( E , F ) ECAR ( E ) and OCR ( F ) in control and MYB silenced MiaPaCa cells cultured under normoxia or treated with hypoxia mimetic CoCl2 ( 100 μM for 4 h ) . Thereafter , ECAR was measured in PC cells with sequential injection of glucose , oligomycin , and 2 - DG , while OCR was measured with the serial addition of oligomycin , FCCP , and antimycin A / rotenone .

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

(E, F) ECAR (E) and OCR (F) in control and MYB silenced MiaPaCa cells cultured under normoxia or treated with hypoxia mimetic CoCl2 (100 μM for 4 h). Thereafter, ECAR was measured in PC cells with sequential injection of glucose, oligomycin, and 2-DG, while OCR was measured with the serial addition of oligomycin, FCCP, and antimycin A/rotenone.


( A ) MYB expression was analyzed by immunoblotting in pancreatic cancer cells cultured under hypoxia for different time intervals . HIF1α accumulation was examined as a positive control .

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(A) MYB expression was analyzed by immunoblotting in pancreatic cancer cells cultured under hypoxia for different time intervals. HIF1α accumulation was examined as a positive control.


MYB protein ( C ) levels were measured in the prostate ( LNCaP ) , lung ( A549 ) , breast ( MB231 ) , and colon ( HCT116 ) cancer cells cultured under normoxia and hypoxia ( 6 h ) by immunoblotting respectively . β - actin served as an internal control . N - normoxia and H - hypoxia .

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MYB protein (C) levels were measured in the prostate (LNCaP), lung (A549), breast (MB231), and colon (HCT116) cancer cells cultured under normoxia and hypoxia (6 h) by immunoblotting respectively. β-actin served as an internal control. N- normoxia and H- hypoxia.


( E ) Pancreatic cancer cells were transiently transfected either with non - targeted control siRNA ( siCtrl ) or HIF1α - targeting siRNA ( siHIF1α ) for 24 h and then incubated under hypoxia for 6 h . MYB expression was analyzed at protein and mRNA levels .

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(E) Pancreatic cancer cells were transiently transfected either with non-targeted control siRNA (siCtrl) or HIF1α -targeting siRNA (siHIF1α) for 24 h and then incubated under hypoxia for 6 h. MYB expression was analyzed at protein and mRNA levels.


( F ) Cells were treated with either vehicle or CoCl2 ( 100µM ) for 6 h , and total protein and RNA were isolated . The expression of HIF1α and MYB was examined . β - actin was used as an internal control .

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(F) Cells were treated with either vehicle or CoCl2 (100µM) for 6 h, and total protein and RNA were isolated. The expression of HIF1α and MYB was examined. β-actin was used as an internal control.


( B ) HuR localization was examined by immunofluorescence microscopy in cells exposed to hypoxia for 1 h . HuR ( green ) , and nucleus ( blue ) . Scale bar , 25 μm ( lower left corner ) . Lower panels are digitally magnified views of the marked rectangles .

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(B) HuR localization was examined by immunofluorescence microscopy in cells exposed to hypoxia for 1 h. HuR (green), and nucleus (blue). Scale bar, 25 μm (lower left corner). Lower panels are digitally magnified views of the marked rectangles.


( C ) Cytosolic and nuclear protein fractions were prepared from cells cultured under hypoxia for 1h and HuR expression was examined by immunoblotting . α - Tubulin ( nuclear ) and Lamin A ( cytoplasm ) were used as loading controls .

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(C) Cytosolic and nuclear protein fractions were prepared from cells cultured under hypoxia for 1h and HuR expression was examined by immunoblotting. α-Tubulin (nuclear) and Lamin A (cytoplasm) were used as loading controls.


( G ) Cells transfected with HuR or control siRNA for 48 h were exposed to hypoxia for 6 h , and MYB and HuR expression was determined by immunoblotting or qRT - PCR .

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(G) Cells transfected with HuR or control siRNA for 48 h were exposed to hypoxia for 6 h, and MYB and HuR expression was determined by immunoblotting or qRT-PCR.


( A ) MYB - silenced ( MiaPaCa - shMYB and Panc1 - shMYB ) and MYB - overexpressing ( BxPC3 - MYB ) PC cells , along with their control cell lines ( MiaPaCa - Scr , Panc1 - Scr , and BxPC3 - Neo ) were exposed to hypoxia for 6 h . Protein lysates were made , and the expression of MYB and HIF1α was examined by immunoblotting . β - actin served as a loading control .

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(A) MYB-silenced (MiaPaCa-shMYB and Panc1-shMYB) and MYB-overexpressing (BxPC3-MYB) PC cells, along with their control cell lines (MiaPaCa-Scr, Panc1-Scr, and BxPC3-Neo) were exposed to hypoxia for 6 h. Protein lysates were made, and the expression of MYB and HIF1α was examined by immunoblotting. β-actin served as a loading control.


( C ) The transcriptional activity of the HIF1α promoter was determined by performing the promoter - reporter assay . MYB - modulated PC cell lines were transfected with the reporter plasmid , and after 24 h of transfection , cells were cultured under hypoxia or normoxia for 24 h , and the supernatant was collected . HIF1α promoter - driven Gaussia luciferase activity was normalized with SEAP activity , and the data was presented as a bar diagram .

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(C) The transcriptional activity of the HIF1α promoter was determined by performing the promoter-reporter assay. MYB-modulated PC cell lines were transfected with the reporter plasmid, and after 24 h of transfection, cells were cultured under hypoxia or normoxia for 24 h, and the supernatant was collected. HIF1α promoter-driven Gaussia luciferase activity was normalized with SEAP activity, and the data was presented as a bar diagram.


( E ) MYB - silenced or - overexpressing PC cell lines were exposed to normoxia or hypoxia for 6 h and subjected to chromatin immunoprecipitation using anti - MYB antibodies or IgG control followed by qPCR analysis using site - specific primers .

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(E) MYB-silenced or -overexpressing PC cell lines were exposed to normoxia or hypoxia for 6 h and subjected to chromatin immunoprecipitation using anti-MYB antibodies or IgG control followed by qPCR analysis using site-specific primers.


( A ) Immunoblot analysis was performed to analyze HIF1α and MYB expression in MYB - silenced ( shMYB ) and those having a forced overexpression of HIF1α ( shMYB - HIF1αOE ) along with their respective controls ( Scr and shMYB - Neo ) cultured under hypoxia . β - actin served as a loading control .

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(A) Immunoblot analysis was performed to analyze HIF1α and MYB expression in MYB-silenced (shMYB) and those having a forced overexpression of HIF1α (shMYB-HIF1αOE) along with their respective controls (Scr and shMYB-Neo) cultured under hypoxia. β-actin served as a loading control.


( C ) Live / Dead staining was performed in MYB - silenced MiaPaCa and Panc - 1 or MYB silenced - HIF1α overexpressed cells cultured under hypoxia for 48 h . Cells were visualized under a fluorescence microscope . Representative fluorescence micrographs are presented . The number of live and dead cells was counted from the captured random images . Scale bar , 150 μm ( lower right corner ) .

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(C) Live/Dead staining was performed in MYB-silenced MiaPaCa and Panc-1 or MYB silenced- HIF1α overexpressed cells cultured under hypoxia for 48 h. Cells were visualized under a fluorescence microscope. Representative fluorescence micrographs are presented. The number of live and dead cells was counted from the captured random images. Scale bar, 150 μm (lower right corner).


( F ) MYB - silenced and HIF1α overexpressing MYB knockdown PC cells and control cells were cultured under hypoxia for 48 h . Conditioned media was collected and analyzed to assess glucose consumption and lactate secretion by measuring their levels . The data was normalized with the number of cells and presented as relative fold changes .

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(F) MYB-silenced and HIF1α overexpressing MYB knockdown PC cells and control cells were cultured under hypoxia for 48 h. Conditioned media was collected and analyzed to assess glucose consumption and lactate secretion by measuring their levels. The data was normalized with the number of cells and presented as relative fold changes.


( C ) Quantitative ChIP - qPCR was performed to analyze the occupancy of MYB under normoxia and hypoxia at promoter regions of selected glycolytic genes ( GLUT3 , HK2 , PFKL , ENO2 , and MCT4 ) using anti - MYB or IgG control antibodies . The enrichment of promoter regions was compared between cells cultured under normoxia or hypoxia .

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(C) Quantitative ChIP-qPCR was performed to analyze the occupancy of MYB under normoxia and hypoxia at promoter regions of selected glycolytic genes (GLUT3, HK2, PFKL, ENO2, and MCT4) using anti-MYB or IgG control antibodies. The enrichment of promoter regions was compared between cells cultured under normoxia or hypoxia.


p300 ( E ) , and H3K9ac ( F ) on the promoter regions were also confirmed by ChIP - qPCR using specific antibodies and fold enrichment calculated and presented as a bar graph after normalization with control IgG .

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p300 (E), and H3K9ac (F) on the promoter regions were also confirmed by ChIP- qPCR using specific antibodies and fold enrichment calculated and presented as a bar graph after normalization with control IgG.


( A , B ) Interaction of MYB with HIF1α ( A ) and p300 ( B ) was examined by co - immunoprecipitation assays in the lysates of pancreatic cancer cells cultured under hypoxia for 6 h . Immunoprecipitation with IgG isotype antibody was used as a control . 10 % of total cell lysate was used as input for western blot analysis .

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(A, B) Interaction of MYB with HIF1α (A) and p300 (B) was examined by co-immunoprecipitation assays in the lysates of pancreatic cancer cells cultured under hypoxia for 6 h. Immunoprecipitation with IgG isotype antibody was used as a control. 10% of total cell lysate was used as input for western blot analysis.


( C , D ) Co - localization of MYB with HIF1α ( C ) and p300 ( D ) was examined in pancreatic cancer cells grown under hypoxia for 6 h by immunofluorescence assays followed by confocal microscopy . Scale bar , 30 μm ( lower left corner ) . Lower panels are digitally magnified views of the marked rectangles . For ease of visualization , a pseudo color ( purple ) was adopted for p300 staining .

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(C, D) Co-localization of MYB with HIF1α (C) and p300 (D) was examined in pancreatic cancer cells grown under hypoxia for 6 h by immunofluorescence assays followed by confocal microscopy. Scale bar, 30 μm (lower left corner). Lower panels are digitally magnified views of the marked rectangles. For ease of visualization, a pseudo color (purple) was adopted for p300 staining.


( B , C ) Tumor growth was monitored by non - invasive bioluminescence imaging . Tumors were resected at the experimental endpoint , and their weights ( B ) and volumes ( C ) were recorded .

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(B, C) Tumor growth was monitored by non-invasive bioluminescence imaging. Tumors were resected at the experimental endpoint, and their weights (B) and volumes (C) were recorded.


( G ) Tumor xenograft serial sections were immunostained for pimonidazole ( PDZ ) adduct , MYB , HK2 , GLUT3 , and MCT4 using specific antibodies . Images were taken at 40X magnification . Scale bar , 60 μm ( lower left corner ) . The dashed lines demarcate the hypoxic region based on the PDZ staining pattern .

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(G) Tumor xenograft serial sections were immunostained for pimonidazole (PDZ) adduct, MYB, HK2, GLUT3, and MCT4 using specific antibodies. Images were taken at 40X magnification. Scale bar, 60 μm (lower left corner). The dashed lines demarcate the hypoxic region based on the PDZ staining pattern.


A . U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes , at which point 4 mM HU was added and cells further incubated for the indicated times . Whole cell extracts were then analysed by western blotting with the indicated antibodies . Reduction of phosphorylation of Ser40 / 41 on MCM2 is indicative of CDC7 inhibition by XL413 . Representative of two independent experiments .

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A. U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes, at which point 4 mM HU was added and cells further incubated for the indicated times. Whole cell extracts were then analysed by western blotting with the indicated antibodies. Reduction of phosphorylation of Ser40/41 on MCM2 is indicative of CDC7 inhibition by XL413. Representative of two independent experiments.


B . U2OS cells were either mock treated or treated with 10 μM XL413 , 4 mM HU or both for 24 hours before performing neutral comet assays . Representative images of comets are shown . Scale bar = 100 μM . In the dot plots ~ 800 comets per each condition were analysed , means are indicated with a red line and their value shown above the plot . Data are from two independent experiments .

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B. U2OS cells were either mock treated or treated with 10 μM XL413, 4 mM HU or both for 24 hours before performing neutral comet assays. Representative images of comets are shown. Scale bar = 100 μM. In the dot plots ~800 comets per each condition were analysed, means are indicated with a red line and their value shown above the plot. Data are from two independent experiments.


C . U2OS cells were either untreated or treated with 10 μM XL413 or with 5 µM AZD6738 alone or in combination and , where indicated , after 30 minutes 4 mM HU was added for a further 2 hours . Whole cell extracts were analysed by western blotting with the indicated antibodies . Total protein stain ( TPS ) as a loading control . Representative of two independent experiments .

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C. U2OS cells were either untreated or treated with 10 μM XL413 or with 5 µM AZD6738 alone or in combination and, where indicated, after 30 minutes 4 mM HU was added for a further 2 hours. Whole cell extracts were analysed by western blotting with the indicated antibodies. Total protein stain (TPS) as a loading control. Representative of two independent experiments.


D . U2OS cells were either untreated or treated with 4 mM HU for 5 hours in the presence or absence of 10 μM XL413 which had either been added to cells 30 minutes prior ( pre - treatment ) or at the same time ( co - treatment ) to the addition of HU . Whole cell extracts were analysed by western blotting with the indicated antibodies and total protein stain ( TPS ) is displayed as a loading control . Representative of two independent experiments .

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D. U2OS cells were either untreated or treated with 4 mM HU for 5 hours in the presence or absence of 10 μM XL413 which had either been added to cells 30 minutes prior (pre-treatment) or at the same time (co-treatment) to the addition of HU. Whole cell extracts were analysed by western blotting with the indicated antibodies and total protein stain (TPS) is displayed as a loading control. Representative of two independent experiments.


B . CSK soluble and chromatin - enriched protein fractions were analysed by western blotting with the indicated antibodies . Representative of two independent experiments .

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B. CSK soluble and chromatin-enriched protein fractions were analysed by western blotting with the indicated antibodies. Representative of two independent experiments.


C - D . Flow cytometry analysis to assess levels of pS139 Histone H2AX ( c ) . Histograms show the mono - parametric analysis of cell count against pS139 Histone H2AX intensity . Histograms are overlaid to appreciate changes in pS139 Histone H2AX intensity upon treatment ( Red lines ) relative to appropriate experimental baseline controls ( Grey lines ) . Data are representative of two independent experiments .

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C-D. Flow cytometry analysis to assess levels of pS139 Histone H2AX (c). Histograms show the mono-parametric analysis of cell count against pS139 Histone H2AX intensity. Histograms are overlaid to appreciate changes in pS139 Histone H2AX intensity upon treatment (Red lines) relative to appropriate experimental baseline controls (Grey lines). Data are representative of two independent experiments.


A . U2OS cells were labelled with EdU for 30 minutes , then EdU was washed and cells were further incubated for either 1 or 2 hours in presence of thymidine . At the indicated time points cells were fixed and proteins binding to EdU - labelled DNA captured by DNA mediated chromatin Pull down technique ( DmChP ) . Graphical experimental outline is shown above the analysis by western blot of relevant proteins in both input and captured material .

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A. U2OS cells were labelled with EdU for 30 minutes, then EdU was washed and cells were further incubated for either 1 or 2 hours in presence of thymidine. At the indicated time points cells were fixed and proteins binding to EdU-labelled DNA captured by DNA mediated chromatin Pull down technique (DmChP). Graphical experimental outline is shown above the analysis by western blot of relevant proteins in both input and captured material.


B . U2OS cells were labelled with EdU for 30 minutes and then treated with 4 mM HU for 2 hours in the presence or absence of 10 μM XL413 . Proteins binding to EdU - labelled DNA captured by DmChP were then analysed by western blot as above .

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B. U2OS cells were labelled with EdU for 30 minutes and then treated with 4 mM HU for 2 hours in the presence or absence of 10 μM XL413. Proteins binding to EdU-labelled DNA captured by DmChP were then analysed by western blot as above.


C . As in panel B but incubation with HU was extended to 24 hours . Black arrow indicates MRE11 electrophoretic - mobility shift .

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C. As in panel B but incubation with HU was extended to 24 hours. Black arrow indicates MRE11 electrophoretic-mobility shift.


D . U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes , at which point 4 mM HU was added and cells incubated for further 24 hours . Extracts prepared from HU treated cells were then incubated in presence or absence of λ - phosphatase . Proteins were analysed by western blotting with anti - MRE11 antibodies . Total protein stain ( TPS ) is used as a loading control . Black arrow indicates MRE11 electrophoretic - mobility shift .

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D. U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes, at which point 4 mM HU was added and cells incubated for further 24 hours. Extracts prepared from HU treated cells were then incubated in presence or absence of λ-phosphatase. Proteins were analysed by western blotting with anti-MRE11 antibodies. Total protein stain (TPS) is used as a loading control. Black arrow indicates MRE11 electrophoretic-mobility shift.


E . U2OS cells were treated with 4 mM HU in the presence or absence of 10 μM XL413 for 24 hours . PCNA ( green ) and MRE11 ( red ) were detected by immunofluorescence . Inset I - II represent enlargements of selected region of the merged images .

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E. U2OS cells were treated with 4 mM HU in the presence or absence of 10 μM XL413 for 24 hours. PCNA (green) and MRE11 (red) were detected by immunofluorescence. Inset I-II represent enlargements of selected region of the merged images.


F . Quantification of PCNA and MRE11 colocalization was assessed with ImageJ in ~ 70 randomly selected cells for each condition from four biological replicates and expressed as Pearson ' s correlation coefficient . Error bars represent SEM . Statistical significance was assessed by student t test ( * p ˂ 0 .

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F. Quantification of PCNA and MRE11 colocalization was assessed with ImageJ in ~70 randomly selected cells for each condition from four biological replicates and expressed as Pearson's correlation coefficient. Error bars represent SEM. Statistical significance was assessed by student t test (*p˂0.05


A . MCF10A cells were either mock or treated with 10 μM XL413 and labelled with IdU ( red ) for 30 minutes at which point 4 mM HU was added for 2 hours . HU was then washed off and cells labelled with CldU ( green ) in the continued presence or absence of XL413 . A set of representative DNA fibres from each condition is shown and the percentage of IdU ( red ) only tracts is plotted . At least 200 replication forks were analysed for each condition . Error bars represent the SEM from three biological repeats and statistical significance was assessed by the student t test ( * * p ˂ 0 .

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A. MCF10A cells were either mock or treated with 10 μM XL413 and labelled with IdU (red) for 30 minutes at which point 4 mM HU was added for 2 hours. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413. A set of representative DNA fibres from each condition is shown and the percentage of IdU (red) only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent the SEM from three biological repeats and statistical significance was assessed by the student t test (** p˂0.01


B . U2OS cells were either mock or treated with 10 μM XL413 , 50 μM Mirin or both , and labelled with IdU ( red ) for 30 minutes at which point 4 mM HU was added for 2 hours . HU was then washed off and cells labelled with CldU ( green ) in the continued presence or absence of XL413 and / or Mirin . A set of representative DNA fibres from each condition is shown and the percentage of IdU ( red ) only tracts is plotted . At least 200 replication forks were analysed for each condition . Error bars represent the SEM from four biological repeats and statistical significance was assessed by the student t test ( * p < 0 . 05 , * * p ˂ 0 .

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B. U2OS cells were either mock or treated with 10 μM XL413, 50 μM Mirin or both, and labelled with IdU (red) for 30 minutes at which point 4 mM HU was added for 2 hours. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413 and/or Mirin. A set of representative DNA fibres from each condition is shown and the percentage of IdU (red) only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent the SEM from four biological repeats and statistical significance was assessed by the student t test (*p<0.05, ** p˂0.01


C . U2OS cells were either mock or pretreated for the indicated times with 10 μM XL413 , 50 μM Mirin or both , and labelled with CldU ( red ) . Then 50 nM CPT was added and cells labelled with of IdU ( green ) for further 30 minutes . A set of representative DNA fibres from each condition is shown and the IdU / CIdU tract length ratio is plotted . The box extends from the 25th to the 75th percentile with the line in the box representing the median . Whiskers indicate the 10 - 90 percentiles with data outside this range for individual outliers being plotted as dots . At least 100 replication forks were analyzed for each condition . P values were calculated using one way ANOVA Kruskal - Wallis test and Dunn ' s multiple comparison post test ( ns , not significant , * * * * p < 0 . 0001 ) and are related to the experiment shown . Similar results were obtained in a second independent experiment .

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C. U2OS cells were either mock or pretreated for the indicated times with 10 μM XL413, 50 μM Mirin or both, and labelled with CldU (red). Then 50 nM CPT was added and cells labelled with of IdU (green) for further 30 minutes. A set of representative DNA fibres from each condition is shown and the IdU/CIdU tract length ratio is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentiles with data outside this range for individual outliers being plotted as dots. At least 100 replication forks were analyzed for each condition. P values were calculated using one way ANOVA Kruskal-Wallis test and Dunn's multiple comparison post test (ns, not significant, **** p<0.0001) and are related to the experiment shown. Similar results were obtained in a second independent experiment.


D . U2OS cells were transfected with control ( siLuc ) or MRE11 targeting siRNA ( siMRE11 ) . After 48 hours cells were either mock or pretreated with 10 μM XL413 and labelled with CldU ( red ) for 30 minutes . Where indicated 50 nM CPT was added and then cells were labelled with IdU ( green ) . A set of representative DNA fibres from each condition is shown and the IdU / CIdU tract length ratio is plotted . The box extends from the 25th to the 75th percentile with the line in the box representing the median . Whiskers indicate the 10 - 90 percentileswith data outside this range for individual outliers being plotted as dots . At least 100 replication forks were analyzed for each condition . P values were calculated using one way ANOVA Kruskal - Wallis test and Dunn ' s multiple comparison post test ( ns , not significant , * * * * p < 0 . 0001 ) and are related to the experiment shown . Similar results were obtained in a second independent experiment .

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

D. U2OS cells were transfected with control (siLuc) or MRE11 targeting siRNA (siMRE11). After 48 hours cells were either mock or pretreated with 10 μM XL413 and labelled with CldU (red) for 30 minutes. Where indicated 50 nM CPT was added and then cells were labelled with IdU (green). A set of representative DNA fibres from each condition is shown and the IdU/CIdU tract length ratio is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentileswith data outside this range for individual outliers being plotted as dots. At least 100 replication forks were analyzed for each condition. P values were calculated using one way ANOVA Kruskal-Wallis test and Dunn's multiple comparison post test (ns, not significant, **** p<0.0001) and are related to the experiment shown. Similar results were obtained in a second independent experiment.


B . Representative western blot of indicated proteins obtained from whole cell extracts . pS40 / 41 on MCM2 indicates effective CDC7 inhibition by XL413 .

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B. Representative western blot of indicated proteins obtained from whole cell extracts. pS40/41 on MCM2 indicates effective CDC7 inhibition by XL413.


C . Ratios of IdU / CldU tracts length is plotted . The box extends from the 25th to the 75th percentile with the line in the box representing the median . Whiskers indicate the 10 - 90 percentiles with data outside this range for individual outliers being plotted as dots . Mean values of IdU / CldU ratios are indicated above the plot . At least 100 replication forks were analyzed for each condition .

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

C. Ratios of IdU/CldU tracts length is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentiles with data outside this range for individual outliers being plotted as dots. Mean values of IdU/CldU ratios are indicated above the plot. At least 100 replication forks were analyzed for each condition.


D . The mean values of IdU / CldU tract length ratios from three independent experiments are plotted , with the standard deviation ( blue lines ) and mean ( red line ) . Statistical analysis : student t - test ; ns , not significant ; * * P < 0 . 01 , * * * P < 0 .

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D. The mean values of IdU/CldU tract length ratios from three independent experiments are plotted, with the standard deviation (blue lines) and mean (red line). Statistical analysis: student t-test; ns, not significant; **P<0.01, ***P < 0.001


E . Electron micrographs of representative replication forks from U2OS cells A regressed arm is labelled : P = parental strand ; D = daughter strand ; and R = reversed fork ; the four - way junction at the reversed fork is magnified in the inset . The red arrows indicates single stranded DNA at the fork . Scale bar is 200 nm ( = 460 bp ) , 100 nm ( = 230 bp ) in the inset .

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E. Electron micrographs of representative replication forks from U2OS cells A regressed arm is labelled: P=parental strand; D=daughter strand; and R=reversed fork; the four-way junction at the reversed fork is magnified in the inset. The red arrows indicates single stranded DNA at the fork. Scale bar is 200 nm ( = 460 bp), 100 nm ( = 230 bp) in the inset.


F . Frequency of reversed replication forks isolated from mock - depleted ( siLuc ) and BRCA2 - depleted ( siBRCA2 ) U2OS cells upon 5 hour treatment with 4 mM HU in the presence and absence of 10 μM XL413 . The number of replication intermediates analyzed is indicated in parentheses . Similar results were obtained in two independent experiments ( Appendix

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F. Frequency of reversed replication forks isolated from mock-depleted (siLuc) and BRCA2-depleted (siBRCA2) U2OS cells upon 5 hour treatment with 4 mM HU in the presence and absence of 10 μM XL413. The number of replication intermediates analyzed is indicated in parentheses. Similar results were obtained in two independent experiments (Appendix Table


G . Amount of ssDNA length at the junction ( Red arrows in Figure 4D ) in siLuc and siBRCA2 U2OS cells treated with 4 mM of HU for 5 hours in the presence or absence of XL413 . N indicates the number of forks observed , and only molecules with detectable ssDNA stretches are included in the analysis . The lines show the mean length of ssDNA regions at the fork and the value is displayed above the

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G. Amount of ssDNA length at the junction (Red arrows in Figure 4D) in siLuc and siBRCA2 U2OS cells treated with 4 mM of HU for 5 hours in the presence or absence of XL413. N indicates the number of forks observed, and only molecules with detectable ssDNA stretches are included in the analysis. The lines show the mean length of ssDNA regions at the fork and the value is displayed above the plot


A . Representative metaphase spread from BRCA2 depleted U2OS cell treated with HU . Scale bar = 5 μM . Arrows indicate the chromosomes enlarged in the insets , two of which show chromatid breaks ( *

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A. Representative metaphase spread from BRCA2 depleted U2OS cell treated with HU. Scale bar = 5 μM. Arrows indicate the chromosomes enlarged in the insets, two of which show chromatid breaks (*


B - C . The graphs show the average number of chromatid breaks per spread . In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed . Error bars represent SEM . Statistical significance assessed by student t test ( * * p ˂ 0 . 01 , * * * p < 0 .

B-C. The graphs show the average number of chromatid breaks per spread. In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance assessed by student t test (** p˂0.01, *** p<0.001


D - E . U2OS and AS - CDC7 cells were transfected with control ( siCon ) or BRCA2 ( siBRCA2 ) targeting siRNAs . 48 hours post transfection cells were either mock - treated or treated with 10 μM XL413 or 10 μM 3MB - PP1 respectively for 24 hours with Nocodazole added for the last 16 hours of the experiment . The graph shows the average number of chromatid breaks per spread . In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed . Error bars represent SEM . Statistical significance assessed by student t test ( * p < 0 . 05 , * * * p < 0 .

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D-E. U2OS and AS-CDC7 cells were transfected with control (siCon) or BRCA2 (siBRCA2) targeting siRNAs. 48 hours post transfection cells were either mock-treated or treated with 10 μM XL413 or 10 μM 3MB-PP1 respectively for 24 hours with Nocodazole added for the last 16 hours of the experiment. The graph shows the average number of chromatid breaks per spread. In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance assessed by student t test (*p<0.05, *** p<0.001


A . Hematoxylin and eosin ( H & E ) - stained cross section of a human liver slice . Scale bar is representative of A - F . Image is also used in Fig . S3A . B . Spatial distribution and relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from A . The image displays a heatmap gradient of intensity from white ( least abundant ) to red ( most abundant ) which is representative of B , D , and F . C . H & E - stained cross section of a human lung slice . Image is also used in Fig . S3D . D . Spatial distribution and relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from C . E . H & E - stained cross section of frontal cortical region of a human brain tissue . Image is also used in Fig . S3G . F . Spatial distribution and relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from E . G . Quantification of relative abundance of total glycogen calculated from the sum of CL3 - CL18 in human liver , lung , and brain . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * 0 . 01 < P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 , analyzed by one - way ANOVA with Tukey ' s multiple comparison for total glycogen . H . Glycogen structure represented by normalized chain length distribution ( ratio to CL3 ) of each human liver , lung and brain from G . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) .

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A. Hematoxylin and eosin (H&E)-stained cross section of a human liver slice. Scale bar is representative of A-F. Image is also used in Fig. S3A. B. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from A. The image displays a heatmap gradient of intensity from white (least abundant) to red (most abundant) which is representative of B, D, and F. C. H&E-stained cross section of a human lung slice. Image is also used in Fig. S3D. D. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from C. E.H&E-stained cross section of frontal cortical region of a human brain tissue. Image is also used in Fig. S3G. F. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from E. G. Quantification of relative abundance of total glycogen calculated from the sum of CL3-CL18 in human liver, lung, and brain. Values are presented as mean +/- standard error (n=3 technical replicates per region). *0.01 < P < 0.05; ***P < 0.001; ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison for total glycogen. H. Glycogen structure represented by normalized chain length distribution (ratio to CL3) of each human liver, lung and brain from G. Values are presented as mean +/- standard error (n=3 technical replicates per region).


A . Hematoxylin and eosin ( H & E ) - stained cross section of adenocarcinoma of the prostate , lung squamous cell carcinoma ( LUSC ) , lung adenocarcinoma ( LUAD ) , and Ewing sarcoma ( ES ) of the abdomen tumor . Annotated regions are T - tumor , S - stroma , D / G - duct / glandular , and A - adipose tissue . Scale bar is representative of A and B . Images are also used in Fig . S4A , 5D and S6G . B . Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section shown in A . The image displays a heatmap gradient of intensity with white ( least abundant ) to red ( most abundant ) . Images are also used in Fig . S4E , 5D , and S6G . C . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length ( CL ) distribution from tumor regions shown in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) .

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

A. Hematoxylin and eosin (H&E)-stained cross section of adenocarcinoma of the prostate, lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), and Ewing sarcoma (ES) of the abdomen tumor. Annotated regions are T-tumor, S-stroma, D/G-duct/glandular, and A-adipose tissue. Scale bar is representative of A and B. Images are also used in Fig. S4A, 5D and S6G. B. Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Images are also used in Fig. S4E, 5D, and S6G. C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from tumor regions shown in A. Values are presented as mean +/- standard error (n=3 technical replicates per region).


A . ( Left ) Hematoxylin and eosin ( H & E ) - stained cross section of an Ewing sarcoma ( ES ) of the shoulder tumor . Annotated regions are annotated as T - tumor and M - muscle . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . The image displays a heatmap gradient of intensity from white ( least abundant ) to red ( most abundant ) . Scale bar and intensity gradient below are representative of all images in A - E . B . ( Left ) H & E stained cross section of an ES of the chest wall tumor . Annotated regions are annotated as T - tumor and N - necrosis . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . C . ( Left ) H & E stained cross section of an ES of the rib tumor . Annotated regions are annotated as T - tumor and N - necrosis . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . D . ( Left ) H & E stained cross section of an ES of the abdomen tumor . Annotated regions are T - tumor , S - stroma , and A - adipose tissue . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . E . ( Left ) H & E stained cross section of an ES of the bladder tumor . Annotated regions are T - tumor and S - stroma . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . F . Magnified H & E cross section of ( top , left ) tumor , and ( top , right ) muscle tissue from ES rib with magnified image of CL7 ( m / z = 1175 ) of ( bottom , left ) tumor , and ( bottom , right ) muscle tissues . G . Magnified H & E cross section of ( top , left ) tumor , ( top , middle ) stroma , and ( top , right ) adipose tissues from ES abdomen with magnified image of CL7 ( m / z = 1175 ) of ( bottom , left ) tumor , ( bottom , middle ) stroma and ( bottom , right ) adipose tissues .

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A. (Left) Hematoxylin and eosin (H&E)-stained cross section of an Ewing sarcoma (ES) of the shoulder tumor. Annotated regions are annotated as T-tumor and M-muscle. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. The image displays a heatmap gradient of intensity from white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A-E. B. (Left) H&E stained cross section of an ES of the chest wall tumor. Annotated regions are annotated as T-tumor and N-necrosis. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. C. (Left) H&E stained cross section of an ES of the rib tumor. Annotated regions are annotated as T-tumor and N-necrosis. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. D. (Left) H&E stained cross section of an ES of the abdomen tumor. Annotated regions are T-tumor, S-stroma, and A-adipose tissue. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. E. (Left) H&E stained cross section of an ES of the bladder tumor. Annotated regions are T-tumor and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. F. Magnified H&E cross section of (top, left) tumor, and (top, right) muscle tissue from ES rib with magnified image of CL7 (m/z=1175) of (bottom, left) tumor, and (bottom, right) muscle tissues. G. Magnified H&E cross section of (top, left) tumor, (top, middle) stroma, and (top, right) adipose tissues from ES abdomen with magnified image of CL7 (m/z=1175) of (bottom, left) tumor, (bottom, middle) stroma and (bottom, right) adipose tissues.


A . ( Left ) Hematoxylin and eosin ( H & E ) stained cross section of a normal human tibia ( Top ) resected at the same time as the Ewing sarcoma ( ES ) of the tibia tumor ( Bottom ) . Annotated regions are CT - connective tissue , B - decalcified bone , BM - bone marrow , T - tumor , and S - stroma . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section shown in A . The image displays a heatmap gradient of intensity with white ( least abundant ) to red ( most abundant ) . Scale bar and intensity gradient below are representative of all images in A . B . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length ( CL ) distribution from normal tibia in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . # 0 . 01 < P < 0 . 05 ; * * * * P < 0 . 0001 , analyzed by two - way ANOVA with Tukey ' s multiple comparison for each glycogen chain length . C . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length ( CL ) distribution from the ES of the tibia tumor in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * * 0 . 001 < P < 0 . 01 ; * * * * P < 0 . 0001 , analyzed by two - tailed t - test for each glycogen chain length .

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A. (Left) Hematoxylin and eosin (H&E) stained cross section of a normal human tibia (Top) resected at the same time as the Ewing sarcoma (ES) of the tibia tumor (Bottom). Annotated regions are CT-connective tissue, B-decalcified bone, BM-bone marrow, T-tumor, and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A. B. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from normal tibia in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). #0.01 < P < 0.05; ****P<0.0001, analyzed by two-way ANOVA with Tukey's multiple comparison for each glycogen chain length.C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from the ES of the tibia tumor in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). **0.001 < P < 0.01; ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.


F . In vivo tumor growth of A673 EV or GYS1 - KO injected in the flanks of athymic nude mice . Values are presented as mean + / - standard error ( n = 8 biological replicates per group ) . * * * * P < 0 . 0001 , analyzed by two - tailed t - test .

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F. In vivo tumor growth of A673 EV or GYS1-KO injected in the flanks of athymic nude mice. Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.


G . Half maximal effective concentration ( EC50 ) of the small molecular inhibitor guaiacol in A673 cells in vitro .

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G. Half maximal effective concentration (EC50) of the small molecular inhibitor guaiacol in A673 cells in vitro.


J . Spatial distribution of phosphorylated CL5 of human tibia ( top ) and tibial ES tumor ( bottom ) . The image displays a heatmap gradient of intensity with black ( least abundant ) to yellow ( most abundant ) . K . Phosphorylated chain length distribution in normal human tibia and tibial ES tumor in J . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * * * * P < 0 . 0001 , analyzed by two - tailed t - test for each glycogen chain length .

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J. Spatial distribution of phosphorylated CL5 of human tibia (top) and tibial ES tumor (bottom). The image displays a heatmap gradient of intensity with black (least abundant) to yellow (most abundant). K. Phosphorylated chain length distribution in normal human tibia and tibial ES tumor in J. Values are presented as mean +/- standard error (n=3 technical replicates per region). ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.


N . Representatives image of immunohistochemical staining of laforin in ES patient samples with annotation between tumor ( T ) and stroma ( S ) tissues . Tissues were scanned digitally using the Axio Scan . Z1 side scanner . Scale bar is shown below the image . O . Quantification of immunohistochemical laforin staining between normal tissue ( n = 3 technical replicates per region ) , tibial ES tumor ( n = 7 technical replicates per region ) , and stroma of five ES tissues ( n = 7 ) . Values are presented as mean + / - standard error . * * 0 . 001 < P < 0 . 0 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 , analyzed by one - way ANOVA with Tukey ' s multiple comparison .

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

N. Representatives image of immunohistochemical staining of laforin in ES patient samples with annotation between tumor (T) and stroma (S) tissues. Tissues were scanned digitally using the Axio Scan.Z1 side scanner. Scale bar is shown below the image. O. Quantification of immunohistochemical laforin staining between normal tissue (n=3 technical replicates per region), tibial ES tumor (n=7 technical replicates per region), and stroma of five ES tissues (n=7). Values are presented as mean +/- standard error. **0.001 < P < 0.0; ***P < 0.001; ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison.


P . ( Top ) Western blot showing confirmation of laforin overexpression in A673 xenograft tumors . ( Bottom ) Tumor growth of xenografts of A673 empty vector ( EV ) and laforin overexpression ( LAOE ) . Values are presented as mean + / - standard error ( n = 8 biological replicates per group ) . * * * * P < 0 . 0001 , analyzed by two - tailed t - test .

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

P. (Top) Western blot showing confirmation of laforin overexpression in A673 xenograft tumors. (Bottom) Tumor growth of xenografts of A673 empty vector (EV) and laforin overexpression (LAOE). Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.


( B ) HEK293T cells transiently expressing FLAG and FLAG - tagged TEX264 were subjected to immunoprecipitation ( IP ) . Inputs ( 20 % of the lysates ) and immunoprecipitates ( from 80 % of the lysates ) were analyzed by immunoblotting using the indicated antibodies .

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

(B) HEK293T cells transiently expressing FLAG and FLAG-tagged TEX264 were subjected to immunoprecipitation (IP). Inputs (20% of the lysates) and immunoprecipitates (from 80% of the lysates) were analyzed by immunoblotting using the indicated antibodies.


( C ) HEK293T cells transiently expressing WT or mutated TEX264 - FLAG were subjected to immunoprecipitation with an anti - FLAG antibody and detected with the indicated antibodies .

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

(C) HEK293T cells transiently expressing WT or mutated TEX264-FLAG were subjected to immunoprecipitation with an anti-FLAG antibody and detected with the indicated antibodies.


( D and E ) MEFs stably expressing TEX264 - GFP or its mutant were cultured in starvation media and immunostained with an anti - LC3 antibody . Bars : 10 µm and 1 µm ( insets ) ( D ) . Quantification of the number of TEX264 puncta per cell . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test . Data were collected from 48 cells for each cell type ( E ) .

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

(D and E) MEFs stably expressing TEX264-GFP or its mutant were cultured in starvation media and immunostained with an anti-LC3 antibody. Bars: 10 µm and 1 µm (insets) (D). Quantification of the number of TEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 48 cells for each cell type (E).


( F and G ) WT and TEX264 - KO HeLa cells ( with or without the indicated TEX264 mutants with a C - terminal FLAG tag ) stably expressing the ER - phagy reporter ( ss - RFP - GFP - KDEL ) were cultured in the presence of doxycycline for 24 h to induce the reporter . After doxycycline was removed , cells were cultured in starvation medium lacking amino acids and serum for 9 h ( F ) . The band intensities of RFP and RFP - GFP were quantified and the ratio of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent the mean ± standard error of the mean ( SEM ) of three independent experiments . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test ( G ) .

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

(F and G) WT and TEX264-KO HeLa cells (with or without the indicated TEX264 mutants with a C-terminal FLAG tag) stably expressing the ER-phagy reporter (ss-RFP-GFP-KDEL) were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, cells were cultured in starvation medium lacking amino acids and serum for 9 h (F). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± standard error of the mean (SEM) of three independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (G).


( A and B ) MEFs stably expressing TEX264 - GFP were cultured in starvation media with or without bafilomycin A1 ( Baf A1 ) and immunostained with anti - pTEX264 and anti - LC3 antibodies . Bars : 10 µm and 1 µm ( insets ) ( A ) . Quantification of the number of pTEX264 puncta per cell . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test . Data were collected from 35 cells for each cell type ( B ) .

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

(A and B) MEFs stably expressing TEX264-GFP were cultured in starvation media with or without bafilomycin A1 (Baf A1) and immunostained with anti-pTEX264 and anti-LC3 antibodies. Bars: 10 µm and 1 µm (insets) (A). Quantification of the number of pTEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 35 cells for each cell type (B).


( C ) HeLa cells were treated with 250 nM Torin 1 and Baf A1 for 16 h , and cell homogenates were subjected to an OptiPrep membrane flotation analysis .

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

(C) HeLa cells were treated with 250 nM Torin 1 and Baf A1 for 16 h, and cell homogenates were subjected to an OptiPrep membrane flotation analysis.


( D , E and F ) WT , ATG3 - KO , and FIP200 - KO HeLa cells were cultured in starvation media lacking amino acids and serum with or without Baf A1 for 1 or 5 h . Cell lysates were analyzed by immunoblotting using the indicated antibodies ( D ) . Relative changes of the ratio of band intensities of phosphorylated TEX264 to HSP90 and total TEX264 during starvation are shown ( E and F ) . Data represent the mean ± SEM of three independent experiments .

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

(D, E and F) WT, ATG3-KO, and FIP200-KO HeLa cells were cultured in starvation media lacking amino acids and serum with or without Baf A1 for 1 or 5 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies (D). Relative changes of the ratio of band intensities of phosphorylated TEX264 to HSP90 and total TEX264 during starvation are shown (E and F). Data represent the mean ± SEM of three independent experiments.


( A ) HeLa cells were cultured with 20 nM CK1 inhibitor ( D4476 ) or CK2 inhibitor ( CX4945 ) for 3 h , followed by starvation ( with the inhibitors ) for 3 h . Cell lysates were analyzed by immunoblotting using the indicated antibodies .

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

(A) HeLa cells were cultured with 20 nM CK1 inhibitor (D4476) or CK2 inhibitor (CX4945) for 3 h, followed by starvation (with the inhibitors) for 3 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies.


( B ) Recombinant TEX264 - FLAG was incubated with or without CK2 and the CK2 inhibitor ( CX4945 ) in the presence of 200 µM ATP for 1 h .

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

(B) Recombinant TEX264-FLAG was incubated with or without CK2 and the CK2 inhibitor (CX4945) in the presence of 200 µM ATP for 1 h.


( C ) HEK293T cells transiently expressing TEX264 - FLAG and one of the three Halo - CK2A isoforms or Halo - CK2B were subjected to immunoprecipitation with an anti - FLAG antibody and detected with anti - Halo and anti - FLAG antibodies .

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

(C) HEK293T cells transiently expressing TEX264-FLAG and one of the three Halo-CK2A isoforms or Halo-CK2B were subjected to immunoprecipitation with an anti-FLAG antibody and detected with anti-Halo and anti-FLAG antibodies.


( D and E ) HEK293T cells transiently expressing WT or TEX264 - FLAG mutants were subjected to immunoprecipitation ( IP ) . Inputs ( 20 % of the lysates ) and immunoprecipitates ( from 80 % of the lysates ) were analyzed by immunoblotting using the indicated antibodies ( D ) . The band intensities in the IP fractions were quantified , and the phospho - serine : FLAG ratio was calculated . Data represent the mean ± SEM of five independent experiments . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test ( E ) .

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

(D and E) HEK293T cells transiently expressing WT or TEX264-FLAG mutants were subjected to immunoprecipitation (IP). Inputs (20% of the lysates) and immunoprecipitates (from 80% of the lysates) were analyzed by immunoblotting using the indicated antibodies (D). The band intensities in the IP fractions were quantified, and the phospho-serine:FLAG ratio was calculated. Data represent the mean ± SEM of five independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (E).


( F and G ) MEFs stably expressing TEX264 - GFP were cultured with the CK2 inhibitor for 3 h followed by starvation ( with the CK2 inhibitor ) and immunostained with an anti - LC3 antibody . Scale bars represent 10 μm and 1 μm ( insets ) ( F ) . Quantification of the number of TEX264 puncta per cell . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by an unpaired two - tailed Student ' s t - test . Data were collected from 85 cells for each cell type ( G ) .

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

(F and G) MEFs stably expressing TEX264-GFP were cultured with the CK2 inhibitor for 3 h followed by starvation (with the CK2 inhibitor) and immunostained with an anti-LC3 antibody. Scale bars represent 10 μm and 1 μm (insets) (F). Quantification of the number of TEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by an unpaired two-tailed Student's t-test. Data were collected from 85 cells for each cell type (G).


( H and I ) WT HeLa cells stably expressing the ER - phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter . After doxycycline was removed , cells were cultured with 20 nM CK2 inhibitor for 3 h followed by starvation ( with the CK2 inhibitor ) for 6 h ( H ) . The band intensities of RFP and RFP - GFP were quantified and the ratio of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent the mean ± SEM of six independent experiments . Differences were statistically analyzed by an unpaired two - tailed Student ' s t - test ( I ) .

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

(H and I) WT HeLa cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, cells were cultured with 20 nM CK2 inhibitor for 3 h followed by starvation (with the CK2 inhibitor) for 6 h (H). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± SEM of six independent experiments. Differences were statistically analyzed by an unpaired two-tailed Student's t-test (I).


( B ) HEK293T cells transiently expressing WT or mutated TEX264 - FLAG were subjected to immunoprecipitation with an anti - FLAG antibody and detected with the indicated antibodies .

(B) HEK293T cells transiently expressing WT or mutated TEX264-FLAG were subjected to immunoprecipitation with an anti-FLAG antibody and detected with the indicated antibodies.


( C and D ) MEFs stably expressing TEX264 - GFP or its mutants were cultured in starvation media and immunostained with an anti - LC3 antibody . Bars : 10 µm and 5 µm ( insets ) ( C ) . The intensity of the GFP signal under starvation conditions was quantified at TEX264 - GFP puncta and the ER . The puncta : ER signal ratio was calculated . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test . Data were collected from 70 puncta for each cell type ( D ) .

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

(C and D) MEFs stably expressing TEX264-GFP or its mutants were cultured in starvation media and immunostained with an anti-LC3 antibody. Bars: 10 µm and 5 µm (insets) (C). The intensity of the GFP signal under starvation conditions was quantified at TEX264-GFP puncta and the ER. The puncta:ER signal ratio was calculated. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 70 puncta for each cell type (D).