B ) Scanning electron microscopy of endothelial - denuded mesenteric resistance arteries show a comparable topography of endothelial basement in wild type , Tek - Cre : : Lama5 - / - Lama4 - / - arteries . arrows indicate artificial ruptures due to preparation procedure . Scale bars are 100 nm .

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B) Scanning electron microscopy images of endothelial cell-denuded mesenteric resistance arteries show a comparable topography of the endothelial basement membrane in wild type, Tek-Cre::Lama5-/- and Lama4-/- arteries. The arrows indicate artificial ruptures due to preparation procedure. Scale bars are 100 nm.


C ) Intravital microscopic of mesenteric arteries sizes in vivo show smaller diameters ( - 24 . 3 % ) in Tek - Cre : : Lama5 - / - arteries larger diameters ( + 27 . 6 % ) in Lama4 - / - arteries compared to wild type controls . Auto - of internal elastic lamina allowed a good approximation of mesenteric arteries lumen diameter . Scale bar is 100 µm . Data are means ± s . e . m from a minimum of 4 first order mesenteric arteries imaged at least in 4 mice per genotype . * P < 0 . 05 unpaired t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Intravital microscopic quantification of mesenteric arteries sizes in vivo show smaller diameters (-24.3%) in Tek-Cre::Lama5-/- arteries and larger diameters (+27.6%) in Lama4-/- arteries compared to wild type controls. Auto-fluorescence of the internal elastic lamina allowed a good approximation of mesenteric arteries lumen diameter. Scale bar is 100 µm. Data are means ± s.e.m from a minimum of 4 first order mesenteric arteries imaged at least in 4 mice per genotype. * P<0.05 unpaired t-test.


A ) In vitro adhesion assays employing HUAECs plated on increasing concentrations of purified laminin 411 511 , compared to non - endothelial laminin 111 fibronectin , showing high of HUAECs adhesion to laminin 511 low binding to laminin 411 .

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A) In vitro cell adhesion assays employing HUAECs plated on increasing concentrations of purified laminin 411 and 511, compared to the non-endothelial cell laminin 111 and fibronectin, showing high levels of HUAECs adhesion to laminin 511 and low binding to laminin 411.


B ) Enhanced fold change ( Δ ) in COX2 mRNA expression in HUAECs plated on laminin 511 versus laminin 111 in response to shear . mRNA data are means ± s . e . m of 4 independent experiments with triplicates / experiment , paired t - .

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B) Enhanced fold change (Δ) in COX2 mRNA expression in HUAECs plated on laminin 511 versus laminin 111 in response to shear. mRNA data are means ± s.e.m of 4 independent experiments with triplicates/ experiment, paired t-test.


B ) Enhanced fold change ( Δ ) in COX2 in HUAECs plated on laminin 511 versus laminin 111 in response to shear . data are means ± s . e . m of 7 independent experiments , Mann - Whitney .

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B) Enhanced fold change (Δ) in COX2 protein levels in HUAECs plated on laminin 511 versus laminin 111 in response to shear. Protein quantification data are means ± s.e.m of 7 independent experiments, Mann-Whitney test.


C ) Angle histogram of HUAEC orientation after 120 min of 10dyn / cm2 shear stress showing at 10 - 90 ° in relation to direction of flow . were plated on 25 nM laminin 511 laminin 111 . Data are means ± s . e . m of 6 independent experiments , t - .

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C) Angle histogram of HUAEC orientation after 120 min of 10dyn/cm2 shear stress showing the percent cells at 10-90° in relation to the direction of flow. Cells were plated on 25 nM laminin 511 and laminin 111. Data are means ± s.e.m of 6 independent experiments, t-test.


D , E ) Inhibition assays performed at 25nM laminin 511 ( D ) in presence or absence of function blocking integrin antibodies . Data are means ± s . e . m of 4 experiments with triplicates / experiment , unpaired t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E) Inhibition assays performed at 25nM laminin 511 (D) in the presence or absence of function blocking integrin antibodies. Data are means ± s.e.m of 4 experiments with triplicates/ experiment, unpaired t-test . *P<0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.


D , E ) Inhibition assays performed at 25nM laminin 111 , laminin 411 or fibronectin ( E ) in presence or absence of function blocking integrin antibodies . Data are means ± s . e . m of 4 experiments with triplicates / experiment , unpaired t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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D, E) Inhibition assays performed at 25nM laminin 111, laminin 411 or fibronectin (E) in the presence or absence of function blocking integrin antibodies. Data are means ± s.e.m of 4 experiments with triplicates/ experiment, unpaired t-test . *P<0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.


( A ) Double immunofluorescence of whole mount wild type mesenteric resistance arteries for β1 integrin vinculin show colocalization in focal adhesions ( arrow heads ) . Scale bar is 10 µm .

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(A) Double immunofluorescence staining of whole mount wild type mesenteric resistance arteries for β1 integrin and vinculin show colocalization in focal adhesions (arrow heads). Scale bar is 10 µm.


B ) Vinculin immunofluorescence of whole mount wild type , Tek - Cre : : Lama5 - / - Lama4 - / - mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek - Cre : : Lama5 - / - ( arrow heads ) larger adhesion complexes in Lama4 - / - vessels ( arrow heads ) . Scale bar is 10 µm . Data are means ± s . e . m from 300 from 9 wild type 9 KO arteries isolated from 3 mice / genotype . * * * P < 0 . 001 , * * * * P < 0 . 0001 , unpaired t - .

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

B) Vinculin immunofluorescence staining of whole mount wild type, Tek-Cre::Lama5-/- and Lama4-/- mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek-Cre::Lama5-/- (arrow heads) and larger adhesion complexes in Lama4-/- vessels (arrow heads). Scale bar is 10 µm. Data are means ± s.e.m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice/genotype. ***P<0.001, ****P<0.0001, unpaired t-test.


B ) Vinculin immunofluorescence of whole mount wild type , Tek - Cre : : Lama5 - / - Lama4 - / - mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek - Cre : : Lama5 - / - ( arrow heads ) larger adhesion complexes in Lama4 - / - vessels ( arrow heads ) . Scale bar is 10 µm . Correpsonding of sizes ( C ) frequency distribution ( D ) of adhesion complexes per endothelial . Data are means ± s . e . m from 300 from 9 wild type 9 KO arteries isolated from 3 mice / genotype . * * * P < 0 . 001 , * * * * P < 0 . 0001 , unpaired t - .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Vinculin immunofluorescence staining of whole mount wild type, Tek-Cre::Lama5-/- and Lama4-/- mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek-Cre::Lama5-/- (arrow heads) and larger adhesion complexes in Lama4-/- vessels (arrow heads). Scale bar is 10 µm. Correpsonding quantification of sizes (C) and frequency distribution (D) of adhesion complexes per endothelial cell. Data are means ± s.e.m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice/genotype. ***P<0.001, ****P<0.0001, unpaired t-test.


E , F ) endothelium of excised wild type , Tek - Cre : : Lama5 - / - aortae were analysed by AFM , revealing reduced cortical stiffness in Tek - Cre : : Lama5 - / - vessels ( - 9 . 5 % ) . Data are means ± s . e . m from 4 experiments employing 4 KO arteries 4 wild type controls in each experiment . * P < 0 . 05 , * * * * P < 0 . 0001 , unpaired t - .

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E, F) The endothelium of excised wild type, Tek-Cre::Lama5-/- aortae were analysed by AFM, revealing reduced cortical stiffness in Tek-Cre::Lama5-/- vessels (-9.5%). Data are means ± s.e.m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment. *P<0.05, ****P<0.0001, unpaired t-test.


E , F ) endothelium of excised wild type Lama4 - / - aortae were analysed by AFM , revealing increased cortical stiffness in Lama4 - / - vessels ( + 2 % ) . Data are means ± s . e . m from 4 experiments employing 4 KO arteries 4 wild type controls in each experiment . * P < 0 . 05 , * * * * P < 0 . 0001 , unpaired t - .

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E, F) The endothelium of excised wild type and Lama4-/- aortae were analysed by AFM, revealing increased cortical stiffness in Lama4-/- vessels (+2%). Data are means ± s.e.m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment. *P<0.05, ****P<0.0001, unpaired t-test.


G ) In vitro AFM measurements of cortical stiffness performed on HUAECs plated on 30 nM purified laminin 511 or 111 , reveal increased cortical stiffness in on laminin 511 . Data are mean ± s . e . m from 3 experiments with triplicates / experiments , * P < 0 . 05 , unpaired t - .

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G) In vitro AFM measurements of cortical stiffness performed on HUAECs plated on 30 nM purified laminin 511 or 111, reveal increased cortical stiffness in cells on laminin 511. Data are mean ± s.e.m from 3 experiments with triplicates/experiments, *P<0.05, unpaired t-test.


A ) A dual pipette - pulling was employed to measure - adhesion strength in HUAECs bound to laminin - coated beads ( asterisk ) or in absence of beads . Scale bar is 10 µm . B ) shows higher adhesion strength between bound to laminin 511 - coated beads compared to laminin 111 - coated beads or not incubated with beads . Measurements could not be made with incubated with laminin 411 - coated beads since it was impossible to find - bead complexes . Values are means ± s . e . m from 15 from 3 independent experiments , unpaired t - with Welch ' s correction .

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

A) A dual pipette-pulling assay was employed to measure cell-cell adhesion strength in HUAECs bound to laminin-coated beads (asterisk) or in the absence of beads. Scale bar is 10 µm. B) Quantification shows higher adhesion strength between cells bound to laminin 511-coated beads compared to laminin 111-coated beads or cells not incubated with beads. Measurements could not be made with cells incubated with laminin 411-coated beads since it was impossible to find cell-bead complexes. Values are means ± s.e.m from 15 cells from 3 independent experiments, unpaired t-test with Welch's correction.


C ) Dual pipette - pulling performed in presence of VE - cadherin blocking antibody , isotope control soluble laminin 511 , showing almost complete ablation of adhesion strength in presence of blocking antibody in conditions . Similar adhesion strengths were measured between in presence of soluble laminin 511 bound to laminin 511 - coated beads .

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

C) Dual pipette-pulling assay performed in the presence of VE-cadherin blocking antibody, isotope control and soluble laminin 511, showing almost complete ablation of adhesion strength in the presence of the blocking antibody in all conditions. Similar adhesion strengths were measured between cells in the presence of soluble laminin 511 and cells bound to laminin 511-coated beads.


D ) Adhesion strength in presence of soluble laminin 511 was significantly reduced in presence of integrin β1 blocking antibody ; - adhesion strength between bound to laminin 511 coated beads was significantly reduced in presence of ROCK , SRC or FAK inhibitors . Values are means ± s . e . m from 11 from 3 independent experiments , unpaired t - with Welch ' s correction . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , n . s . = not significant , n . d . = not determined .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Adhesion strength in the presence of soluble laminin 511 was significantly reduced in the presence of integrin β1 blocking antibody; cell-cell adhesion strength between cells bound to laminin 511 coated beads was significantly reduced in the presence of ROCK, SRC or FAK inhibitors. Values are means ± s.e.m from 11 cells from 3 independent experiments, unpaired t-test with Welch's correction. *P <0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s. = not significant, n.d. = not determined.


A ) Immunofluorescence - antibody feeding performed using HUAECs seeded on laminin 411 , 511 or 111 - coated coverslips , reveals more VE - cadherin at junctions less in vesicles in plated on laminin 511 ( arrow heads mark VE - cadherin positive vesicles ) . Scale bar is 10 µm . B ) of VE - cadherin positive vesicles / in HUAECs plated on laminin 411 , 511 or 111 . Values are means ± s . e . m from 300 from 3 independent experiments , unpaired t - .

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

A) Immunofluorescence-based antibody feeding assay performed using HUAECs seeded on laminin 411, 511 or 111-coated coverslips, reveals more VE-cadherin at junctions and less in vesicles in cells plated on laminin 511 (arrow heads mark VE-cadherin positive vesicles). Scale bar is 10 µm. B) Quantification of VE-cadherin positive vesicles/ cell in HUAECs plated on laminin 411, 511 or 111. Values are means ± s.e.m from 300 cells from 3 independent experiments, unpaired t-test.


C ) Western blot of biotinylated ( surface ) VE - cadherin in HUAECs plated on different laminins versus total VE - cadherin ( input ) confirms more junctionally located VE - cadherin in bound to laminin 511 .

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C) Western blot of biotinylated (surface) VE-cadherin in HUAECs plated on different laminins versus total VE-cadherin (input) confirms more junctionally located VE-cadherin in cells bound to laminin 511.


D ) of Western blots expressed as relative signal proportions ( surface VE - cadherin / total VE - cadherin ) . Values shown are means ± s . e . m of 6 experiments , unpaired t - .

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D) Quantification of Western blots expressed as relative signal proportions (surface VE-cadherin/ total VE-cadherin). Values shown are means ± s.e.m of 6 experiments, unpaired t-test.


E ) Western blot of p120 catenincoimmunoprecipitated with VE - cadherin ( input ) from HUAECs plated on different laminins Asterisks are isotope controls . Values shown are means ± s . e . m of 4 experiments , Mann - Whitney .

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E) Western blot of p120 catenincoimmunoprecipitated with VE-cadherin (input) from HUAECs plated on different laminins Asterisks are isotope controls. Values shown are means ± s.e.m of 4 experiments, Mann-Whitney test.


E ) Western blot of p120 catenin coimmunoprecipitated with VE - cadherin ( input ) from HUAECs plated on different laminins , ( F ) corresponding of p120 signal relative to total VE - cadherin signal . Asterisks are isotope controls . Values shown are means ± s . e . m of 4 experiments , Mann - Whitney .

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E) Western blot of p120 catenin coimmunoprecipitated with VE-cadherin (input) from HUAECs plated on different laminins, and (F) corresponding quantification of the p120 signal intensity relative to the total VE-cadherin signal. Asterisks are isotope controls. Values shown are means ± s.e.m of 4 experiments, Mann-Whitney test.


A ) Double immunofluorescence for phosphorylated myosin chain II ( pMLC II ) VE - cadherin in mesenteric resistance artery endothelium from wild type , Tek - Cre : : Lama5 - / - Lama4 - / - mice ( arrow heads ) . Scale bar is 10µm . B ) of pMLC II signal along VE - cadherin signal revealed significantly less overlap in Tek - Cre : : Lama5 - / - more overlap in Lama4 - / - endothelium , compared with wild type controls . data are normalized to skeletal of VE - cadherin signal . Values shown are means ± s . e . m resulting from of 4 field of view per arteries , 4 mice / genotype . * P < 0 . 05 , * * P < 0 . 01 , paired t - .

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

A) Double immunofluorescence staining for phosphorylated myosin light chain II (pMLC II) and VE-cadherin in mesenteric resistance artery endothelium from wild type, Tek-Cre::Lama5-/- and Lama4-/- mice (arrow heads). Scale bar is 10µm. B) Quantification of the pMLC II signal along VE-cadherin signal revealed significantly less overlap in Tek-Cre::Lama5-/- and more overlap in Lama4-/- endothelium, compared with wild type controls. The data are normalized to the skeletal length of the VE-cadherin signal. Values shown are means ± s.e.m resulting from quantification of 4 field of view per arteries, 4 mice /genotype. *P<0.05, **P<0.01, paired t-test.


A , B . Aggregate number of detected undetected cysteines ( A ) lysines ( B ) in 3 , 840 CpDAA - containing .

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A, B. Aggregate number of detected and undetected cysteines (A) and lysines (B) in 3,840 CpDAA-containing proteins.


C . Shows CADD38 max codon scores for nonsynonymous SNVs at residues 220 - 479 of CASP8 ( UniProt ID Q14790 ) . Dashed horizontal marks deleterious threshold of 25 .

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C. Shows CADD38 max codon scores for nonsynonymous SNVs at residues 220-479 of CASP8 (UniProt ID Q14790). Dashed horizontal line marks deleterious threshold of 25.


E . Activity of recombinant caspase - 8 assayed using fluorogenic IETD - AFC substrate . activity shown relative to wild - type ( WT ) for three replicate experiments , bars error bars as mean ± SD .

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E. Activity of recombinant caspase-8 protein assayed using fluorogenic IETD-AFC substrate. Percentage activity shown relative to wild-type (WT) protein for three replicate experiments, bars and error bars as mean ± SD.


( A ) Amylose pulldown from benzonase - treated HEK293T lysates expressing 2xMBP - BRCA2NT in untreated or irradiated ( 6Gy ; + IR ) . DDX5 BRCA2NT ( MBP ) detected by immunoblot . StainFree of gels before transfer were used as loading

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(A) Amylose pulldown from benzonase-treated HEK293T cell lysates expressing 2xMBP-BRCA2NT in untreated or irradiated cells (6Gy; +IR). DDX5 and BRCA2NT (MBP) detected by immunoblot. StainFree images of the gels before transfer were used as loading control


( B ) Immunoprecipitation ( IP ) of endogenous BRCA2 from benzonase - treated HEK293T lysates left untreated or treated with IR ( 6Gy ) harvested 4h post - IR , as indicated . Mouse IgG was used as negative control . Immunoblot of DDX5 BRCA2 . StainFree image of gels before transfer were used as loading control ( cropped image is shown ) . Asterisk ( * ) indicate a non - specific band detected by anti - DDX5 antibody .

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(B) Immunoprecipitation (IP) of endogenous BRCA2 from benzonase-treated HEK293T cell lysates left untreated or treated with IR (6Gy) and harvested 4h post-IR, as indicated. Mouse IgG was used as negative control. Immunoblot of DDX5 and BRCA2. StainFree image of the gels before transfer were used as loading control (cropped image is shown). Asterisk (*) indicate a non-specific band detected by anti-DDX5 antibody.


( C ) Left : Representative of in situ proximity ligation ( PLA ) between BRCA2 DDX5 antibodies in U2OS either left untreated ( - ) or irradiated ( 4h post - IR ; 6 Gy ) . Nuclei as defined by auto threshold plugin on DAPI image ( Image J ) our outlined in yellow . When indicated , were transfected with a plasmid expressing RNase H1 ( RH ) 24h before or treated with Cordycepin ( Cordy ) for 2h at 37 ° C before fixation . Single antibody controls from untreated siC are shown . Scale bar indicates 10 µm . Right : of number of PLA spots per nucleus . For statistical comparison of differences between samples we applied a Kruskal - Wallis followed by Dunn ' s multiple comparison p - values show significant differences . red in plot indicates median each symbol represents a single PLA spot .

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(C) Left: Representative images of in situ proximity ligation assay (PLA) between BRCA2 and DDX5 antibodies in U2OS cells either left untreated (-) or irradiated (4h post-IR; 6 Gy). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls from untreated siC cells are shown. Scale bar indicates 10 µm. Right: Quantification of the number of PLA spots per nucleus. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( D ) Diagram showing BRCA2 N - terminal truncations used in this study amylose pulldown from HEK293T whole extracts overexpressing indicated BRCA2 N - terminal truncations ( BRCA2T1 , BRCA2LT2 , BRCA2LT3 ) or 2xMBP tag . DDX5 BRCA2 truncations were detected using specific antibodies against DDX5 MBP , respectively . StainFree of gels before transfer were used as loading

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(D) Diagram showing the BRCA2 N-terminal truncations used in this study and amylose pulldown from HEK293T whole cells extracts overexpressing the indicated BRCA2 N-terminal truncations (BRCA2T1, BRCA2LT2, BRCA2LT3) or the 2xMBP tag. DDX5 and BRCA2 truncations were detected using specific antibodies against DDX5 and MBP, respectively. StainFree images of the gels before transfer were used as loading control


( E ) Left : GST pulldown using purified BRCA2T1 DDX5 ; MBP antibody was used for detection of both . UB : unbound ; E : eluate . Right : SDS - PAGE showing 300 ng of purified MBP - DDX5 - GST of 2xMBP - tagged BRCA2T1 used in pulldown .

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(E) Left: GST pulldown assay using purified BRCA2T1 and DDX5; MBP antibody was used for the detection of both proteins. UB: unbound; E: eluate. Right: SDS-PAGE showing 300 ng of purified MBP-DDX5-GST and of 2xMBP-tagged BRCA2T1 used in the pulldown assay.


( A ) Left : Representative of S9 . 6 immunofluorescence of U2OS depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control ( siC ) after transfection with either an empty plasmid or a plasmid expressing DDX5 . merged show signal of S9 . 6 , nucleolin ( nucleoli ) antibodies DAPI . Scale bar indicates 25 µm . Right : of S9 . 6 average of U2OS depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control ( siC ) after transfection with either an empty plasmid or a plasmid expressing DDX5 . red in plot indicates median each symbol represents value of a single . statistical significance of difference was calculated with Mann - Whitney U - p - values show significant difference . data represent at least 235 per condition from three independent experiments .

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(A) Left: Representative images of S9.6 immunofluorescence of U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing DDX5. The merged images show the signal of S9.6, nucleolin (nucleoli) antibodies and DAPI staining. Scale bar indicates 25 µm. Right: Quantification of S9.6 average nuclear intensity of U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing DDX5. The red line in the plot indicates the median and each symbol represents the value of a single cell. The statistical significance of the difference was calculated with Mann-Whitney U-test and the p-values show the significant difference. The data represent at least 235 cells per condition from three independent experiments.


( B ) Top : Representative of in situ PLA performed with anti - DDX5 S9 . 6 antibodies in EdU - labelled U2OS . Where indicated , were transfected with a plasmid expressing RNase H1 ( RH ) . Nuclei as defined by auto threshold plugin on DAPI image ( Image J ) our outlined in yellow . Bottom : of PLA spots per nucleus in each condition as indicated . At least 300 per condition were counted from three independent experiments . For statistical comparison of differences between samples we applied a Kruskal - Wallis followed by Dunn ' s multiple comparison , p - values show significant differences . red in plot indicates median each symbol represents a single PLA spot .

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(B) Top: Representative images of in situ PLA performed with anti-DDX5 and S9.6 antibodies in EdU-labelled U2OS cells. Where indicated, cells were transfected with a plasmid expressing RNase H1 (RH). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Bottom: Quantification of PLA spots per nucleus in each condition as indicated. At least 300 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test, the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( C ) Representative screenshot of a specific genomic region showing DRIPc - seq profiles at Watson ( W ) Crick ( C ) strands in K562 depleted of DDX5 ( siDDX5 ) or control ( siC ) from two independent experiments .

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(C) Representative screenshot of a specific genomic region showing DRIPc-seq profiles at Watson (W) and Crick (C) strands in K562 cells depleted of DDX5 (siDDX5) or control cells (siC) from two independent experiments.


( D ) - hybrid distribution along - coding genes containing DRIPc - seq peaks in both conditions ( siC siDDX5 ) replicates . Gene metaplots represent mean of antisense or sense mean DRIPc - seq signal from two independent experiments in K562 depleted of DDX5 ( siDDX5 ) or control ( siC ) as indicated .

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(D) DNA-RNA hybrid distribution along protein-coding genes containing DRIPc-seq peaks in both conditions (siC and siDDX5) and replicates. Gene metaplots represent the mean of antisense or sense mean DRIPc-seq signal from two independent experiments in K562 cells depleted of DDX5 (siDDX5) or control cells (siC) as indicated.


( A ) Left : Representative of in situ PLA between S9 . 6 γH2AX antibodies in U2OS depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control ( siC ) . When indicated , were transfected with a plasmid expressing RNase H1 ( RH ) 24h before or treated with Cordycepin ( Cordy ) for 2h at 37 ° C before fixation . Single antibody controls from non - irradiated siC are shown . Scale bar indicates 10 µm . Nuclei as defined by auto threshold plugin on DAPI image ( Image J ) our outlined in yellow . Right : of PLA spots per nucleus in each condition as indicated . At least 500 per condition were counted from three independent experiments . For statistical comparison of differences between samples we applied a Kruskal - Wallis followed by Dunn ' s multiple comparison p - values show significant differences . red in plot indicates median each symbol represents a single PLA spot .

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(A) Left: Representative images of in situ PLA between S9.6 and γH2AX antibodies in U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC). When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls from non-irradiated siC cells are shown. Scale bar indicates 10 µm. Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Right: Quantification of PLA spots per nucleus in each condition as indicated. At least 500 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( B ) DRIP - qPCR signal values at RBMXL1 HIST1H2BG loci in U2OS DIvA transfected with indicated siRNAs treated in vitro with RNase H1 ( RH ) pre - immunoprecipitation where indicated . experiment was performed in both untreated ( - OHT ) after tamoxifen addition ( + OHT ) . data represent mean ± from at least four independent experiments . statistical significance of difference was calculated with unpaired one - tail t - p - values show significant difference .

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(B) DRIP-qPCR signal values at RBMXL1 and HIST1H2BG loci in U2OS DIvA cells transfected with the indicated siRNAs and treated in vitro with RNase H1 (RH) pre-immunoprecipitation where indicated. The experiment was performed in both untreated cells (-OHT) and after tamoxifen addition (+OHT). The data represent the mean ± SEM from at least four independent experiments. The statistical significance of the difference was calculated with unpaired one-tail t-test and the p-values show the significant difference.


( A ) Top : Scheme showing experimental set up for irradiation in DDX5 - GFP transfected U2OS depleted of BRCA2 ( siBRCA2 ) or control ( siC ) . Middle : Live imaging of recruitment of GFP - 53BP1 or DDX5 - GFP to damage tracks at different time points as indicated . Exposure processing were adjusted to best demonstrate stripes anti - stripes . Scale bar indicates 10 µm . Bottom left : Western blot showing siRNA mediated knock - down of BRCA2 from U2OS transfected with DDX5 - GFP . Bottom right : of of transfected that exhibit DDX5 - GFP " anti - stripe " pattern ( reduced GFP signal at damage tracks compared to signal in nucleus ) at times indicated in depleted of BRCA2 ( siBRCA2 ) or treated with control siRNA ( siC ) . data represent mean ± from three independent experiments .

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(A) Top: Scheme showing the experimental set up for laser irradiation in DDX5-GFP transfected U2OS cells depleted of BRCA2 (siBRCA2) or control cells (siC). Middle: Live cell imaging of the recruitment of GFP-53BP1 or DDX5-GFP to DNA damage tracks at different time points as indicated. Exposure and processing were adjusted to best demonstrate stripes and anti-stripes. Scale bar indicates 10 µm. Bottom left: Western blot showing the siRNA mediated knock-down of BRCA2 from U2OS cells transfected with DDX5-GFP. Bottom right: Quantification of the percentage of transfected cells that exhibit DDX5-GFP "anti-stripe" pattern (reduced GFP signal at DNA damage tracks compared to the signal in the nucleus) at the times indicated in cells depleted of BRCA2 (siBRCA2) or treated with control siRNA (siC). The data represent the mean ± SEM from three independent experiments.


( B ) DDX5 ChIP - qPCR signal values at RBMXL1 ASXL1 loci in U2OS DIvA transfected with indicated siRNAs either untreated ( - OHT ) or after tamoxifen addition ( + OHT ) . data represent mean ± from three independent experiments . green represents background of DDX5 signal . statistical significance of difference was calculated with unpaired one - tail t - p - values show significant differences between untreated ( - OHT ) after tamoxifen addition ( + OHT ) .

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(B) DDX5 ChIP-qPCR signal values at RBMXL1 and ASXL1 loci in U2OS DIvA cells transfected with the indicated siRNAs and either untreated cells (-OHT) or after tamoxifen addition (+OHT). The data represent the mean ± SEM from three independent experiments. The green line represents the background levels of DDX5 signal. The statistical significance of the difference was calculated with unpaired one-tail t-test and the p-values show the significant differences between untreated cells (-OHT) and after tamoxifen addition (+OHT).


( C ) Top : Representative of immunofluorescence of U2OS DIvA depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control ( siC ) either untreated ( - OHT ) or after tamoxifen addition ( + OHT ) , as indicated . Scale bar indicates 10 µm . Bottom : of number of γH2AX foci per nucleus ( left ) DDX5 ( right ) . data represent at least 800 per condition from three independent experiments . red in plot indicates median each symbol represents value of a single . statistical significance of difference was calculated with Mann - Whitney U - p - values show significant difference .

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(C) Top: Representative images of immunofluorescence of U2OS DIvA cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) and either untreated cells (-OHT) or after tamoxifen addition (+OHT), as indicated. Scale bar indicates 10 µm. Bottom: Quantification of the number of γH2AX foci per nucleus (left) and DDX5 nuclear intensity (right). The data represent at least 800 cells per condition from three independent experiments. The red line in the plot indicates the median and each symbol represents the value of a single cell. The statistical significance of the difference was calculated with Mann-Whitney U-test and the p-values show the significant difference.


( A ) Top : PAGE gel showing a representative unwinding assays in which purified MBP - DDX5 - GST ( 1 - 5 nM ) was incubated with 32P - labelled synthetic R - loop substrate in presence or absence of 2 nM purified EGFP - MBP - BRCA2 ( top gel left ) or 10 nM purified BRCA2LT3 ( top gel right ) or 50 nM purified BRCA2T1 ( bottom gel ) . Bottom : of unwinding experiments showing of free relative to R - loop substrate ( unwound product ) as a function of DDX5 alone ( black ) or in presence of BRCA2 ( red ) or BRCA2LT3 ( blue ) or BRCA2T1 ( pink ) . data represent mean ± SD of at least three independent experiments . Right : SDS - PAGE showing 500 ng of purified 2XMBP - BRCA2LT3 150 ng of purified BRCA2 used in unwinding .

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(A) Top: PAGE gel showing a representative unwinding assays in which purified MBP-DDX5-GST (1-5 nM) was incubated with 32P-labelled synthetic R-loop substrate in presence or absence of 2 nM purified EGFP-MBP-BRCA2 (top gel left) or 10 nM purified BRCA2LT3 (top gel right) or 50 nM purified BRCA2T1 (bottom gel). Bottom: Quantification of the unwinding experiments showing the percentage of free RNA relative to the R-loop substrate (unwound product) as a function of DDX5 concentration alone (black) or in presence of BRCA2 (red) or BRCA2LT3 (blue) or BRCA2T1 (pink). The data represent the mean ± SD of at least three independent experiments. Right: SDS-PAGE showing 500 ng of purified 2XMBP-BRCA2LT3 and 150 ng of purified BRCA2 used in the unwinding assay.


( B ) Left : Thin layer chromatography ( TLC ) plate showing a representative ATP hydrolysis in which purified MBP - DDX5 - GST ( 50 - 200 nM ) was incubated with R - loop synthetic substrate [ γ32P ] ATP in presence or absence of or 6 nM purified BRCA2LT3 . Right : of ATP hydrolysed in each condition . No control was used as background . data represent mean ± SD from three independent experiments .

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(B) Left: Thin layer chromatography (TLC) plate showing a representative ATP hydrolysis assay in which purified MBP-DDX5-GST (50-200 nM) was incubated with R-loop synthetic substrate and [γ32P] ATP in presence or absence of or 6 nM purified BRCA2LT3. Right: Quantification of the ATP hydrolysed in each condition. No protein control was used as background. The data represent the mean ± SD from three independent experiments.


( A ) Left : GFP pull - down from whole extracts of BRCA2 deficient DLD1 expressing BRCA2 ( WT ) or variant T207A ( T207A ) . DLD1 BRCA2 + / + ( + / + ) extracts were used as control for GFP - trap . DDX5 BRCA2 were detected with specific antibodies against DDX5 BRCA2 , respectively . StainFree of gels before transfer was used as loading control ( cropped image is shown ) . Right : of GFP - trap pull - down experiments calculated as co - immunoprecipitated DDX5 with either BRCA2 WT or BRCA2 - T207A relative to input of DDX5 amount of immunoprecipitated EGFPMBP - BRCA2 . Results are presented as fold change compared to BRCA2 WT clone . data represents mean ± SD of three independent experiments . Statistical significance of difference was calculated with unpaired t p - values show significant difference .

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(A) Left: GFP pull-down assay from whole cells extracts of BRCA2 deficient DLD1 expressing BRCA2 (WT) or the variant T207A (T207A). DLD1 BRCA2+/+ (+/+) cell extracts were used as control for the GFP-trap. DDX5 and BRCA2 were detected with specific antibodies against DDX5 and BRCA2, respectively. StainFree images of the gels before transfer was used as loading control (cropped image is shown). Right: Quantification of the GFP-trap pull-down experiments calculated as the co-immunoprecipitated DDX5 with either BRCA2 WT or BRCA2-T207A relative to the input levels of DDX5 and the amount of immunoprecipitated EGFPMBP-BRCA2. Results are presented as the fold change compared to the BRCA2 WT clone. The data represents the mean ± SD of three independent experiments. Statistical significance of the difference was calculated with unpaired t test and the p-values show the significant difference.


( B ) Left : Representative of S9 . 6 immunofluorescence of DLD1 bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . merged show signal of S9 . 6 , nucleolin ( nucleoli ) antibodies DAPI . Scale bar indicates 25 µm . Right : of average of S9 . 6 antibody in DLD1 BRCA2 - WT ( WT ) or BRCA2 - T207A ( T207A ) transfected with either a plasmid expressing GFP alone ( - RH ) or GFP - RNase H1 ( + RH ) , as indicated . red in plot indicates median each symbol represents value of a single . Data correspond to at least 170 per condition from two independent experiments . statistical significance of difference was calculated with Mann - Whitney U - ; p - values show significant difference .

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(B) Left: Representative images of S9.6 immunofluorescence of DLD1 cells bearing BRCA2 (WT) or BRCA2-T207A (T207A). The merged images show the signal of S9.6, nucleolin (nucleoli) antibodies and DAPI staining. Scale bar indicates 25 µm. Right: Quantification of the average nuclear intensity of S9.6 antibody in DLD1 BRCA2-WT (WT) or BRCA2-T207A (T207A) cells transfected with either a plasmid expressing GFP alone (-RH) or GFP-RNase H1 (+RH), as indicated. The red line in the plot indicates the median and each symbol represents the value of a single cell. Data correspond to at least 170 cells per condition from two independent experiments. The statistical significance of the difference was calculated with Mann-Whitney U-test; p-values show the significant difference.


( C ) Relative DRIP - qPCR signal values ( respect to siC level at each locus ) at MALAT1 , RRPH1 HIST1H2BG loci in DLD1 BRCA2 - / - bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) treated in vitro with RNase H1 ( RH ) pre - immunoprecipitation where indicated . data represent mean ± from seven independent experiments . statistical significance of difference was calculated with paired t - Student ; p - values show significant difference .

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(C) Relative DRIP-qPCR signal values (respect to the siC level at each locus) at the MALAT1, RRPH1 and HIST1H2BG loci in DLD1 BRCA2-/- cells bearing BRCA2 (WT) or BRCA2-T207A (T207A) and treated in vitro with RNase H1 (RH) pre-immunoprecipitation where indicated. The data represent the mean ± SEM from seven independent experiments. The statistical significance of the difference was calculated with paired t-Student test; the p-values show the significant difference.


( D ) Top : Representative of In situ PLA performed between BRCA2 DDX5 antibodies in bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) , as indicated . were fixed directly or 4h post - irradiation ( 6Gy ) . Single antibody controls in non - irradiated BRCA2 WT are shown . Scale bar indicates 10 µm . Bottom : of number of PLA spots per nucleus . At least 750 were counted per condition from three independent experiments . For statistical comparison of differences between samples we applied a Kruskal - Wallis followed by Dunn ' s multiple comparison p - values show significant differences . red in plot indicates median each symbol represents a single PLA spot .

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

(D) Top: Representative images of In situ PLA performed between BRCA2 and DDX5 antibodies in cells bearing BRCA2 (WT) or BRCA2-T207A (T207A), as indicated. Cells were fixed directly or 4h post-irradiation (6Gy). Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Bottom: Quantification of the number of PLA spots per nucleus. At least 750 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( A ) Top : Representative of In situ PLA performed between DDX5 S9 . 6 ( - hybrids ) antibodies in BRCA2 deficient DLD1 ( BRCA2 - / - ) or DLD1 stable clones expressing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . were fixed directly or 4h post - irradiation ( 6Gy ) . When indicated , were transfected / treated with RNase H1 ( RH ) prior to fixation . Single antibody controls in non - irradiated BRCA2 WT are shown . Scale bar indicates 10 µm . Bottom : of number of PLA spots per nucleus . At least 500 were counted per condition from three independent experiments . For statistical comparison of differences between samples we applied a Kruskal - Wallis followed by Dunn ' s multiple comparison p - values show significant differences . red in plot indicates median each symbol represents a single PLA spot . ( B ) Top : Representative of In situ PLA performed between DDX5 γH2AX antibodies in BRCA2 deficient DLD1 ( BRCA2 - / - ) bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . were fixed directly or 4h post - irradiation ( 6Gy ) . When indicated , were transfected / treated with RNase H1 ( RH ) prior to fixation . Single antibody controls in non - irradiated BRCA2 WT are shown . Scale bar indicates 10 µm . Bottom : of number of PLA spots per nucleus . At least 600 were counted per condition from three independent experiments . For statistical comparison of differences between samples we applied a Kruskal - Wallis followed by Dunn ' s multiple comparison p - values show significant differences . red in plot indicates median each symbol represents a single PLA spot .

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

(A) Top: Representative images of In situ PLA performed between DDX5 and S9.6 (DNA-RNA hybrids) antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) or DLD1 stable clones expressing BRCA2 (WT) or BRCA2-T207A (T207A). Cells were fixed directly or 4h post-irradiation (6Gy). When indicated, cells were transfected/treated with RNase H1 (RH) prior to fixation. Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Bottom: Quantification of the number of PLA spots per nucleus. At least 500 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot. (B) Top: Representative images of In situ PLA performed between DDX5 and γH2AX antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) bearing BRCA2 (WT) or BRCA2-T207A (T207A). Cells were fixed directly or 4h post-irradiation (6Gy). When indicated, cells were transfected/treated with RNase H1 (RH) prior to fixation. Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Bottom: Quantification of the number of PLA spots per nucleus. At least 600 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( C ) Left : Representative of recruitment of transient transfected GFP - MBP - BRCA2 ( WT T207A ) to damage tracks at 5 min post - irradiation in U2OS . γH2AX is used as a marker of damage . Scale bar indicates 10 µm . Right : of of GFP signal at tracks . At least 130 were counted per condition from three independent experiments . For statistical comparison of differences between two samples we applied an unpaired t - . red in plot indicates median each symbol represents a single value .

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

(C) Left: Representative images of the recruitment of transient transfected GFP-MBP-BRCA2 (WT and T207A) to DNA damage tracks at 5 min post-irradiation in U2OS cells. γH2AX is used as a marker of DNA damage. Scale bar indicates 10 µm. Right: Quantification of the intensity of GFP signal at the laser tracks. At least 130 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the two samples we applied an unpaired t-test. The red line in the plot indicates the median and each symbol represents a single cell intensity value.


( D ) Top : Representative of in situ PLA performed between S9 . 6 ( - hybrids ) anti - γH2AX antibodies in BRCA2 deficient DLD1 ( BRCA2 - / - ) bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . When indicated , were transfected / treated with RNase H1 ( RH ) or treated with Cordycepin ( Cordy ) for 2h at 37 ° C before fixation . Single antibody controls in non - irradiated BRCA2 WT are shown . Scale bar indicates 10 µm . Nuclei as defined by auto threshold plugin on DAPI image ( Image J ) our outlined in yellow . Bottom : of number of PLA spots per nucleus . At least 600 were counted per condition from three independent experiments . For statistical comparison of differences between samples we applied a Kruskal - Wallis followed by Dunn ' s multiple comparison p - values show significant differences . red in plot indicates median each symbol represents a single PLA spot .

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

(D) Top: Representative images of in situ PLA performed between S9.6 (DNA-RNA hybrids) and anti-γH2AX antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) bearing BRCA2 (WT) or BRCA2-T207A (T207A). When indicated, cells were transfected/treated with RNase H1 (RH) or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Bottom: Quantification of the number of PLA spots per nucleus. At least 600 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( E ) Top : PAGE gel showing a representative unwinding assays in which purified MBP - DDX5 - GST ( 1 - 5 nM ) was incubated with 32P - labelled synthetic R - loop substrate in presence or absence of 50 nM purified BRCA2T1 - T207A ( bottom right ) . Bottom left : of unwinding experiments showing of free relative to R - loop substrate ( unwound product ) as a function of DDX5 alone ( black ) or in presence of BRCA2T1 - T207A ( green ) . data represent mean ± SD of at least three independent experiments . Bottom right : SDS - PAGE showing 650 ng of purified 2XMBP - BRCA2T1 - T207A used in unwinding .

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

(E) Top: PAGE gel showing a representative unwinding assays in which purified MBP-DDX5-GST (1-5 nM) was incubated with 32P-labelled synthetic R-loop substrate in presence or absence of 50 nM purified BRCA2T1-T207A (bottom right). Bottom left: Quantification of the unwinding experiments showing the percentage of free RNA relative to the R-loop substrate (unwound product) as a function of DDX5 concentration alone (black) or in presence of BRCA2T1-T207A (green). The data represent the mean ± SD of at least three independent experiments. Bottom right: SDS-PAGE showing 650 ng of purified 2XMBP-BRCA2T1-T207A used in the unwinding assay.


( A ) Top : Representative immunofluorescence of stained for RAD51 ( green ) in U2OS depleted of DDX5 ( siDDX5 ) in control ( siC ) in non - treated ( - ) or different time points after exposure to IR ( 6Gy ) , as indicated . Scale bar indicates 10 µm . Bottom : Graph showing average number of RAD51 repair foci in both lines . data represent mean ± of three independent experiments .

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

(A) Top: Representative immunofluorescence images of cells stained for RAD51 (green) in U2OS cells depleted of DDX5 (siDDX5) and in control cells (siC) in non-treated (-) or different time points after exposure to IR (6Gy), as indicated. Scale bar indicates 10 µm. Bottom: Graph showing the average number of RAD51 repair foci in both cell lines. The data represent the mean ± SEM of three independent experiments.


( B ) Top : Representative immunofluorescence of DLD1 BRCA2 deficient ( BRCA2 - / - ) or BRCA2 - / - expressing BRCA2 WT ( WT ) or BRCA2 - T207A ( T207A ) in non - treated or at different time points post - IR ( 6 Gy ) , as indicated ; hybridized with anti - RAD51 antibody ( green ) . Scale bar indicates 10 µm . Bottom : Graph showing average number of RAD51 repair foci in three lines . data represent mean ± of three independent experiments .

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

(B) Top: Representative immunofluorescence images of DLD1 BRCA2 deficient cells (BRCA2-/-) or BRCA2-/- cells expressing BRCA2 WT (WT) or BRCA2-T207A (T207A) in non-treated or at different time points post-IR (6 Gy), as indicated; hybridized with anti-RAD51 antibody (green). Scale bar indicates 10 µm. Bottom: Graph showing the average number of RAD51 repair foci in the three cell lines. The data represent the mean ± SEM of three independent experiments.


( C ) Left : Representative immunofluorescence of DLD1 BRCA2 WT or BRCA2 - T207A 4h post - irradiation ( 6Gy ) hybridized with anti - γH2AX anti - RPA antibodies . Right : of number of γH2AX RPA foci per nucleus , as indicated . data represent at least 400 per condition from two independent experiments . For statistical comparison of differences between samples we applied a Kruskal - Wallis followed by Dunn ' s multiple comparison p - values show significant differences . red in plot indicates median each symbol represents a single focus .

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

(C) Left: Representative immunofluorescence images of DLD1 BRCA2 WT or BRCA2-T207A cells 4h post-irradiation (6Gy) hybridized with anti-γH2AX and anti-RPA antibodies. Right: Quantification of the number of γH2AX and RPA foci per nucleus, as indicated. The data represent at least 400 cells per condition from two independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single focus.


( D ) Left : Representative immunofluorescence of DLD1 BRCA2 - T207A 2h post - irradiation ( 6Gy ) , as indicated , hybridized with anti - γH2AX anti - RAD51 antibodies . ( RH ) RNase H . Right : of number of γH2AX foci ( left ) or RAD51 foci ( right ) per nucleus . When indicated , were transfected with a plasmid expressing RNase H1 ( RH ) 48h prior fixation . data shown is from at least 400 per condition from three independent experiments . For statistical comparison of differences between samples we applied a Kruskal - Wallis followed by Dunn ' s multiple comparison p - values show significant differences . red in plot indicates median each symbol represents a single focus .

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 B-MEASURED_VAR O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: Representative immunofluorescence images of DLD1 BRCA2-T207A cells 2h post-irradiation (6Gy), as indicated, hybridized with anti-γH2AX and anti-RAD51 antibodies. (RH) RNase H. Right: Quantification of the number of γH2AX foci (left) or RAD51 foci (right) per nucleus. When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 48h prior fixation. The data shown is from at least 400 cells per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single focus.


( E ) Top : Subcellular fractionation showing amount of RAD51 in DLD1 stable clones expressing BRCA2 - T207A ( T207A ) in fraction . When indicated , were irradiated ( 2h treatment ) / or transfected with a plasmid expressing RNase H1 ( RH ) 48h prior fixation . α - tubulin Histone H3 signals are shown as markers of fraction respectively . Bottom : of RAD51 detected by Western blot with a RAD51 specific antibody in fraction relative to RAD51 in WCE . Results are presented as fold change compared to untreated sample . data represents mean ± SD of three independent experiments . Statistical significance of difference was calculated with unpaired t p - values show significant difference .

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

(E) Top: Subcellular fractionation showing amount of RAD51 in DLD1 stable clones expressing BRCA2-T207A (T207A) in chromatin fraction. When indicated, cells were irradiated (2h treatment) and/or transfected with a plasmid expressing RNase H1 (RH) 48h prior fixation. α-tubulin and Histone H3 signals are shown as markers of cytoplasmic and chromatin fraction respectively. Bottom: quantification of RAD51 levels detected by Western blot with a RAD51 specific antibody in chromatin fraction relative to RAD51 levels in WCE. Results are presented as the fold change compared to the untreated sample. The data represents the mean ± SD of three independent experiments. Statistical significance of the difference was calculated with unpaired t test and the p-values show the significant difference.


TP53INP1 - KO ( − / − ) WT ( + / + ) mice were subjected to a high - fat diet ( HFD , 60 % fat ) or a control diet ( CTRL ) for 16 . Mice drank tap water or tap water supplemented with NAC ( 10 mg / ml or 1 % ) . Curves show mice recorded every week . CTRL : P ( − / − versus + / + ; t = 8w ) = 0 . 047 ; P ( − / − versus + / + ; t = 9w ) = 0 . 023 . HFD : P ( − / − versus + / + ; t = 7w ) = 0 . 039 ; P ( − / − versus + / + ; t = 8w ) = 0 . 029 ; P ( − / − versus + / + ; t = 9w ) = 0 . 021 ; P ( − / − versus + / + ; t = 10w ) = 0 . 014 ; P ( − / − versus + / + ; t = 11w ) = 0 . 0046 ; P ( − / − versus + / + ; t = 12w ) = 0 . 0028 ; P ( − / − versus + / + ; t = 13w ) = 0 . 0025 ; P ( − / − versus + / + ; t = 14w ) = 0 . 00051 ; P ( − / − versus + / + ; t = 15w ) = 0 . 00027 ; P ( − / − versus + / + ; t = 16w ) = 0 . 00013 .

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

TP53INP1-KO (−/−) and WT (+/+) male mice were subjected to a high-fat diet (HFD, 60% fat) or a control diet (CTRL) for 16 weeks. Mice drank tap water or tap water supplemented with NAC (10 mg/ml or 1%).Curves show mice body weight recorded every week. CTRL: P (−/− versus +/+; t = 8w) = 0.047; P (−/− versus +/+; t = 9w) = 0.023. HFD: P (−/− versus +/+; t = 7w) = 0.039; P (−/− versus +/+; t = 8w) = 0.029; P (−/− versus +/+; t = 9w) = 0.021; P (−/− versus +/+; t = 10w) = 0.014; P (−/− versus +/+; t = 11w) = 0.0046; P (−/− versus +/+; t = 12w) = 0.0028; P (−/− versus +/+; t = 13w) = 0.0025; P (−/− versus +/+; t = 14w) = 0.00051; P (−/− versus +/+; t = 15w) = 0.00027; P (−/− versus +/+; t = 16w) = 0.00013.


At end of protocol , mice were sacrificed ; liver epididymal renal fat masses were taken weighed . Histograms show organ . Liver : P ( − / − versus + / + ; HFD ) = 0 . 014 ; P ( HFD versus CTRL ; + / + ) = 0 . 00063 ; P ( HFD versus CTRL ; − / − ) = 0 . 0010 ; P ( HFD versus CTRL ; + / + NAC ) = 0 . 034 ; P ( HFD versus CTRL ; − / − NAC ) = 0 . 027 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 014 . Epididymal fat mass : P ( − / − versus + / + ; HFD ) = 0 . 011 ; P ( HFD versus CTRL ; + / + ) = 0 . 028 ; P ( HFD versus CTRL ; − / − ) = 0 . 000017 ; P ( HFD versus CTRL ; + / + NAC ) = 0 . 019 ; P ( HFD versus CTRL ; − / − NAC ) = 0 . 0054 ; P ( NAC versus no NAC ; + / + CTRL ) = 0 . 037 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 0025 . Renal fat mass : P ( − / − versus + / + ; HFD ) = 0 . 0041 ; P ( HFD versus CTRL ; + / + ) = 0 . 028 ; P ( HFD versus CTRL ; − / − ) = 0 . 000013 ; P ( HFD versus CTRL ; + / + NAC ) = 0 . 019 ; P ( HFD versus CTRL ; − / − NAC ) = 0 . 0078 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 0047 .

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

At the end of protocol, mice were sacrificed; liver and epididymal and renal fat masses were taken and weighed. Histograms show organ weight. Liver: P (−/− versus +/+; HFD) = 0.014; P (HFD versus CTRL; +/+) = 0.00063; P (HFD versus CTRL; −/−) = 0.0010; P (HFD versus CTRL; +/+ NAC) = 0.034; P (HFD versus CTRL; −/− NAC) = 0.027; P (NAC versus no NAC; −/− HFD) = 0.014. Epididymal fat mass: P (−/− versus +/+; HFD) = 0.011; P (HFD versus CTRL; +/+) = 0.028; P (HFD versus CTRL; −/−) = 0.000017; P (HFD versus CTRL; +/+ NAC) = 0.019; P (HFD versus CTRL; −/− NAC) = 0.0054; P (NAC versus no NAC; +/+ CTRL) = 0.037; P (NAC versus no NAC; −/− HFD) = 0.0025. Renal fat mass: P (−/− versus +/+; HFD) = 0.0041; P (HFD versus CTRL; +/+) = 0.028; P (HFD versus CTRL; −/−) = 0.000013; P (HFD versus CTRL; +/+ NAC) = 0.019; P (HFD versus CTRL; −/− NAC) = 0.0078; P (NAC versus no NAC; −/− HFD) = 0.0047.


A , B Histograms show bloodglucose ( A ) or plasmainsulin ( B ) of 6 - h - fasted mice at beginning ( Week 0 ) / or at end of protocol ( Week 16 ) . Fasting bloodglucose week 16 : P ( − / − versus + / + ; HFD ) = 0 . 0052 ; P ( CTRL versus HFD ; − / − ) = 0 . 000081 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 0019 ; P ( w16 versus w0 ; + / + CTRL ) = 0 . 012 ; P ( w16 versus w0 ; − / − CTRL ) = 0 . 050 ; P ( w16 versus w0 ; − / − HFD ) = 0 . 0023 . Fasting plasmainsulin : P ( − / − versus + / + ; HFD ) = 0 . 043 ; P ( CTRL versus HFD ; + / + ) = 0 . 028 ; P ( CTRL versus HFD ; − / − ) = 0 . 013 ; P ( CTRL versus HFD ; + / + NAC ) = 0 . 011 ; P ( CTRL versus HFD ; − / − NAC ) = 0 . 0015 ; P ( NAC versus no NAC ; − / − HFD ) = 0 . 038 .

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

A, B Histograms show bloodglucose (A) or plasmainsulin (B) levels of 6-h-fasted mice at the beginning (Week 0) and/or at the end of the protocol (Week 16). Fasting bloodglucose week 16: P (−/− versus +/+; HFD) = 0.0052; P (CTRL versus HFD; −/−) = 0.000081; P (NAC versus no NAC; −/− HFD) = 0.0019; P (w16 versus w0; +/+ CTRL) = 0.012; P (w16 versus w0; −/− CTRL) = 0.050; P (w16 versus w0; −/− HFD) = 0.0023. Fasting plasmainsulin: P (−/− versus +/+; HFD) = 0.043; P (CTRL versus HFD; +/+) = 0.028; P (CTRL versus HFD; −/−) = 0.013; P (CTRL versus HFD; +/+ NAC) = 0.011; P (CTRL versus HFD; −/− NAC) = 0.0015; P (NAC versus no NAC; −/− HFD) = 0.038.


C Glucose tolerance ( GTT ) was performed on 6 - h - fasted mice during 120 min after injection of 1 g glucose / kg of . Curves on left show blood glucose level monitored after injection of glucose . Histograms on right show under curve ( AUC ) . Fasting blood glucose : P ( − / − versus + / + ; CTRL ; t = 0 min ) = 0 . 045 ; P ( − / − versus + / + ; CTRL ; t = 15 min ) = 0 . 013 ; P ( − / − versus + / + ; CTRL ; t = 30 min ) = 0 . 016 ; P ( − / − versus + / + ; CTRL ; t = 60 min ) = 0 . 030 ; P ( − / − versus + / + ; CTRL ; t = 90 min ) = 0 . 017 ; P ( − / − versus + / + ; HFD ; t = 60 min ) = 0 . 041 ; P ( − / − versus + / + ; HFD ; t = 90 min ) = 0 . 043 ; P ( − / − versus + / + ; HFD ; t = 120 min ) = 0 . 034 ; P ( HFD versus CTRL ; + / + ; t = 15 min ) = 0 . 0076 ; P ( HFD versus CTRL ; + / + ; t = 30 min ) = 0 . 0067 ; P ( HFD versus CTRL ; + / + ; t = 60 min ) = 0 . 00058 ; P ( HFD versus CTRL ; + / + ; t = 90 min ) = 0 . 0010 ; P ( HFD versus CTRL ; + / + ; t = 120 min ) = 0 . 023 ; P ( HFD versus CTRL ; − / − ; t = 60 min ) = 0 . 032 . AUC : P ( − / − versus + / + ; CTRL ) = 0 . 023 ; P ( − / − versus + / + ; HFD ) = 0 . 035 ; P ( HFD versus CTRL ; + / + ) = 0 . 042 . D Insulin tolerance ( ITT ) was performed on 6 - h - fasted mice during 150 min after injection of 0 . 70 U insulin / kg of . Curves on left show blood glucose level monitored after injection of insulin . Histograms on right show above curve ( AAC ) . Fasting blood glucose : P ( − / − versus + / + ; CTRL ; t = 15 min = 0 . 012 ; P ( − / − versus + / + ; CTRL ; t = 30 min ) = 0 . 022 ; P ( − / − versus + / + ; HFD ; t = 0 min ) = 0 . 027 ; P ( − / − versus + / + ; HFD ; t = 15 min ) = 0 . 011 ; P ( − / − versus + / + ; HFD ; t = 30 min ) = 0 . 0037 ; P ( − / − versus + / + ; HFD ; t = 60 min ) = 0 . 0028 ; P ( − / − versus + / + ; HFD ; t = 90 min ) = 0 . 041 ; P ( − / − versus + / + ; HFD ; t = 120 min ) = 0 . 0032 ; P ( − / − versus + / + ; HFD ; t = 150 min ) = 0 . 0025 ; P ( HFD versus CTRL ; + / + ; t = 15 min ) = 0 . 0082 ; P ( HFD versus CTRL ; + / + ; t = 30 min ) = 0 . 033 ; P ( HFD versus CTRL ; + / + ; t = 90 min ) = 0 . 047 ; P ( HFD versus CTRL ; + / + ; t = 150 min ) = 0 . 028 ; P ( HFD versus CTRL ; − / − ; t = 15 min ) = 0 . 026 ; P ( HFD versus CTRL ; − / − ; t = 30 min ) = 0 . 0095 ; P ( HFD versus CTRL ; − / − ; t = 60 min ) = 0 . 0031 ; P ( HFD versus CTRL ; − / − ; t = 90 min ) = 0 . 033 ; P ( HFD versus CTRL ; − / − ; t = 120 min ) = 0 . 0068 ; P ( HFD versus CTRL ; − / − ; t = 150 min ) = 0 . 0082 . AAC : P ( − / − versus + / + ; HFD ) = 0 . 030 ; P ( HFD versus CTRL ; − / − ) = 0 . 037 .

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 B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Glucose tolerance test (GTT) was performed on 6-h-fasted mice during 120 min after injection of 1 g glucose/kg of body weight. Curves on the left show blood glucose level monitored after injection of glucose. Histograms on the right show area under curve (AUC). Fasting blood glucose: P (−/− versus +/+; CTRL; t = 0 min) = 0.045; P (−/− versus +/+; CTRL; t = 15 min) = 0.013; P (−/− versus +/+; CTRL; t = 30 min) = 0.016; P (−/− versus +/+; CTRL; t = 60 min) = 0.030; P (−/− versus +/+; CTRL; t = 90 min) = 0.017; P (−/− versus +/+; HFD; t = 60 min) = 0.041; P (−/− versus +/+; HFD; t = 90 min) = 0.043; P (−/− versus +/+; HFD; t = 120 min) = 0.034; P (HFD versus CTRL; +/+; t = 15 min) = 0.0076; P (HFD versus CTRL; +/+; t = 30 min) = 0.0067; P (HFD versus CTRL; +/+; t = 60 min) = 0.00058; P (HFD versus CTRL; +/+; t = 90 min) = 0.0010; P (HFD versus CTRL; +/+; t = 120 min) = 0.023; P (HFD versus CTRL; −/−; t = 60 min) = 0.032. AUC: P (−/− versus +/+; CTRL) = 0.023; P (−/− versus +/+; HFD) = 0.035; P (HFD versus CTRL; +/+) = 0.042.D Insulin tolerance test (ITT) was performed on 6-h-fasted mice during 150 min after injection of 0.70 U insulin/kg of body weight. Curves on the left show blood glucose level monitored after injection of insulin. Histograms on the right show area above curve (AAC). Fasting blood glucose: P (−/− versus +/+; CTRL; t = 15 min = 0.012; P (−/− versus +/+; CTRL; t = 30 min) = 0.022; P (−/− versus +/+; HFD; t = 0 min) = 0.027; P (−/− versus +/+; HFD; t = 15 min) = 0.011; P (−/− versus +/+; HFD; t = 30 min) = 0.0037; P (−/− versus +/+; HFD; t = 60 min) = 0.0028; P (−/− versus +/+; HFD; t = 90 min) = 0.041; P (−/− versus +/+; HFD; t = 120 min) = 0.0032; P (−/− versus +/+; HFD; t = 150 min) = 0.0025; P (HFD versus CTRL; +/+; t = 15 min) = 0.0082; P (HFD versus CTRL; +/+; t = 30 min) = 0.033; P (HFD versus CTRL; +/+; t = 90 min) = 0.047; P (HFD versus CTRL; +/+; t = 150 min) = 0.028; P (HFD versus CTRL; −/−; t = 15 min) = 0.026; P (HFD versus CTRL; −/−; t = 30 min) = 0.0095; P (HFD versus CTRL; −/−; t = 60 min) = 0.0031; P (HFD versus CTRL; −/−; t = 90 min) = 0.033; P (HFD versus CTRL; −/−; t = 120 min) = 0.0068; P (HFD versus CTRL; −/−; t = 150 min) = 0.0082. AAC: P (−/− versus +/+; HFD) = 0.030; P (HFD versus CTRL; −/−) = 0.037.


A , B ( A , B ) Immunocytofluorescent of TP53INP1 ( red ) insulin ( green ) in mouse pancreatic sections ( A ) single human islet beta ( B ) . Scale bars represent 50 μm ( A ) 10 μm ( B ) .

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

A, B (A, B) Immunocytofluorescent staining of TP53INP1 (red) and insulin (green) in mouse pancreatic sections (A) and single human islet beta cell (B). Scale bars represent 50 μm (A) and 10 μm (B).


C - E Quantitative PCR for Tp53inp1 mRNA in tissues from rat ( C ) C57BL / 6Jmice fed with a normal diet ( 5 % fat ; ND ) or an high - fat diet ( 45 % fat ; HFD ) ( D , E ) . Results are expressed as mean ± are representative of two independent experiments . n = 2 for ratliver , islets mousespleen ; n = 4 for INS - 1E ; n = 5 for mouseexocrine pancreas ; n = 6 for Min6 ND HFD islets ; n = 11 for mouseislets . * P = 0 . 035 for HFD versus ND .

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 O O O O O

C-E Quantitative PCR for Tp53inp1 mRNA levels in tissues and cells from rat (C) and C57BL/6Jmice fed with a normal diet (5% fat; ND) or an high-fat diet (45% fat; HFD) (D, E). Results are expressed as the mean ± SEM and are representative of two independent experiments. n = 2 for ratliver, islets and mousespleen; n = 4 for INS-1E cells; n = 5 for mouseexocrine pancreas; n = 6 for Min6 cells and ND and HFD islets; n = 11 for mouseislets. *P = 0.035 for HFD versus ND.


Immortalized MEFs ( MEFi ) deficient ( − / − ) or not ( + / + ) for TP53INP1 were left untreated ( NT ) or treated with 3 - methyladenine ( 3 - MA , 5 mM ) during 4 h . Histograms show mt ROS level measured by flow cytometry upon MitoSox . P ( − / − versus + / + ; NT ) = 0 . 019 ; P ( − / − versus + / + ; 3 - MA ) = 0 . 025 ; P ( 3 - MA versus NT ; + / + ) = 0 . 031 . Histograms show mt mass evaluated by flow cytometry in KO or WT MEFi using MitoTracker . P ( − / − versus + / + ; NT ) = 0 . 012 ; P ( 3 - MA versus NT ; + / + ) = 0 . 036 . Histogram shows MitoSox normalized with MitoTracker . P ( − / − versus + / + ; NT ) = 0 . 041 ; P ( − / − versus + / + ; 3 - MA ) = 0 . 030 ; P ( 3 - MA versus NT ; + / + ) = 0 . 045 .

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

Immortalized MEFs (MEFi) deficient (−/−) or not (+/+) for TP53INP1 were left untreated (NT) or treated with 3-methyladenine (3-MA, 5 mM) during 4 h.Histograms show mt ROS level measured by flow cytometry upon MitoSox staining. P (−/− versus +/+; NT) = 0.019; P (−/− versus +/+; 3-MA) = 0.025; P (3-MA versus NT; +/+) = 0.031.Histograms show mt mass evaluated by flow cytometry in KO or WT MEFi using MitoTracker staining. P (−/− versus +/+; NT) = 0.012; P (3-MA versus NT; +/+) = 0.036.Histogram shows MitoSox fluorescence normalized with MitoTracker fluorescence. P (−/− versus +/+; NT) = 0.041; P (−/− versus +/+; 3-MA) = 0.030; P (3-MA versus NT; +/+) = 0.045.


After 4 h recovering in normal media , H2O2 ( 1 h , 100 μM ) or non - treated ( NT ) MEFi deficient ( − / − ) or not ( + / + ) for TP53INP1 were observed by transmission electron microscopy ( ) . N = nucleus ; arrow = mitophagic vacuoles . Scale bar represents 0 . 5 μm . Mean of mitochondrion ( ) , number of mitochondria mitophagic vacuoles normalized by surface were quantified . : P ( − / − versus + / + ; H2O2 ) = 0 . 000027 ; P ( H2O2 versus NT ; + / + ) = 0 . 0070 ; P ( H2O2 versus NT ; − / − ) = 0 . 013 . Nb mito . : P ( − / − versus + / + ; NT ) = 0 . 035 ; P ( − / − versus + / + ; H2O2 ) = 0 . 016 . Nb vacuoles . : P ( − / − versus + / + ; NT ) = 0 . 047 .

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

After 4 h recovering in normal media, H2O2 (1 h, 100 μM) or non-treated (NT) MEFi deficient (−/−) or not (+/+) for TP53INP1 were observed by transmission electron microscopy (TEM). N = nucleus; white arrow = mitophagic vacuoles. Scale bar represents 0.5 μm.Mean size of mitochondrion (area), number of mitochondria and mitophagic vacuoles normalized by cytoplasmic surface area were quantified. Size: P (−/− versus +/+; H2O2) = 0.000027; P (H2O2 versus NT; +/+) = 0.0070; P (H2O2 versus NT; −/−) = 0.013. Nb mito.: P (−/− versus +/+; NT) = 0.035; P (−/− versus +/+; H2O2) = 0.016. Nb vacuoles.: P (−/− versus +/+; NT) = 0.047.


After 4 h recovering in normal media , TCLs from H2O2 - ( 1 h , 100 μM ) , NAC - ( 24 h , 10 mM ) or non - treated ( NT ) MEFi deficient ( − / − ) or not ( + / + ) for TP53INP1 were analyzed by immunoblotting for PGC - 1α , PINK1 , PARKIN , BNIP3 , BNIP3L / NIX , VDAC1 β - tubulin .

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

After 4 h recovering in normal media, TCLs from H2O2- (1 h, 100 μM), NAC- (24 h, 10 mM) or non-treated (NT) MEFi deficient (−/−) or not (+/+) for TP53INP1 were analyzed by immunoblotting for PGC-1α, PINK1, PARKIN, BNIP3, BNIP3L/NIX, VDAC1 and β-tubulin.


HEK293T were cotransfected with plasmids encoding TP53INP1α - NTAP or TP53INP1β - NTAP . TP53INP1α - or β - NTAP was precipitated with a streptavidin - containing resin ( P ) , resolved by PAGE Western blots developed with anti - TP53INP1 ( TP53INP1 precipitation control ) , anti - PINK1 , anti - PARKIN , anti - BNIP3 or anti - BNIP3L / NIX antibody . Western blot on TCL ( on right ) served as a transfection control .

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

HEK293T cells were cotransfected with plasmids encoding TP53INP1α-NTAP or TP53INP1β-NTAP. TP53INP1α- or β-NTAP was precipitated with a streptavidin-containing resin (P), resolved by PAGE and Western blots developed with anti-TP53INP1 (TP53INP1 precipitation control), anti-PINK1, anti-PARKIN, anti-BNIP3 or anti-BNIP3L/NIX antibody. Western blot on TCL (on the right) served as a transfection control.


Three - month - old TP53INP1 - deficient WT mice were sacrificed their livers harvested . Mitochondrial lysates ( Mito ) were purified from total liver lysates ( TCL ) , both were analyzed by immunoblotting for TP53INP1 , PINK1 , PARKIN , VDAC1 β - tubulin .

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

Three-month-old TP53INP1-deficient and WT male mice were sacrificed and their livers harvested. Mitochondrial lysates (Mito) were purified from total liver lysates (TCL), and both were analyzed by immunoblotting for TP53INP1, PINK1, PARKIN, VDAC1 and β-tubulin.


A - D High - resolution respirometry was performed on permeabilized MEFi TP53INP1 deficient ( − / − , black bars ) or not ( + / + , bars ) using glucose ( A ) , lipid - related substrates ( C ) or IV substrate ( D ) . High - resolution respirometry was also performed on intact MEFi ( B ) . For different respiratory state details ( routine , 3 , leak , ETS capacity ) , please refer to Materials Methods section . Results are expressed as mean ± are representative of three independent experiments . In ( A ) : P ( − / − versus + / + ; State 3 ) = 0 . 0079 ; P ( − / − versus + / + ; ETS ) = 0 . 0079 ; n = 5 in each group . In ( B ) : P ( − / − versus + / + ; routine ) = 0 . 0159 ; P ( − / − versus + / + ; leak ) = 0 . 0159 ; P ( − / − versus + / + ; ETS ) = 0 . 0159 ; n = 5 in each group . In ( C ) : P ( − / − versus + / + ; State 3 ) = 0 . 029 ; P ( − / − versus + / + ; ETS ) = 0 . 0079 ; n = 5 in each group . In ( D ) : P ( − / − versus + / + ; ETS ) = 0 . 0259 ; n = 5 in each group . * TP53INP1 − / − versus TP53INP1 + / + ; 1 character : P < 0 . 05 ; 2 characters : P < 0 . 01 .

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

A-D High-resolution respirometry was performed on permeabilized MEFi TP53INP1 deficient (−/−, black bars) or not (+/+, white bars) using glucose (A), lipid-related substrates (C) or complex IV substrate (D). High-resolution respirometry was also performed on intact MEFi (B). For different respiratory state details (routine, 3, leak, and ETS capacity), please refer to the Materials and Methods section. Results are expressed as the mean ± SEM and are representative of three independent experiments. In (A): P (−/− versus +/+; State 3) = 0.0079; P (−/− versus +/+; ETS) = 0.0079; n = 5 in each group. In (B): P (−/− versus +/+; routine) = 0.0159; P (−/− versus +/+; leak) = 0.0159; P (−/− versus +/+; ETS) = 0.0159; n = 5 in each group. In (C): P (−/− versus +/+; State 3) = 0.029; P (−/− versus +/+; ETS) = 0.0079; n = 5 in each group. In (D): P (−/− versus +/+; ETS) = 0.0259; n = 5 in each group. *TP53INP1−/− versus TP53INP1 +/+; 1 character: P < 0.05; 2 characters: P < 0.01.


E TCLs from MEFi were analyzed by immunoblotting for CPT1A , CPT1B β - tubulin . Results are representative of three independent experiments .

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

E TCLs from MEFi were analyzed by immunoblotting for CPT1A, CPT1B and β-tubulin. Results are representative of three independent experiments.


MEFi deficient ( − / − ) or not ( + / + ) for TP53INP1 were seeded in media containing or not 10 mM NAC . Forty - eight hours later , MEFi were treated or not ( NT ) with 100 μM hydrogen peroxide ( H2O2 ) during 1 h in serum free media . were left to recover for 4 h in normal media containing or not NAC ( 10 mM ) , GW - 9662 ( PPARγ inhibitor , 10 μM ) or PNU - 74654 ( Wnt / β - catenin pathway inhibitor , 50 μM ) before being harvested . were observed by . Number of LD normalized by surface is shown in both histograms at bottom of figure . Scale bar represents 0 . 5 μm . Results are expressed as mean ± are representative of three independent experiments . * P ( − / − versus + / + ; NT ) = 0 . 00086 ; # P ( H2O2 versus NT ; + / + ) = 0 . 013 .

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

MEFi deficient (−/−) or not (+/+) for TP53INP1 were seeded in media containing or not 10 mM NAC. Forty-eight hours later, MEFi were treated or not (NT) with 100 μM hydrogen peroxide (H2O2) during 1 h in serum free media. Cells were left to recover for 4 h in normal media containing or not NAC (10 mM), GW-9662 (PPARγ inhibitor, 10 μM) or PNU-74654 (Wnt/β-catenin pathway inhibitor, 50 μM) before being harvested. Cells were observed by TEM.Number of LD normalized by cytoplasmic surface area is shown in both histograms at the bottom of the figure. Scale bar represents 0.5 μm. Results are expressed as the mean ± SEM and are representative of three independent experiments. *P (−/− versus +/+; NT) = 0.00086; #P (H2O2 versus NT; +/+) = 0.013.


were also observed by microscopy after Bodipy493 / 503 of LD ( green ) DAPI of nucleus ( blue ) . L = lipid droplets . Scale bar represents 100 μm . Results are representative of three independent experiments .

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Cells were also observed by fluorescence microscopy after Bodipy493/503 staining of LD (green) and DAPI staining of nucleus (blue). L = lipid droplets. Scale bar represents 100 μm. Results are representative of three independent experiments.


TCLs from MEFi were analyzed by immunoblotting for β - catenin , PPARγ , ATGL , MGLL β - tubulin . Results are representative of three independent experiments .

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TCLs from MEFi were analyzed by immunoblotting for β-catenin, PPARγ, ATGL, MGLL and β-tubulin. Results are representative of three independent experiments.


( A ) Third instar larval skeletal musculature stained with Phalloidin ( F - actin ) . In this subsequent figures , we assayed ventral longitudinal ( highlighted in green ) .

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(A) Third instar larval skeletal musculature stained with Phalloidin (F-actin). In this and subsequent figures, we assayed the ventral longitudinal muscles (highlighted in green).


( B - D ) GFP - Atg8 , overexpressed using Dmef2 - Gal4 driver , labels autophagosomes . ( B ) , starved on low - nutrientfood for 6 h ( C ) , or starved on low - nutrientfood + 2 . 5 mg / ml CQ for 6 h ( D ) . GFP - Atg8 - labeled vesicles appeared only in starved ( C - D ) , localizing around nucleus between myofibers . ( D ) CQ treatment caused accumulation of bloated GFP - Atg8 - labeled vesicles .

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(B-D) GFP-Atg8, overexpressed using the Dmef2-Gal4 driver, labels autophagosomes. (B), starved on low-nutrientfood for 6 h (C), or starved on low-nutrientfood +2.5 mg/ml CQ for 6 h (D). GFP-Atg8-labeled vesicles appeared only in the starved animals (C-D), localizing around the nucleus and between myofibers. (D) CQ treatment caused accumulation of bloated GFP-Atg8-labeled vesicles.


( E - G ) Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - HRP - Lamp1 were assayed for Lamp1 Atg8 localization ( anti - HRP , red ; GFP , green ; DAPI , blue ) . . ( E ) High - nutrient food suppressed formation of both GFP - Atg8 HRP - Lamp1 - labeled vesicles . ( F ) Colocalization of GFP - Atg8 HRP - Lamp in starved on low - nutrient food . yellow arrowhead points to a vesicle positive for both Atg8 Lamp . ( G ) Addition of CQ to starvation diet resulted in accumulation of both GFP - Atg8 HRP - Lamp - labeled vesicles , but they failed to colocalize .

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(E-G) Dmef2-Gal4, UAS-GFP-Atg8/UAS-HRP-Lamp1 animals were assayed for Lamp1 and Atg8 localization (anti-HRP, red; GFP, green; DAPI, blue). . (E) High-nutrient food suppressed formation of both GFP-Atg8 and HRP-Lamp1-labeled vesicles. (F) Colocalization of GFP-Atg8 and HRP-Lamp in animals starved on low-nutrient food. The yellow arrowhead points to a vesicle positive for both Atg8 and Lamp. (G) Addition of CQ to the starvation diet resulted in accumulation of both GFP-Atg8 and HRP-Lamp-labeled vesicles, but they failed to colocalize.


( H ) of number of GFP - Atg8 , HRP - Lamp1 , or GFP - Atg8 + HRP - Lamp1 vesicles in starved or starved + CQ .

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(H) Quantification of the number of GFP-Atg8, HRP-Lamp1, or GFP-Atg8+HRP-Lamp1 vesicles in starved or starved +CQ muscles.