( E ) Experimental scheme of - labelling of in shWRNIP1 . were transfected with control siRNA ( shWRNIP1siCtrl ) or FBH1 siRNA ( shWRNIP1siFBH1 ) , 48 h thereafter labelled with IdU , then treated or not with 4 mM HU . ( F ) Representative IdU tract distributions in shWRNIP1siCtrl or shWRNIP1siFBH1 with or without HU treatment . Representative fiber are reported . Scale bars , 10 µm . Western blot shows FBH1 depletion in . was probed with an anti - FBH1 . GAPDH was used as a loading control . Median tract lengths are given in parentheses . See Tables S1 S2 for details on data sets statistical .

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(E) Experimental scheme of pulse-labelling of DNA fibers in shWRNIP1 cells. Cells were transfected with control siRNA (shWRNIP1siCtrl) or FBH1 siRNA (shWRNIP1siFBH1), and 48 h thereafter labelled with IdU, then treated or not with 4 mM HU.(F) Representative IdU tract length distributions in shWRNIP1siCtrl or shWRNIP1siFBH1 cells with or without HU treatment. Representative DNA fiber images are reported. Scale bars, 10 µm. Western blot shows FBH1 depletion in the cells. The membrane was probed with an anti-FBH1. GAPDH was used as a loading control. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test.


( G ) of binding of RAD51 in shWRNIP1 depleted for FBH1 . were transfected with control siRNA ( shWRNIP1siCtrl ) or FBH1 siRNA ( shWRNIP1siFBH1 ) , 48 h treated or not with HU for 4h . fractions of were analysed by immunoblotting . was probed with anti - FBH1 anti - RAD51 antibodies . LAMIN B1 was used as a loading for fraction . Total amount of RAD51 ( Input ) in was determined with relevant antibodies . GAPDH was used as a loading control . of RAD51 / LAMIN B1 signal ( ) is reported below each lane .

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

(G) Analysis of chromatin binding of RAD51 in shWRNIP1 cells depleted for FBH1. Cells were transfected with control siRNA (shWRNIP1siCtrl) or FBH1 siRNA (shWRNIP1siFBH1), and 48 h treated or not with HU for 4h. Chromatin fractions of cells were analysed by immunoblotting. The membrane was probed with the anti-FBH1 and anti-RAD51 antibodies. LAMIN B1 was used as a loading for the chromatin fraction. Total amount of RAD51 (Input) in the cells was determined with the relevant antibodies. GAPDH was used as a loading control. The ratio of the RAD51/LAMIN B1 signal (chromatin) is reported below each lane.


( A ) of damage accumulation . Wild - type ( shWRNIP1WT ) , WRNIP1 - deficient ( shWRNIP1 ) or mutant ( shWRNIP1T294A ) were treated or not with 4 mM HU for 4 h , then subjected to - H2AXimmunofluorescence microscopy . Graph shows data presented as mean of - H2AX - positive SE from three independent experiments ; n = 3 . Error bars represent standard error ( * , p < 0 . 1 ; * * , p < 0 . 01 ; two - Student ' s t ) . Representative of nuclei showing different number of foci per nucleus are reported .

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(A) Analysis of DNA damage accumulation. Wild-type (shWRNIP1WT), WRNIP1-deficient (shWRNIP1) or mutant (shWRNIP1T294A) cells were treated or not with 4 mM HU for 4 h, then subjected to -H2AXimmunofluorescence microscopy. Graph shows data presented as mean of -H2AX-positive cells SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images of nuclei showing the different number of foci per nucleus are reported.


( B ) of breakage accumulation . shWRNIP1WT , shWRNIP1 shWRNIP1T294A were treated as in ( A ) , then subjected to alkaline Comet . Graph shows data presented as mean tail moment SE from three independent experiments ; n = 3 . Error bars represent standard error ( * , p < 0 . 1 ; * * , p < 0 . 01 ; two - Student ' s t ) . Representative are shown .

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(B) Analysis of DNA breakage accumulation. shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells were treated as in (A), then subjected to alkaline Comet assay. Graph shows data presented as mean tail moment SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images are shown.


( C ) Evaluation of . shWRNIP1WT shWRNIP1 shWRNIP1T294A were treated or not with 4 mM HU for 16 h . viability was evaluated by LIVE / DEAD . Data are expressed as mean of dead SE from three independent experiments ; n = 3 . Error bars represent standard error ( * , p < 0 . 1 ; * * , p < 0 . 01 ; two - Student ' s t ) . Representative of double - of viable ( green ) dead ( red ) are shown .

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(C) Evaluation of cell death. shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU for 16 h. Cell viability was evaluated by LIVE/DEAD fluorescent assay. Data are expressed as mean of dead cells SE from three independent experiments; n = 3. Error bars represent standard error (*, p < 0.1; **, p < 0.01; two-tailed Student's t test). Representative images of double-staining of viable (green) and dead (red) cells are shown.


( D ) Experimental scheme for evaluation of chromosomal aberrations is shown . shWRNIP1WT shWRNIP1 shWRNIP1T294A were treated or not with 4 mM HU , then left to recover for 16h in drug - free medium metaphases collected with colcemid . Next , were fixed processed as reported in " Supplemental information " section . Dot plot shows number of chromosomal aberrations per . Horizontal black lines represent mean SE . Error bars represent standard error ( ns , not significant ; * * , p < 0 . 01 ; two - Student ' s t ) . Representative Giemsa - stained metaphases of treated or not with 4 mM HU . Arrows indicate chromosomal aberrations .

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(D) Experimental scheme for evaluation of the chromosomal aberrations is shown. shWRNIP1WT shWRNIP1 and shWRNIP1T294A cells were treated or not with 4 mM HU, then left to recover for 16h in drug-free medium and metaphases collected with colcemid. Next, cells were fixed and processed as reported in "Supplemental information" section. Dot plot shows the number of chromosomal aberrations per cell. Horizontal black lines represent the mean SE. Error bars represent standard error (ns, not significant; **, p < 0.01; two-tailed Student's t test). Representative Giemsa-stained metaphases of cells treated or not with 4 mM HU. Arrows indicate chromosomal aberrations.


( E ) Experimental scheme of Mirin aberration is given . experiment was carried out as in ( D ) but were pre - treated or not with 50 µM Mirin . Dot plot shows effect of Mirin exposure on number of chromosome aberrations per in shWRNIP1 . Horizontal black lines represent mean SE . Error bars represent standard error ( ns , not significant ; * * , p < 0 . 01 ; two - Student ' s t ) . Representative Giemsa - stained metaphases of shWRNIP1 treated with Mirin alone or in combination with HU . Arrows indicate chromosomal aberrations .

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(E) Experimental scheme of the Mirin aberration analysis is given. The experiment was carried out as in (D) but cells were pre-treated or not with 50 µM Mirin. Dot plot shows the effect of Mirin exposure on the number of chromosome aberrations per cell in shWRNIP1 cells. Horizontal black lines represent the mean SE. Error bars represent standard error (ns, not significant; **, p < 0.01; two-tailed Student's t test). Representative Giemsa-stained metaphases of shWRNIP1 cells treated with Mirin alone or in combination with HU. Arrows indicate chromosomal aberrations.


( F ) Experimental design of chromosomal aberration is reported . shWRNIP1 were transfected with control siRNAs ( siCtrl ) or FBH1 siRNA ( siFBH1 ) . Fourth - eight hours thereafter , were treated or not with 4 mM HU then left to recover for 16 h . Metaphases were collected with colcemid prepared as reported in " Supplemental information " section . Dot plot shows number of chromosomal aberrations per . Western blot shows FBH1 depletion in . was probed with an anti - FBH1 . GAPDH was used as a loading control . Horizontal black lines represent mean SE . Error bars represent standard error . ( * * , p < 0 . 01 ; two - Student ' s t ) .

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

(F) Experimental design of the chromosomal aberration assay is reported. shWRNIP1 cells were transfected with control siRNAs (siCtrl) or FBH1 siRNA (siFBH1). Fourth-eight hours thereafter, cells were treated or not with 4 mM HU and then left to recover for 16 h. Metaphases were collected with colcemid and prepared as reported in "Supplemental information" section. Dot plot shows the number of chromosomal aberrations per cell. Western blot shows FBH1 depletion in the cells. The membrane was probed with an anti-FBH1. GAPDH was used as a loading control. Horizontal black lines represent the mean SE. Error bars represent standard error. (**, p < 0.01; two-tailed Student's t test).


A representative nuclease with MR , MBP - NBS1 - his ( denoted MBP - NBS1 ) , MRN pCtIP on 5 ' - end labeled 70 - bp dsDNA with ends blocked with streptavidin . Samples were separated on 15 % denaturing polyacrylamide gel . Quantitation of nuclease assays such as in ( A ) . Averages shown ; n ≥ 3 ; error bars , .

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A representative nuclease assay with MR, MBP-NBS1-his (denoted MBP-NBS1), MRN and pCtIP on 5'-end labeled 70-bp dsDNA with all ends blocked with streptavidin. Samples were separated on 15 % denaturing polyacrylamide gel. Quantitation of nuclease assays such as in (A). Averages shown; n ≥ 3; error bars, SEM.


effect of mutations in FHA / or BRCT domains of NBS1 on nuclease of MR pCtIP . Quantitation of experiments such as shown in Fig EV2D . Averages shown ; n ≥ 4 , error bars , . Statistical significance denotations represent between wild type full - NBS1 ( in black ) corresponding concentrations of NBS1 variants ; ns ( p > 0 . 05 , not significant ) , * ( p < 0 . 05 ) , * * ( p < 0 . 01 ) , * * * ( p < 0 . 001 ) , two - t - .

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The effect of mutations in FHA and/or BRCT domains of NBS1 on the nuclease of MR and pCtIP. Quantitation of experiments such as shown in Fig EV2D. Averages shown; n ≥ 4, error bars, SEM. Statistical significance denotations represent the analysis between wild type full-length NBS1 (in black) and the corresponding concentrations of the NBS1 variants; ns (p > 0.05, not significant), * (p < 0.05), ** (p < 0.01), *** (p < 0.001), two-tailed t-test.


of pCtIP binding to NBS1 variants . Anti CtIP antibody was immobilized on G agarose , bound to pCtIP , tested for interactions with indicated NBS1 variants ( see cartoon at top ) . Western blot of eluates ( right part ) was performed with anti - CtIP anti - NBS1 antibodies . Left part ( input ) indicates that anti - NBS1 antibody recognizes NBS1 constructs to a similar level .

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Analysis of pCtIP binding to NBS1 variants. Anti CtIP antibody was immobilized on Protein G agarose, bound to pCtIP, and tested for interactions with the indicated NBS1 variants (see cartoon at the top). Western blot of eluates (right part) was performed with anti-CtIP and anti-NBS1 antibodies. Left part (input) indicates that the anti-NBS1 antibody recognizes the NBS1 constructs to a similar level.


of phosphorylated CtIP ( pCtIP , mock - treated ) or λ phosphatase treated CtIP ( λCtIP ) interactions with NBS1 . CtIP variants were immobilized on G agarose , incubated with NBS1 ( see cartoon on left ) . western blot was performed with anti CtIP anti NBS1 antibodies .

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Analysis of phosphorylated CtIP (pCtIP, mock-treated) or λ phosphatase treated CtIP (λCtIP) interactions with NBS1. The CtIP variants were immobilized on protein G agarose, and incubated with NBS1 (see cartoon on the left). The western blot was performed with anti CtIP and anti NBS1 antibodies.


effect of MRE11 interaction region ( MIR ) of NBS1 on nuclease of MR pCtIP . Quantitation of experiments such as shown in Fig EV2E . Averages shown , n ≥ 3 , error bars , . quantitation of wild type NBS1 ( NBS1 WT , in black ) is same as in panel ( B ) is shown again for reference . Statistical significance denotations represent between wild type full - NBS1 ( in black ) corresponding concentrations of NBS1 variants ; ns ( p > 0 . 05 , not significant ) , * * * ( p < 0 . 001 ) , two - t - .

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The effect of the MRE11 interaction region (MIR) of NBS1 on the nuclease of MR and pCtIP. Quantitation of experiments such as shown in Fig EV2E. Averages shown, n ≥ 3, error bars, SEM. The quantitation of wild type NBS1 (NBS1 WT, in black) is the same as in panel (B) and is shown again for reference. Statistical significance denotations represent the analysis between wild type full-length NBS1 (in black) and the corresponding concentrations of the NBS1 variants; ns (p > 0.05, not significant), *** (p < 0.001), two-tailed t-test.


Nuclease assays with MR various NBS1 fragments ( 50 nM ) on oligonucleotide - 5 ' - end labeled dsDNA . reaction buffer contained 60 mM NaCl reactions were incubated for 2 h . Quantitation of experiments such as shown in ( A ) . Averages shown ; n ≥ 3 , error bars , .

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Nuclease assays with MR and various NBS1 fragments (50 nM) on oligonucleotide-based 5'-end labeled dsDNA. The reaction buffer contained 60 mM NaCl and reactions were incubated for 2 h.   Quantitation of experiments such as shown in (A). Averages shown; n ≥ 3, error bars, SEM.


In vivo resection using BrdU under native conditions to directly detect ssDNA at sites of DSBs . Shown are representative .

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In vivo resection assay using BrdU staining under native conditions to directly detect ssDNA at sites of DSBs. Shown are representative images.


In vivo resection using RPA2 as a marker for ssDNA present at sites of DSBs . Shown are representative .

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In vivo resection assay using RPA2 staining as a marker for ssDNA present at sites of DSBs. Shown are representative images.


Quantitation of experiment shown in ( B ) . At least 100 were assessed per condition . horizontal represents a median , box spans from 25th to 75th percentile whiskers from 10th to 90th percentile .

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Quantitation of the experiment shown in (B). At least 100 cells were assessed per condition. The horizontal line represents a median, the box spans from 25th to 75th percentile and the whiskers from 10th to 90th percentile.


Quantitation of experiment shown in ( C ) . At least 100 were assessed per condition . horizontal represents a median , box spans from 25th to 75th percentile whiskers from 10th to 90th percentile .

Quantitation of the experiment shown in (C). At least 100 cells were assessed per condition. The horizontal line represents a median, the box spans from 25th to 75th percentile and the whiskers from 10th to 90th percentile.


Quantitation of single strand annealing ( SSA ) reporter in U2OS . were treated with DMSO ; where indicated , both PFM03 ( 50 μM ) PFM39 ( 50 μM ) MRE11 inhibitors were added . Averages shown ; n = 4 , error bars , . Insert , western blot of NBS1 CtIP upon depletion with siRNA .

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Quantitation of the single strand annealing (SSA) reporter assay in U2OS cells. All cells were treated with DMSO; where indicated, both PFM03 (50 μM) and PFM39 (50 μM) MRE11 inhibitors were added. Averages shown; n = 4, error bars, SEM. Insert, western blot analysis of NBS1 and CtIP protein levels upon depletion with siRNA.


Quantitation of resection activity in NBS1 ∆ N complemented with wild type ( WT ) or NBS1 R28A K160M mutant either mock depleted ( siCtrl ) or depleted of CtIP ( siCtIP ) . At least 300 were assessed per condition . horizontal represents a median , box spans from 25th to 75th percentile whiskers from 10th to 90th percentile . Statistical significance was determined using ordinary one - way ANOVA with Tukey ' s multiple comparison , * * * * ( p < 0 . 0001 ) .

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Quantitation of resection activity in NBS1∆N cells complemented with wild type (WT) or NBS1 R28A K160M mutant and either mock depleted (siCtrl) or depleted of CtIP (siCtIP). At least 300 cells were assessed per condition. The horizontal line represents a median, the box spans from 25th to 75th percentile and the whiskers from 10th to 90th percentile. Statistical significance was determined using ordinary one-way ANOVA with Tukey's multiple comparison test, **** (p<0.0001).


Nuclease assays with MR pCtIP on 3 ' - end labeled dsDNA , incubated for 2 h . Quantitation of experiments such as shown in ( A ) . Averages shown ; n = 2 ; error bars , range .

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Nuclease assays with MR and pCtIP on 3'-end labeled dsDNA, incubated for 2 h.   Quantitation of experiments such as shown in (A). Averages shown; n = 2; error bars, range.


Nuclease assays with MRN , MR various concentrations of pCtIP ( mock - treated ) or λCtIP on 5 ' - end labeled dsDNA , incubated for 2 h .

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Nuclease assays with MRN, MR and various concentrations of pCtIP (mock-treated) or λCtIP on 5'-end labeled dsDNA, incubated for 2 h.


Quantitation of experiments such as shown in ( C ) . Averages shown ; n = 3 ; error bars , .

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Quantitation of experiments such as shown in (C). Averages shown; n = 3; error bars, SEM.


Phosphorylated CtIP was mock - incubated ( pCtIP , lane 2 ) or treated with λ phosphatase ( λCtIP , lane 3 ) . were separated on a 10 % polyacrylamide gel stained with Coomassie brilliant blue .

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Phosphorylated CtIP was mock-incubated (pCtIP, lane 2) or treated with λ phosphatase (λCtIP, lane 3). The proteins were separated on a 10% polyacrylamide gel and stained with Coomassie brilliant blue.


Quantitation of kinetic nuclease assays with MRN pCtIP ( mock - treated ) or λCtIP . Averages shown ; n = 3 ; error bars , . Statistical denotations , * * ( p < 0 . 01 ) , two - t - .

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Quantitation of kinetic nuclease assays with MRN and pCtIP (mock-treated) or λCtIP. Averages shown; n = 3; error bars, SEM. Statistical denotations, ** (p < 0.01), two-tailed t-test.


Quantitation of nuclease assays as in ( F ) , but with MR instead of MRN . Averages shown ; n = 3 ; error bars , . Statistical denotations , ns ( p > 0 . 05 ) , two - t - .

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Quantitation of nuclease assays as in (F), but with MR complex instead of MRN. Averages shown; n = 3; error bars, SEM. Statistical denotations, ns (p > 0.05), two-tailed t-test.


Nuclease assays with MR , MRN , pCtIP or pCtIP T847A , as indicated , on 5 ' - end labeled dsDNA . Reactions were incubated for 2 h . Quantitation of experiments such as shown in ( H ) . Averages shown ; n = 4 ; error bars , .

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Nuclease assays with MR, MRN, pCtIP or pCtIP T847A, as indicated, on 5'-end labeled dsDNA. Reactions were incubated for 2 h.   Quantitation of experiments such as shown in (H). Averages shown; n = 4; error bars, SEM.


Interaction between MRE11 CtIP . MRE11 was immobilized on antibody - coupled G agarose , incubated with pCtIP or λCtIP ( see cartoon ) . Western blot of bound was performed with anti MRE11 anti CtIP antibodies .

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Interaction between MRE11 and CtIP. MRE11 was immobilized on antibody-coupled Protein G agarose, and incubated with pCtIP or λCtIP (see cartoon). Western blot analysis of bound proteins was performed with anti MRE11 and anti CtIP antibodies.


Interaction between RAD50 CtIP . RAD50 - FLAG was immobilized on anti - FLAG affinity resin incubated with pCtIP λCtIP ( see cartoon ) . Western blot of bound was performed with anti FLAG anti CtIP antibodies .

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Interaction between RAD50 and CtIP. RAD50-FLAG was immobilized on anti-FLAG affinity resin and incubated with pCtIP and λCtIP (see cartoon). Western blot analysis of bound proteins was performed with anti FLAG and anti CtIP antibodies.


Interaction between MRE11 , MR or MRN pCtIP or λCtIP , as indicated . Phosphorylated ( pCtIP ) or λ phosphatase - treated CtIP ( λCtIP ) were immobilized on antibody - coupled G agarose , incubated with MRE11 , MR or MRN complexes ( see cartoon ) . Bound were analyzed by western blot using anti CtIP anti MRE11 antibodies .

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

Interaction between MRE11, MR or MRN and pCtIP or λCtIP, as indicated. Phosphorylated (pCtIP) or λ phosphatase-treated CtIP (λCtIP) were immobilized on antibody-coupled Protein G agarose, and incubated with MRE11, MR or MRN complexes (see cartoon). Bound proteins were analyzed by western blot using anti CtIP and anti MRE11 antibodies.


Representative nuclease assays with MRE11 , MR , MRN pCtIP , as indicated , on 5 ' - end labeled dsDNA with a single streptavidin - blocked end ( S ) . substrate contained 5 phosphorothioate bonds ( PTO ) at both 3 ' - ends to prevent exonucleolytic degradation . Reactions were incubated for 2 h .

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

Representative nuclease assays with MRE11, MR, MRN and pCtIP, as indicated, on 5'-end labeled dsDNA with a single streptavidin-blocked end (S). The substrate contained 5 phosphorothioate bonds (PTO) at both 3'-ends to prevent exonucleolytic DNA degradation. Reactions were incubated for 2 h.


Representative nuclease assays as in ( A ) , but with a substrate containing a nick in top oligonucleotide ( 20 nt away from 3 ' - end , see cartoon ) .

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

Representative nuclease assays as in (A), but with a substrate containing a nick in the top oligonucleotide (20 nt away from the 3'-end, see cartoon).


Quantitation of experiments such as shown in ( A ) , with pCtIP . Averages shown ; n ≥ 4 ; error bars , .

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Quantitation of experiments such as shown in (A), with pCtIP. Averages shown; n ≥ 4; error bars, SEM.


Quantitation of experiments such as shown in ( B ) , with without pCtIP . Averages shown ; n = 3 ; error bars , .

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

Quantitation of experiments such as shown in (B), with and without pCtIP. Averages shown; n = 3; error bars, SEM.


Population doublings ( PDs ) determined in BJ cultured with without folate for 28 days .

O O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O

Population doublings (PDs) determined in BJ cells cultured with and without folate for 28 days.


Example of a single combed molecule labeled with IdU ( green ) CldU ( red ) , showing replication from three adjacent origins . Horizontal arrows indicate fork orientation .

O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR O O

Example of a single combed DNA molecule labeled with IdU (green) and CldU (red), showing replication from three adjacent origins. Horizontal white arrows indicate fork orientation.


Representative examples of single combed molecules from control grown for 7 days in a folate - free medium .

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

Representative examples of single combed DNA molecules from control cells and cells grown for 7 days in a folate-free medium.


Fork rate ( Kb / min ) distribution . blue bars : BJ ( n = 126 ) ; black bars : BJ that were cultured for 7 days in a folate - free medium ( n = 131 ) ; blue bars : BJ cultured for 7 days in a folate - free medium supplemented with A , G , C , T nucleosides for last 48 h of experiment ( n = 138 ) . Box plot summarizing fork rate distribution ( Kb / min ) of three independent experiments . Control ( n = 360 ) ; − folate ( n = 372 ) ; − folate + AGCT ( n = 361 ) .

B-SUBCELLULAR O O O O O O O O O O O B-CELL_LINE O O O O O O O O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O

Fork rate (Kb/min) distribution. Light blue bars: BJ cells (n = 126); black bars: BJ cells that were cultured for 7 days in a folate-free medium (n = 131); blue bars: BJ cells cultured for 7 days in a folate-free medium and supplemented with A, G, C, and T nucleosides for the last 48 h of the experiment (n = 138).Box plot summarizing the fork rate distribution (Kb/min) of three independent experiments. Control (n = 360); −folate (n = 372); −folate + AGCT (n = 361).


Fork ( Kb ) distribution . color code is as in ( D ) . Control ( n = 72 ) ; − folate ( n = 71 ) ; − folate + AGCT ( n = 75 ) . Box plot summarizing fork distribution ( Kb ) of three independent experiments . Control ( n = 212 ) ; − folate ( n = 215 ) ; − folate + AGCT ( n = 209 ) .

B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O

Fork distance (Kb) distribution. The color code is as in (D). Control (n = 72); −folate (n = 71); −folate + AGCT (n = 75).Box plot summarizing the fork distance distribution (Kb) of three independent experiments. Control (n = 212); −folate (n = 215); −folate + AGCT (n = 209).


A Population doublings ( PDs ) determined in BJ cultured at indicated folate concentrations for 48 days .

O O O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O

A Population doublings (PDs) determined in BJ cells cultured at the indicated folate concentrations for 48 days.


B , C average replication rate ± ( B ) average fork ± ( C ) in indicated folate concentrations at 14 21 days . At least 115 were analyzed at each at each time point to determine average replication rate . At least 71 replication forks were analyzed at each at each time point to determine average fork .

O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O

B, C The average replication rate ± SEM (B) and the average fork distance ± SEM (C) in the indicated folate concentrations at 14 and 21 days. At least 115 DNA fibers were analyzed at each concentration and at each time point to determine the average replication rate. At least 71 replication forks were analyzed at each concentration and at each time point to determine the average fork distance.


D - G BJ were grown for 14 days in 100 nM folate with without nucleoside supplementation . ( D ) Fork rate ( Kb / min ) distribution . blue bars : BJ ( n = 115 ) ; gray bars : BJ that were cultured for 14 days in 100 nM folate ( n = 117 ) ; blue bars : BJ cultured for 14 days in 100 nM folate supplemented with A , G , C , T nucleosides for last 48 h of experiment ( n = 117 ) . ( E ) Box plot summarizing fork rate distribution ( Kb / min ) of three independent experiments . Control ( n = 352 ) ; 100 nM folate ( n = 364 ) ; 100 nM folate + AGCT ( n = 355 ) . Main box represents values from lower to upper quartile ( 25th to 75th percentile ) . middle represents median . ( F ) Fork ( Kb ) distribution . color code is as in ( D ) . Control ( n = 69 ) ; 100 nM folate ( n = 74 ) ; 100 nM folate + AGCT ( n = 72 ) . ( G ) Box plot summarizing fork distribution ( Kb ) of three independent experiments . Control ( n = 201 ) ; 100 nM folate ( n = 220 ) ; 100 nM folate + AGCT ( n = 228 ) . Main box represents values from lower to upper quartile ( 25th to 75th percentile ) . middle represents median . * * P < 0 . 001 .

O O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-CELL_LINE O O O O O O O O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-G BJ cells were grown for 14 days in 100 nM folate with and without nucleoside supplementation. (D) Fork rate (Kb/min) distribution. Light blue bars: BJ cells (n = 115); gray bars: BJ cells that were cultured for 14 days in 100 nM folate (n = 117); blue bars: BJ cells cultured for 14 days in 100 nM folate and supplemented with A, G, C, and T nucleosides for the last 48 h of the experiment (n = 117). (E) Box plot summarizing the fork rate distribution (Kb/min) of three independent experiments. Control (n = 352); 100 nM folate (n = 364); 100 nM folate + AGCT (n = 355). Main box represents the values from the lower to upper quartile (25th to 75th percentile). The middle line represents the median. (F) Fork distance (Kb) distribution. The color code is as in (D). Control (n = 69); 100 nM folate (n = 74); 100 nM folate + AGCT (n = 72). (G) Box plot summarizing the fork distance distribution (Kb) of three independent experiments. Control (n = 201); 100 nM folate (n = 220); 100 nM folate + AGCT (n = 228). Main box represents the values from the lower to upper quartile (25th to 75th percentile). The middle line represents the median. **P < 0.001.


Cyclin E - expressing BJ were grown for 7 days with without folate . Fork rate ( Kb / min ) distribution . bars : BJ expressing an empty vector ( n = 145 ) ; gray bars : BJ expressing cyclin E oncogene ( n = 147 ) ; gray bars : BJ cultured for 7 days in a folate - free medium ( n = 135 ) ; black bars : BJ expressing cyclin E oncogene cultured for 7 days in a folate - free medium ( n = 138 ) .

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

Cyclin E-expressing BJ cells were grown for 7 days with and without folate.Fork rate (Kb/min) distribution. White bars: BJ cells expressing an empty vector (n = 145); dark gray bars: BJ cells expressing the cyclin E oncogene (n = 147); light gray bars: BJ cells cultured for 7 days in a folate-free medium (n = 135); black bars: BJ cells expressing the cyclin E oncogene cultured for 7 days in a folate-free medium (n = 138).


Cyclin E - expressing BJ were grown for 7 days with without folate . Fork distribution ( Kb ) . color code is as in ( A ) . Empty vector ( n = 78 ) ; CycE ( n = 79 ) ; empty vector − folate ( n = 71 ) ; CycE − folate ( n = 80 ) .

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

Cyclin E-expressing BJ cells were grown for 7 days with and without folate. Fork distance distribution (Kb). The color code is as in (A). Empty vector (n = 78); CycE (n = 79); empty vector −folate (n = 71); CycE −folate (n = 80).


Cyclin E - expressing BJ were grown for 7 days with without folate . of origins with indicated progression between sister forks . Empty vector ( n = 158 ) ; CycE ( n = 155 ) ; empty vector − folate ( n = 160 ) ; CycE − folate ( n = 154 ) . * P < 0 . 05 .

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

Cyclin E-expressing BJ cells were grown for 7 days with and without folate. Percent of origins with the indicated progression ratio between sister forks. Empty vector (n = 158); CycE (n = 155); empty vector −folate (n = 160); CycE −folate (n = 154). *P < 0.05.


Cyclin E - expressing BJ were grown for 7 days with without folate . Examples of nuclei with γH2AX 53BP1 foci following cyclin E expression ( CycE ) ( n = 65 ) , empty vector ( n = 65 ) , folate - free medium for 7 days ( empty vector − folate ) ( n = 67 ) or oncogene expression under folate - free conditions ( CycE − folate ) ( n = 70 ) . Red : γH2AX ; green : 53BP1 ; blue : DAPI . of nuclei with indicated number of γH2AX - 53BP1 co - localized foci . * * P < 0 . 01 .

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

Cyclin E-expressing BJ cells were grown for 7 days with and without folate.Examples of nuclei with γH2AX and 53BP1 foci following cyclin E expression (CycE) (n = 65), empty vector (n = 65), folate-free medium for 7 days (empty vector −folate) (n = 67) or oncogene expression under folate-free conditions (CycE −folate) (n = 70). Red: γH2AX; green: 53BP1; blue: DAPI staining.Percent of nuclei with the indicated number of γH2AX-53BP1 co-localized foci. **P < 0.01.


Cyclin E - expressing BJ grown for 7 days with without folate . Immunoblotting with anti - phosphorylated ATM anti - phosphorylated CHK1 antibodies . Anti - β - catenin anti - actin antibodies were used as loading controls . level .

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

Cyclin E-expressing BJ cells grown for 7 days with and without folate.Immunoblotting with anti-phosphorylated ATM and anti-phosphorylated CHK1 antibodies. Anti-β-catenin and anti-actin antibodies were used as loading controls.Protein level quantification.


Cyclin E - expressing BJ grown for 7 days with without folate . Examples of nuclei with RAD51 foci following cyclin E expression ( CycE ) ( n = 67 ) , empty vector ( n = 65 ) , folate - free medium for 7 days ( − folate ) ( n = 71 ) or oncogene expression under folate - free conditions ( CycE − folate ) ( n = 75 ) . Green : RAD51 , blue : DAPI . of nuclei with indicated number of RAD51 foci ( n = 66 ) . * * P < 0 . 01 .

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

Cyclin E-expressing BJ cells grown for 7 days with and without folate.Examples of nuclei with RAD51 foci following cyclin E expression (CycE) (n = 67), empty vector (n = 65), folate-free medium for 7 days (−folate) (n = 71) or oncogene expression under folate-free conditions (CycE −folate) (n = 75). Green: RAD51, blue: DAPI staining.Percent of nuclei with the indicated number of RAD51 foci (n = 66). **P < 0.01.


A , B Cyclin E - Ras ( H - RasV12 ) - expressing 3T3 grown in 100 nM folate for 4 then two additional in a normal medium . Control were grown in a normal medium for whole period . ( A ) Examples of anchorage - independent growth in soft agar of 3T3 . ( B ) Average number of colonies per soft agar plate of 3T3 . number of colonies per plate is expressed as average ± from three independent experiments .

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

A, B Cyclin E- and Ras (H-RasV12)-expressing 3T3 cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium. Control cells were grown in a normal medium for the whole period. (A) Examples of anchorage-independent growth in soft agar of 3T3 cells. (B) Average number of colonies per soft agar plate of 3T3 cells. The number of colonies per plate is expressed as the average ± SEM from three independent experiments.


C - E Ras ( H - RasV12 ) - expressing MCF10A grown in 100 nM folate for 4 then two additional in a normal medium . Control were grown in a normal medium for whole period . ( C ) Examples of anchorage - independent growth in soft agar of MCF10A . ( D ) Average number of colonies per soft agar plate of MCF10A . number of colonies per plate is expressed as average ± from three independent experiments . ( E ) of - free flanks at indicated time points after injection . Ten mice were injected in both sides in each group .

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

C-E Ras (H-RasV12)-expressing MCF10A cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium. Control cells were grown in a normal medium for the whole period. (C) Examples of anchorage-independent growth in soft agar of MCF10A cells. (D) Average number of colonies per soft agar plate of MCF10A cells. The number of colonies per plate is expressed as the average ± SEM from three independent experiments. (E) Percentage of tumor-free flanks at the indicated time points after cell injection. Ten mice were injected in both sides in each group.


GFP - ATG8a expressing seedlings in Murashige & Skoog ( MS ) growth medium or 30 min after treatment with MS containing ACC , ABA , ATP , BL , 6 - BA , Flg22 , NAA or PEP1 . ( A ) Representative maximum projection of 10 Z - stacks per image . Scale bar : 10 µm .

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GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (A) Representative maximum intensity projection images of 10 Z-stacks per image. Scale bar: 10 µm.


GFP - ATG8a expressing seedlings in Murashige & Skoog ( MS ) growth medium or 30 min after treatment with MS containing ACC , ABA , ATP , BL , 6 - BA , Flg22 , NAA or PEP1 . ( B ) of GFP foci per 0 . 0025 mm2 . Values are presented as mean ± standard deviation of mean were calculated from at least three independent experiments with 3 individuals per replicate . Bars marked with an asterisk ( * ) are statistically significant ( P < 0 . 05 ) according to T - .

B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (B) Quantification of GFP foci per 0.0025 mm2. Values are presented as mean ± standard deviation of the mean and were calculated from at least three independent experiments with 3 individuals per replicate. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.


GFP - ATG8a expressing seedlings in Murashige & Skoog ( MS ) growth medium or 30 min after treatment with MS containing ACC , ABA , ATP , BL , 6 - BA , Flg22 , NAA or PEP1 . ( C ) GFP - ATG8a cleavage immunoblot for exposed to same treatments as in ( A ) . Numbers below blots represent for given sample normalized to input relative to non - treated control . Experiments were repeated minimum 3 times with similar results .

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

GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (C) GFP-ATG8a cleavage immunoblot for plants exposed to the same treatments as in (A). Numbers below the blots represent ratio for given sample normalized to input and relative to non-treated control. Experiments were repeated minimum 3 times with similar results.


GFP - ATG8a expressing seedlings in Murashige & Skoog ( MS ) growth medium or 30 min after treatment with MS containing ACC , ABA , ATP , BL , 6 - BA , Flg22 , NAA or PEP1 . ( D ) NBR1 immunoblot for atg2 - 2 samples for given treatments . Numbers below blots represent for given sample normalized to input relative to non - treated control . Experiments were repeated minimum 3 times with similar results .

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

GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (D) NBR1 immunoblot for atg2-2 samples for given treatments. Numbers below the blots represent ratio for given sample normalized to input and relative to non-treated control. Experiments were repeated minimum 3 times with similar results.


( A ) Seedlings were acclimated for 16 h in MS containing ABA then imaged 30 min after being swapped to MS containing ABA / NAA ( control ) , MS or MS containing flg22 . are representative maximum projection of 10 Z - stacks per condition . Experiments were repeated 3 times independently with similar results . Scale bar : 10 µm . ( B ) of GFP foci per 0 . 0025 mm2 , for samples treated as described in A . Values are presented as mean ± standard deviation of mean are on 3 independent experiments , with 3 individuals per condition . Bars marked with an asterisk ( * ) are statistically significant ( P < 0 . 05 ) according to T - .

O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Seedlings were acclimated for 16 h in MS containing ABA and then imaged 30 min after being swapped to MS containing ABA/NAA (control), MS or MS containing flg22. Images are representative maximum intensity projection of 10 Z-stacks per condition. Experiments were repeated 3 times independently with similar results. Scale bar: 10 µm. (B) Quantification of GFP foci per 0.0025 mm2, for samples treated as described in A. Values are presented as mean ± standard deviation of the mean and are based on 3 independent experiments, with 3 individuals per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.


( C ) GFP - ATG8a cleavage immunoblot for given treatments . of free GFP to loading control , normalized to 16h pre - treated sample ( set to 1 ) is provided below each band .

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(C) GFP-ATG8a cleavage immunoblot for given treatments. The ratio of free GFP to loading control, normalized to the 16h pre-treated sample (set to 1) is provided below each band.


( D ) Seedlings were acclimated for 16 h in MS containing NAA then imaged 30 min after being swapped to MS containing ABA / NAA ( control ) , MS or MS containing 6 - BA . are representative maximum projection of 10 Z - stacks per condition . Experiments were repeated 3 times independently with similar results . Scale bar : 10 µm . ( E ) of GFP foci per 0 . 0025 mm2 , for samples treated as described in ( D ) . Values are presented as mean ± standard deviation of mean are on 3 independent experiments , with 3 individuals per condition . Bars marked with an asterisk ( * ) are statistically significant ( P < 0 . 05 ) according to T - .

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

(D) Seedlings were acclimated for 16 h in MS containing NAA and then imaged 30 min after being swapped to MS containing ABA/NAA (control), MS or MS containing 6-BA. Images are representative maximum intensity projection of 10 Z-stacks per condition. Experiments were repeated 3 times independently with similar results. Scale bar: 10 µm. (E) Quantification of GFP foci per 0.0025 mm2, for samples treated as described in (D). Values are presented as mean ± standard deviation of the mean and are based on 3 independent experiments, with 3 individuals per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.


( F ) GFP - ATG8a cleavage immunoblot for given treatments . of free GFP to loading control , normalized to 16h pre - treated sample ( set to 1 ) is provided below each band .

(F) GFP-ATG8a cleavage immunoblot for given treatments. The ratio of free GFP to loading control, normalized to the 16h pre-treated sample (set to 1) is provided below each band.


( B ) Pattern correlation used to find that accumulate upon ABA / NAA treatment are removed in WT but not in atg2 after swapping to flg22 / 6 - BA .

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(B) Pattern correlation used to find proteins that accumulate upon ABA/NAA treatment and are removed in WT but not in atg2 after swapping to flg22/6-BA.


clusters obtained after quantitative proteomics of WT ( green ) atg2 - 2 ( magenta ) samples treated as described ( C ) cluster fitting pattern displayed ( D ) cluster for that accumulate to higher in atg2 than WT upon treatment with flg22 .

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Protein clusters obtained after quantitative proteomics of WT (green) and atg2-2 (magenta) samples treated as described (C) Protein cluster fitting the pattern displayed (D) Protein cluster for proteins that accumulate to higher levels in atg2 than WT upon treatment with flg22.


clusters obtained after quantitative proteomics of WT ( green ) atg2 - 2 ( magenta ) samples treated as described ( E ) cluster of which accumulate upon NAA treatment are removed in WT but not in atg2 after swapping to 6 - BA ( F ) cluster for that accumulate to higher in atg2 than WT upon treatment with 6 - BA .

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Protein clusters obtained after quantitative proteomics of WT (green) and atg2-2 (magenta) samples treated as described (E) Protein cluster of proteins which accumulate upon NAA treatment and are removed in WT but not in atg2 after swapping to 6-BA (F) Protein cluster for proteins that accumulate to higher levels in atg2 than WT upon treatment with 6-BA.


( B ) Dry of WT , atg5 - 1 or atg2 - 2 grown under stable ( 21 / 16 ºC 16 / 8 - h photoperiod ) or following Swedish Spring of 2013 . Box plots : centerlines show medians ; box limits indicate 25th 75th percentiles ; whiskers extend to minimum maximum . Significance calculated by Kruskal - Wallis or Feltz Miller for equality of coefficients of variation as explained in materials methods . Asterisks represent significant pairwise differences ( ∗ ∗ ∗ ∗ P < 0 . 0001 ) ( C ) Box plots displaying heterogeneity of samples in ( B ) . Box plots : centerlines show medians ; box limits indicate 25th 75th percentiles ; whiskers extend to minimum maximum . Significance calculated by Kruskal - Wallis or Feltz Miller for equality of coefficients of variation as explained in materials methods . Asterisks represent significant pairwise differences ( ∗ ∗ P < 0 . 01 , ∗ ∗ ∗ ∗ P < 0 . 0001 )

O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dry weight of WT, atg5-1 or atg2-2 plants grown under stable (21/16 ºC 16/8-h photoperiod) or following the Swedish Spring of 2013. Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. Significance calculated by Kruskal-Wallis or Feltz and Miller test for the equality of coefficients of variation as explained in materials and methods. Asterisks represent significant pairwise differences ( ∗∗∗∗P< 0.0001) (C) Box plots displaying the heterogeneity of samples in (B). Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. Significance calculated by Kruskal-Wallis or Feltz and Miller test for the equality of coefficients of variation as explained in materials and methods. Asterisks represent significant pairwise differences (∗∗P < 0.01, ∗∗∗∗P< 0.0001)


( A ) Representative of root explants from WT , atg2 - 2 atg5 - 1 in CIM ( 6 days ) or CIM + SIM ( 6 + 21 days ) . Scale bar : 2

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(A) Representative images of root explants from WT, atg2-2 and atg5-1 in CIM (6 days) or CIM + SIM (6 + 21 days). Scale bar: 2 cm


( B ) ( % ) of explants presenting shoots after 21 days after incubation on SIM . Results were obtained from 3 independent experiments with at least 20 calli per condition . Bars marked with an asterisk ( * ) are statistically significant ( P < 0 . 05 ) according to T - .

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(B) Quantification (%) of explants presenting shoots after 21 days after incubation on SIM. Results were obtained from 3 independent experiments with at least 20 calli per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.


( A ) Representative of root explants from WT , atg2 - 2 atg5 - 1 in CIM ( 21 days ) or CIM + SIM ( 21 + 21 days or 21 + 35 days ) . Scale bar : 2

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(A) Representative images of root explants from WT, atg2-2 and atg5-1 in CIM (21 days) or CIM + SIM (21+ 21 days or 21+ 35 days). Scale bar: 2 cm


( B C ) Fresh weights of calli after incubation on CIM ( B ) or CIM + SIM ( 21 + 21 ) ( C ) . Box plots : centerlines show medians ; box limits indicate 25th 75th percentiles ; whiskers extend to minimum maximum . Results were obtained from 3 independent experiments with at least 20 calli per condition , asteriscs mark statistical significance to WT according to T - ( * * P < 0 . 001 ; * * * P < 0 . 0001 ) .

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(B and C) Fresh weights of calli after incubation on CIM (B) or CIM + SIM (21+ 21) (C). Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. Results were obtained from 3 independent experiments with at least 20 calli per condition, asteriscs mark statistical significance to WT according to the T -test (** P<0.001; *** P<0.0001).


A . of P4 ( top ) P21 ( bottom ) Mpdz + / + Mpdz - / - mice . Arrowheads point to domed foreheads of latter .

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A. Images of P4 (top) and P21 (bottom) Mpdz+/+ and Mpdz-/- mice. Arrowheads point to the domed foreheads of the latter.


B . Mean weights of P18 - P21 Mpdz + / + Mpdz - / - mice ( n = 8 ÷ 12 , mean ± SD ) .

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B. Mean weights of P18-P21 Mpdz+/+ and Mpdz-/- mice (n=8÷12, mean ± SD).


C . Coronal , axial , sagittal ( top to bottom ) PET of P18 - P21 Mpdz + / + Mpdz - / - mice . emission is shown as a 7 - point scale from black ( 0 ) to ( 7 ) .

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C. Coronal, axial, and sagittal (top to bottom) PET images of P18-P21 Mpdz+/+ and Mpdz-/- mice. The emission intensity is shown as a 7-point temperature scale from black (0) to white (7).


D . Mean PET emission intensities in indicated brain sections ( n = 4 , mean ± SD ) .

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D. Mean PET emission intensities in the indicated brain sections (n=4, mean ± SD).


A . Coronal , sagittal , axial ( clockwise from top left corner image of each genotype ) MR of Mpdz + / + Mpdz - / - P18 - P21 mice . Ventricles are pseudo - colored in red 3D - reconstructed in lower left panels .

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A. Coronal, sagittal, and axial (clockwise from top left corner image of each genotype) MR images of Mpdz+/+ and Mpdz-/- P18-P21 mice. Ventricles are pseudo-colored in red and 3D-reconstructed in the lower left panels.


B . Means of total ventricle volumes total brain volumes of Mpdz + / + Mpdz - / - mice ( n = 8 , mean ± SD ) .

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B. Means of total ventricle volumes and total brain volumes of Mpdz+/+ and Mpdz-/- mice (n=8, mean ± SD).


C . A coronal HE - stained section ( center ) flanked by anatomically - corresponding coronal T2 - weighted MR of Mpdz - / - P18 - P21 mice . Arrows or arrowheads show match between lateral ventricle CP in HE section in MR .

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C. A coronal HE-stained section (center) flanked by anatomically-corresponding coronal T2-weighted MR images of Mpdz-/- P18-P21 mice. Arrows or arrowheads show the match between the lateral ventricle CP in the HE section and in the MR images.


D . Duplicate rows of T2 - weighted coronal anatomically - corresponding T1 - weighted coronal at 1 min at peak - signal time point after contrast - medium injection ; bottom row shows a similar set of axial . Areas surrounded by squares are magnified in insets ; arrows mark contrast - medium signal in T1 - weighted . Each row corresponds to one mouse aged 18 - 21 - days . E . Time courses of normalized T1 - weighted image intensities corresponding to areas surrounded by numbered squares on T2 - weighted . Scale bars , 1 mm ; insets , 0 . 5 mm .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O

D. Duplicate rows of T2-weighted coronal images and anatomically-corresponding T1-weighted coronal images at 1 min and at the peak-signal time point after contrast-medium injection; the bottom row shows a similar set of axial images. Areas surrounded by squares are magnified in the insets; arrows mark the contrast-medium signal in the T1-weighted images. Each row corresponds to one mouse aged 18-21-days. E. Time courses of the normalized T1-weighted image intensities corresponding to the areas surrounded by numbered squares on the T2-weighted images. Scale bars, 1 mm; insets, 0.5 mm.


A . Coronal HE - stained brain sections of a P18 Mpdz + / + mouse ( out of a total of three ) showing 3rd ventricle , proximal distal sections of aqueduct of Sylvius ; these features are marked by arrows in insets . B . same , for P18 Mpdz - / - mice .

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A. Coronal HE-stained brain sections of a P18 Mpdz+/+ mouse (out of a total of three) showing the 3rd ventricle, and the proximal and distal sections of the aqueduct of Sylvius; these features are marked by arrows in the insets. B. The same, for P18 Mpdz-/- mice.


C . Top views of brains of P7 Mpdz + / + Mpdz - / - mice that show sites of Evans blue injection , midline planes ( dashed ) sectioned to produce sagittal below . They show extent of Evans blue spread in ventricles surrounding tissue ( one out of three experiments ) . In , injection ; LV , lateral ventricle ; SA , Sylvian Aqueduct ; V3 , third ventricle ; V4 , fourth ventricle . Scale bars , 1 mm ; insets , 0 . 25 mm .

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C. Top views of brains of P7 Mpdz+/+ and Mpdz-/- mice that show the sites of Evans blue injection, and the midline planes (dashed line) sectioned to produce the sagittal images below. They show the extent of Evans blue spread in the ventricles and surrounding tissue (one out of three experiments). In, injection; LV, lateral ventricle; SA, Sylvian Aqueduct; V3, third ventricle; V4, fourth ventricle. Scale bars, 1 mm; insets, 0.25 mm.


A . Immunofluorescence of 10 μm - thick sections of CP from third ventricle villi of Mpdz + / + Mpdz - / - mice were immunolabeled as shown . areas in square frames are magnified in bottom panels .

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A. Immunofluorescence images of 10 μm-thick sections of CP from third ventricle villi of Mpdz+/+ and Mpdz-/- mice were immunolabeled as shown. The areas in the square frames are magnified in the bottom panels.


CP sections from lateral ventricle villi immunolabeled for ZO1 ( B ) are representative of 2 Mpdz + / + mice 3 Mpdz - / - mice . Scale bars , top panels , 100 μm ; bottom panels , 10 μm .

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CP sections from lateral ventricle villi immunolabeled for ZO1 (B) The images are representative of 2 Mpdz+/+ mice and 3 Mpdz-/- mice. Scale bars, top panels, 100 μm; bottom panels, 10 μm.


CP sections from lateral ventricle villi immunolabeled for E - cadherin ( E - cad ; C ) . are representative of 2 Mpdz + / + mice 3 Mpdz - / - mice . Scale bars , top panels , 100 μm ; bottom panels , 10 μm .

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CP sections from lateral ventricle villi immunolabeled for E-cadherin (E-cad; C). The images are representative of 2 Mpdz+/+ mice and 3 Mpdz-/- mice. Scale bars, top panels, 100 μm; bottom panels, 10 μm.


D . Immunoblots with indicated antibodies ( top ) their quantifications . densitometry measurements were normalized relative to signal of samples transduced by non - targeting shRNA ( Ctrl ) . Note that same β - actin immunoblot was used twice because Jam - C E - cadherin samples were immunoblotted on same ( mean ± SD , n = 3 ) .

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D. Immunoblots with the indicated antibodies (top) and their quantifications. The densitometry measurements were normalized relative to the signal of the samples transduced by non-targeting shRNA (Ctrl). Note that the same β-actin immunoblot was used twice because the Jam-C and E-cadherin samples were immunoblotted on the same membrane (mean ± SD, n=3).


A . of longitudinal sections of lateral ventricle CP villi of P18 - P21 Mpdz + / + Mpdz - / - mice . Scale bar , 2 μm . B . A large number of voids of varying sizes was evident in CPECs of Mpdz - / - mice . Higher magnification of areas in rectangles are shown underneath panels . Adherens junctions are denoted by horizontal lines arrows ; voids are indicated by arrows . Scale bar , 1 μm ; insets , 200 nm .

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A. TEM images of longitudinal sections of lateral ventricle CP villi of P18-P21 Mpdz+/+ and Mpdz-/- mice. Scale bar, 2 μm. B. A large number of voids of varying sizes was evident in the CPECs of Mpdz-/- mice. Higher magnification images of the areas in the rectangles are shown underneath the panels. Adherens junctions are denoted by horizontal lines and arrows; voids are indicated by arrows. Scale bar, 1 μm; insets, 200 nm.


C . Triplicate of tight junctions proximal to apical faces of CPECs in Mpdz + / + Mpdz - / - mice . Scale bar , 100

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C. Triplicate images of tight junctions proximal to the apical faces of CPECs in Mpdz+/+ and Mpdz-/- mice. Scale bar, 100 nm


D . Mitochondria were smaller frequently lacked large portions of their cristae . Autophagosomes are present in some of them ( arrows ) . Scale bar , 200 nm . are representative of 2 Mpdz + / + mice 2 Mpdz - / - mice .

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D. Mitochondria were smaller and frequently lacked large portions of their cristae. Autophagosomes are present in some of them (arrows). Scale bar, 200 nm. The images are representative of 2 Mpdz+/+ mice and 2 Mpdz-/- mice.


E . Time course standard deviations of impedance of confluent hpCPEC monolayers that were transduced by either MPDZ or non - targeting ( Ctrl ) shRNA . Each record represents 4 wells . MPDZ immunoblot of each group is shown in inset .

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E. Time course and standard deviations of the impedance of confluent hpCPEC monolayers that were transduced by either MPDZ or non-targeting (Ctrl) shRNA. Each record represents 4 wells. MPDZ immunoblot of each cell group is shown in the inset.


A . of CPEC sections from lateral ventricle villi of two Mpdz + / + two Mpdz - / - P14 - P16 mice injected with HRP . CPs were reacted with hydrogen peroxide DAB ex - vivo . particles are DAB deposits internalized by micropinocytosis . magnified fields to right show particles engulfed in macropinosomes . Note layered structure of particles , macropinosome that is open to ventricular space in Mpdz - / - section . Scale bars , 1 μm ; insets , 100 nm . B . Mean number of engulfed DAB particles per in Mpdz + / + Mpdz - / - mice ( mean ± SD , n = 22 ) .

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

A. TEM images of CPEC sections from lateral ventricle villi of two Mpdz+/+ and two Mpdz-/- P14-P16 mice injected with HRP. The CPs were reacted with hydrogen peroxide and DAB ex-vivo. The dark particles are DAB deposits internalized by micropinocytosis. The magnified fields to the right show individual particles engulfed in macropinosomes. Note the layered structure of the particles, and the macropinosome that is open to the ventricular space in the Mpdz-/- section. Scale bars, 1 μm; insets, 100 nm. B. Mean number of engulfed DAB particles per cell in Mpdz+/+ and Mpdz-/- mice (mean ± SD, n=22).


C . A CPEC section showing engulfment of a DAB particle by its basal ruffles in magnified field . Scale bars in panels C D , 1 μm ; insets , 200 nm . D . A CPEC section showing a preponderance of macropinosomes close to apical face of , a magnified field that contains several macropinosomes . E . Mean numbers of DAB - containing macropinosomes close to apical or basal sides of CPECs from Mpdz + / + or Mpdz - / - mice ( mean ± SD , n = 20 ) .

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C. A CPEC section showing the engulfment of a DAB particle by its basal ruffles in the magnified field. Scale bars in panels C and D, 1 μm; insets, 200 nm. D. A CPEC section showing a preponderance of macropinosomes close to the apical face of the cell, and a magnified field that contains several macropinosomes. E. Mean numbers of DAB-containing macropinosomes close to the apical or basal sides of CPECs from Mpdz+/+ or Mpdz-/- mice (mean ± SD, n=20).


A . Immunofluorescence of 10 μm - thick sections from lateral ventricle CP villi of Mpdz + / + Mpdz - / - P14 - P16 mice immunolabeled by anti - LDLR . Scale bars , 50 ( top ) 25 ( bottom ) μm . B . Mean intensities ( normalized relative to highest recorded ) per in several LDLR - immunolabeled CP sections ( mean ± SD , n = 101 ) .

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A. Immunofluorescence images of 10 μm-thick sections from lateral ventricle CP villi of Mpdz+/+ and Mpdz-/- P14-P16 mice immunolabeled by anti-LDLR. Scale bars, 50 (top) and 25 (bottom) μm. B. Mean fluorescence intensities (normalized relative to the highest recorded intensity) per cell in several LDLR-immunolabeled CP sections (mean ± SD, n=101).


C . Immunoblots showing abundances of LDLR MPDZ in hCPECs transduced by MPDZ - targeting or non - targeting shRNA . Mean abundances are quantified by densitometry of LDLR bands , normalized relative to β - actin bands ( mean ± SD , n = 3 ) .

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C. Immunoblots showing the abundances of LDLR and MPDZ in hCPECs transduced by MPDZ-targeting or non-targeting shRNA. Mean abundances are quantified by densitometry of the LDLR bands, normalized relative to the β-actin bands (mean ± SD, n=3).


D . of hCPECs transduced by either MPDZ or non - targeting ( Ctrl ) shRNA immunolabeled by anti - LDLR either before ( 0 min ) or after 8 min of constitutive endocytosis of LDLR . histograms below show mean intensities per hCPEC in each group , normalized relative to highest recorded ( mean ± SD , n = 41 ÷ 53 ) .

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D. Fluorescence images of hCPECs transduced by either MPDZ or non-targeting (Ctrl) shRNA and immunolabeled by anti-LDLR either before (0 min) or after 8 min of constitutive endocytosis of LDLR. The histograms below show the mean fluorescence intensities per hCPEC in each cell group, normalized relative to the highest recorded intensity (mean ± SD, n=41÷53).


A . Mean in CSF of Mpdz + / + Mpdz - / - P14 - P21 mice ( mean ± SD , n = 2 ÷ 3 ) .

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A. Mean protein concentration in the CSF of Mpdz+/+ and Mpdz-/- P14-P21 mice (mean ± SD, n=2÷3).


B . Volcano plot showing that contents in sera of P15 - P21 Mpdz + / + Mpdz - / - mice are similar to each other .

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B. Volcano plot showing that the protein contents in sera of P15-P21 Mpdz+/+ and Mpdz-/- mice are similar to each other.


C . A volcano plot showing that 23 are at least two - fold significantly more abundant in CSF of P15 - 21 Mpdz - / - mice ( red circles , names are indicated ) whereas only two were more abundant in CSF of Mpdz + / + mice ( green circles ) . CSF composition was analyzed in 3 Mpdz - / - 3 Mpdz + / + mice .

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C. A volcano plot showing that 23 proteins are at least two-fold significantly more abundant in the CSF of P15-21 Mpdz-/- mice (red circles, protein names are indicated) whereas only two proteins were more abundant in the CSF of Mpdz+/+ mice (green circles). The CSF composition was analyzed in 3 Mpdz-/- and 3 Mpdz+/+ mice.


D . of 10 μm - thick sections from lateral ventricle CP villi of Mpdz + / + Mpdz - / - mice , immunolabeled for Nkcc1 . areas in marked squares are magnified below . Scale bars , top , 50 μm , bottom 20 μm .

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D. Images of 10 μm-thick sections from lateral ventricle CP villi of Mpdz+/+ and Mpdz-/- mice, immunolabeled for Nkcc1. The areas in the marked squares are magnified below. Scale bars, top, 50 μm, bottom 20 μm.


E . Immunoblots of LDLR Mpdz , with β - actin as loading control . MPDZ immunoblot shows efficiency of shRNA - mediated knockdown . Bands from three immunoblots were quantified by normalization to β - actin bands ( mean ± SD ) .

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E. Immunoblots of LDLR and Mpdz, with β-actin as loading control. The MPDZ immunoblot shows the efficiency of the shRNA-mediated knockdown. Bands from three immunoblots were quantified by normalization to β-actin bands (mean ± SD).


( A ) Immunoblots of mitochondrial fractions from siRNA ‐ transfected HEK293T ± CCCP .

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(A) Immunoblots of mitochondrial fractions from siRNA‐transfected HEK293T cells±CCCP.


( B ) Immunoblots against indicated proteases in siRNA ‐ transfected .

(B) Immunoblots against indicated proteases in siRNA‐transfected cells.


( C ) of full ‐ PINK1 ( n = 4 ) knockdown efficiency ( n = 2 ) compared with non ‐ targeting ( NT ) siRNA from experiments performed as in B . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 . CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase tensin homologue ‐ induced kinase 1 ; siRNA , short interfering .

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(C) Quantification of full‐length PINK1 (n=4) and knockdown efficiency (n=2) compared with non‐targeting (NT) siRNA from experiments performed as in B. *P0.05; **P0.01; ***P0.001. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.


( A ) Live confocal of siRNA ‐ transfected HEK293T ± CCCP stained with MitoTracker ( MTR ) Green Deep Red ( DR ) ; Scale bar , 5 μm .

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(A) Live confocal micrographs of siRNA‐transfected HEK293T cells±CCCP stained with MitoTracker (MTR) Green and Deep Red (DR); Scale bar, 5 μm.


( B ) MTR DR / Green from as in A . N = 3 ; * P0 . 05 compared with NT siRNA .

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(B) MTR DR/Green ratio from images as in A. N=3; *P0.05 compared with NT siRNA.


( C ) Mitochondrial interconnectivity from as in A , N = 3 .

(C) Mitochondrial interconnectivity from images as in A, N=3.


( D ) Oxygen consumption in state 3 respiration in mitochondrial fractions from siRNA ‐ transfected . Antimycin A with NT siRNA was used as a control . A . U . , arbitrary unit ; CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; siRNA , short interfering .

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(D) Oxygen consumption in state 3 respiration in mitochondrial fractions from siRNA‐transfected cells. Antimycin A with NT siRNA was used as a control. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; siRNA, short interfering RNA.


( A ) Immunoblots of mitochondrial ( M ) ( C ) fractions from siRNA ‐ transfected HEK293T ± CCCP for 3 . 5 h , ± MG132 for final hour .

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(A) Immunoblots of mitochondrial (M) and cytosolic (C) fractions from siRNA‐transfected HEK293T cells±CCCP for 3.5 h, ±MG132 for the final hour.


( B ) of 52 ‐ kDa PINK1 from A ( with MG132 ) , n = 3 ; * P0 . 05 ; * * P0 . 001 compared with NT siRNA .

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(B) Quantification of 52‐kDa PINK1 from A (with MG132), n=3; *P0.05; **P0.001 compared with NT siRNA.


( C ) Mitochondria from siRNA ‐ transfected treated with 10 μM CCCP or dimethylsulphoxide were incubated with or without respiration substrate ( Resp . mix ) for 30 min at 37 ° C . al hour .

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(C) Mitochondria from siRNA‐transfected cells treated with 10 μM CCCP or dimethylsulphoxide were incubated with or without respiration substrate (Resp. mix) for 30 min at 37°C.al hour.


( A ) Mitochondria from CCCP ‐ treated or siRNA ‐ transfected HEK293T were incubated with increasing concentrations of proteinase K ( PK ) .

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(A) Mitochondria from CCCP‐treated or siRNA‐transfected HEK293T cells were incubated with increasing concentrations of proteinase K (PK).


( B ) Mitochondria from CCCP ‐ treated or MPPβ knockdown were incubated ± 0 . 1 M Na2CO3 before supernatant ( S ) pellet ( P ) were separated by centrifugation . CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase tensin homologue ‐ induced kinase 1 ; siRNA , short interfering ; WB , western blot .

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(B) Mitochondria from CCCP‐treated or MPPβ knockdown cells were incubated ±0.1 M Na2CO3 before supernatant (S) and pellet (P) were separated by centrifugation. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA; WB, western blot.


( A ) Live confocal of siRNA ‐ , GFP ‐ Parkin ‐ OCT ‐ DsRed ‐ transfected HEK293T ± CCCP ; Scale bars , 20 μm ( low magnification ) 5 μm ( high magnification ) .

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(A) Live confocal micrographs of siRNA‐, GFP‐Parkin‐ and OCT‐DsRed‐transfected HEK293T cells ±CCCP; Scale bars, 20 μm (low magnification) and 5 μm (high magnification).


( B ) of transfected displaying a punctate mitochondrial pattern of GFP ‐ Parkin distribution , n = 3 ; * P0 . 05 ; * * P0 . 01 compared with NT siRNA .

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(B) Percentage of transfected cells displaying a punctate mitochondrial pattern of GFP‐Parkin distribution, n=3; *P0.05; **P0.01 compared with NT siRNA.


( C ) Immunoblots of endogenous Parkin in mitochondrial fractions from siRNA ‐ transfected ± CCCP .

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(C) Immunoblots of endogenous Parkin in mitochondrial fractions from siRNA‐transfected cells ±CCCP.


( D ) Mitochondrial mass measured by flow cytometry of MTR Green in siRNA ‐ transfected treated ± CCCP for 24 h , n = 3 . A . U . , arbitrary unit ; CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; GFP , green ; MPP , mitochondrial processing peptidase ; MTR , MitoTracker ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase tensin homologue ‐ induced kinase 1 ; siRNA , short interfering .

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(D) Mitochondrial mass measured by flow cytometry of MTR Green fluorescence in siRNA‐transfected cells treated ±CCCP for 24 h, n=3. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; GFP, green fluorescent protein; MPP, mitochondrial processing peptidase; MTR, MitoTracker; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.


( a ) Transverse slice of live E10 . 5 Venus : : HES5 homozygous knock - in mouse showing ventral HES5 domain in spinal cord ex vivo ( left panel ) ; Draq5 live stain with segmentation overlay ( right panel ) ; scale bar 30 μm .

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(a) Transverse slice of live E10.5 Venus::HES5 homozygous knock-in mouse showing the ventral HES5 domain in spinal cord ex vivo (left panel); Draq5 live nuclear stain with nuclear segmentation overlay (right panel); scale bar 30 μm.


( a ) 20x snapshot of an ex - vivo slice culture of E10 . 5 spinal cord from Venus : : HES5 heterozygous knock - in mouse , transverse section ; delineated region ( blue ) correspond to data shown in ( b - c ) . D - dorsal , V - ventral . ( b ) Spatial profile of Venus : : HES5 averaged over 2 . 5hrs with 0 representing ventral end of kymograph ; black represents trend in Venus : : HES5 data across domain produced using an polynomial order 6 ( see Materials Methods ) . ( c ) Detrended spatial profile of Venus : : HES5 corresponding to data shown in ( b ) .

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(a) 20x snapshot of an ex-vivo slice culture of E10.5 spinal cord from Venus::HES5 heterozygous knock-in mouse, transverse section; delineated region (blue) correspond to data shown in (b-c). D- dorsal, V- ventral. (b) Spatial profile of Venus::HES5 intensity averaged over 2.5hrs with 0 distance representing the ventral end of kymograph; black line represents the trend in Venus::HES5 data across the domain produced using an polynomial order 6 (see Materials and Methods). (c) Detrended spatial profile of Venus::HES5 corresponding to data shown in (b).


( a ) Start : Finish Venus : : HES5 in E10 . 5 Venus : : HES5 heterozygous spinal cord slices treated with control ( DMSO ) Notch inhibitor DBZ ( 2 μM ) observed over 16h ; bars indicate mean standard deviation of DMS0 ( n = 3 experiments ) DBZ ( n = 4 experiments ) ; 2 - t - * * * * p = 0 . 0001 .

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(a) Start:Finish Venus::HES5 intensity ratio in E10.5 Venus::HES5 heterozygous spinal cord slices treated with control (DMSO) and Notch inhibitor DBZ (2 μM) observed over 16h; bars indicate mean and standard deviation of DMS0 (n=3 experiments) and DBZ (n=4 experiments); 2-tailed t-test ****p=0.0001.


( a ) Transverse cryosection of E10 . 5 Venus : : HES5 spinal cord . Venus : : HES5 endogenous signal , OLIG2 - motorneuron progenitor marker , NGN2 - early marker of neuronal commitment ; scale bar 20 μm . ( b ) Relative intensities of Venus : : HES5 NGN2 in motorneuron interneuron progenitors ; bars show mean SD of at least 494 per domain from 5 slices in 2 experiments ; Kruskal - Wallis with Dunn ' s multiple comparison adjusted p - values * * p = 0 . 0032 , * * * p < 0 . 001 .

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(a) Transverse cryosection of E10.5 Venus::HES5 spinal cord. Venus::HES5 endogenous signal, OLIG2 - motorneuron progenitor marker, NGN2 - early marker of neuronal commitment; scale bar 20 μm. (b) Relative nuclear intensities of Venus::HES5 and NGN2 in motorneuron and interneuron progenitors; bars show mean and SD of at least 494 cells per domain from 5 slices in 2 experiments; Kruskal-Wallis with Dunn's multiple comparison test adjusted p-values **p=0.0032, ***p<0.001.


( a ) Detrended spatial profile of NGN2 : : mScarlet - I from transverse slice of E10 . 5 spinal cord from heterozygous knock - in mouse in ventral - dorsal direction ; red indicates motorneuron ( MN ) domain , blue interneuron domain ( IN ) .

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(a) Detrended spatial profile of NGN2::mScarlet-I intensity from transverse slice of E10.5 spinal cord from heterozygous knock-in mouse in ventral-dorsal direction;red indicates motorneuron (MN) domain, blue interneuron domain (IN).


A Combined IF FISH of RAD54 ( IF ) telomeres ( FISH ) in ALT non - ALT lines . arrows indicate RAD54 foci that colocalize with telomeres . Scale bars = 10 µm B of data in A . A was counted positive if it contained 1 or more colocalization event between RAD54 telomere . At least 100 were counted per per repeat . For SaOS2 , NOS , SJSA1 , HeLa LT n = 3 . For Cal72 , U2OS , HuO9 n = 4 . Values shown are mean ± sd

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A Combined IF and DNA FISH analysis of RAD54 (IF) and telomeres (FISH) in ALT and non-ALT cell lines. White arrows indicate RAD54 foci that colocalize with telomeres. Scale bars = 10 µm B Quantification of data in A. A cell was counted positive if it contained 1 or more colocalization event between RAD54 and the telomere. At least 100 cells were counted per cell line per repeat. For SaOS2, NOS, SJSA1, HeLa LT n = 3. For Cal72, U2OS, HuO9 n = 4. Values shown are mean±sd


Combined IF FISH of RAD54 ( IF ) , PML ( IF ) telomeres ( FISH ) in ALT lines SaOS2 HuO9 . Scale bars = 10

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Combined IF and DNA FISH analysis of RAD54 (IF), PML (IF) and telomeres (FISH) in ALT cell lines SaOS2 and HuO9. Scale bars = 10 µm


Western blot showing expression of TRF1 - FOK1 - mCherry fusion following doxycycline induction , both WT D450A ( nuclease dead ) . were treated with 40 ng / mL doxycycline for 16 hrs then 1µM Shield - 1 1µM 4 - OHT for 4

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Western blot showing expression of TRF1-FOK1-mCherry fusion protein following doxycycline induction, both WT and D450A (nuclease dead). Cells were treated with 40 ng/mL doxycycline for 16 hrs and then 1µM Shield-1 and 1µM 4-OHT for 4 hrs


Combined IF FISH for RAD54 ( IF ) telomeres ( FISH ) in U2OS - TRF1 - FOK1 - WT or U2OS - TRF1 - FOK1 - D450A ( nuclease dead ) . were treated as in E immediately prior to . Scale bars = 10 µm of data from F . RAD54 foci were detected using particle in Fiji . At least a total of 300 were counted from 3 repeated experiments . Values shown are mean ± . Values were compared using Kruskal - Wallis followed by Dunn ' s multiple comparison . of data from F . RAD54 foci were detected using particle in Fiji . were counted as positive if they counted 8 or more RAD54 foci that colocalized with telomeres . Values shown are mean ± of n = 3 biological replicates . Values were compared using standard two - way ANOVA followed by Sidak ' s multiple comparison .

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

Combined IF and DNA FISH for RAD54 (IF) and telomeres (FISH) in U2OS-TRF1-FOK1-WT or U2OS-TRF1-FOK1-D450A (nuclease dead) cells. Cells were treated as in E immediately prior to staining. Scale bars = 10 µm Quantification of data from F. RAD54 foci were detected using particle analysis in Fiji. At least a total of 300 cells were counted from 3 repeated experiments. Values shown are mean±sem. Values were compared using Kruskal-Wallis test followed by Dunn's multiple comparison test. Quantification of data from F. RAD54 foci were detected using particle analysis in Fiji. Cells were counted as positive if they counted 8 or more RAD54 foci that colocalized with telomeres. Values shown are mean±sem of n=3 biological replicates. Values were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.


Combined IF FISH for RAD51 ( IF ) telomeres ( FISH ) in SaOS2 or HuO9 . were transfected with 20nM siRAD54 # 2 for 48 hours immediately prior to . Scale bars = 10 µm of A . A was counted as positive it is contained at least 1 colocalization event between RAD51 telomeres . At least 100 were counted per condition per repeat , n = 3 . Values shown are mean ± sd . Data were compared using standard two - way ANOVA followed by Sidak ' s multiple comparison .

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

Combined IF and DNA FISH for RAD51 (IF) and telomeres (FISH) in SaOS2 or HuO9 cells. Cells were transfected with 20nM siRAD54#2 for 48 hours immediately prior to staining. Scale bars = 10 µm Quantification of A. A cell was counted as positive it is contained at least 1 colocalization event between RAD51 and telomeres. At least 100 cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.


ChIP for telomeric associated with RAD51 . dot blot probed with DIG labeled telomere probe or Alu probe . HuO9 were transfected with 20nM siRAD54 # 2 , or 15µg myc - TRF2 for 72 hours prior to immunoprecipitation with RAD51 antibody ( Mock , siRAD54 conditions ) or myc antibody ( myc - TRF2 positive control ) . of ( CCCTAA ) 4 dot blot performed in C . IP values were normalized to loading ( IP / input ) then normalized to mock control . Values shown are mean ± sd of n = 3 biological replicates . Values were compared using two - unpaired student ' s t - .

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

ChIP for telomeric DNA associated with RAD51. DNA dot blot probed with DIG labeled telomere probe or Alu probe. HuO9 cells were transfected with 20nM siRAD54 #2, or 15µg myc-TRF2 for 72 hours prior to immunoprecipitation with RAD51 antibody (Mock, siRAD54 conditions) or myc antibody (myc-TRF2 positive control). Quantification of (CCCTAA)4 dot blot performed in C. IP values were normalized to loading (IP/input) and then normalized to mock control. Values shown are mean±sd of n=3 biological replicates. Values were compared using two-tailed unpaired student's t-test.


Western blot of samples used for ChIP in C - D .

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Western blot of samples used for ChIP in C-D.