Figure 6 ( 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 ) 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 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 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 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 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 ) .

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Figure 6(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) 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 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 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 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 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).


Figure 1A 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 .

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Figure 1A 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.


Figure 2The 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 . 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 . 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 . 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 .

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Figure 2The 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.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.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.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.


Figure 3Nuclease 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 .

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Figure 3Nuclease 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.


Figure 4In vivo resection assay using BrdU staining under native conditions to directly detect ssDNA at sites of DSBs . Shown are representative images . In vivo resection assay using RPA2 staining as a marker for ssDNA present at sites of DSBs . Shown are representative images . 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 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 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 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 ) .

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Figure 4In vivo resection assay using BrdU staining under native conditions to directly detect ssDNA at sites of DSBs. Shown are representative images.In vivo resection assay using RPA2 staining as a marker for ssDNA present at sites of DSBs. Shown are representative images.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 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 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 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).


Figure 5Nuclease 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 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 , SEM . 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 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 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 , SEM .

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Figure 5Nuclease 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 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, SEM.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 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 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, SEM.


Figure 6Interaction 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 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 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 .

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Figure 6Interaction 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 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 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.


Figure 7Representative 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 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 , SEM . Quantitation of experiments such as shown in ( B ) , with and without pCtIP . Averages shown ; n = 3 ; error bars , SEM .

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Figure 7Representative 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 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, SEM.Quantitation of experiments such as shown in (B), with and without pCtIP. Averages shown; n = 3; error bars, SEM.


Figure 1Population doublings ( PDs ) determined in BJ cells cultured with and without folate for 28 days . 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 DNA molecules from control cells and cells grown for 7 days in a folate - free medium . 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 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 ) .

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Figure 1Population doublings (PDs) determined in BJ cells cultured with and without folate for 28 days.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 DNA molecules from control cells and cells grown for 7 days in a folate-free medium.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 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).


Figure 2A Population doublings ( PDs ) determined in BJ cells cultured at the indicated folate concentrations for 48 days . 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 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 .

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

Figure 2A Population doublings (PDs) determined in BJ cells cultured at the indicated folate concentrations for 48 days.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 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.


Figure 3Cyclin 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 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 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 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 .

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

Figure 3Cyclin 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 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 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 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.


Figure 4Cyclin 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 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 .

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

Figure 4Cyclin 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 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.


Figure 5A , 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 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 .

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

Figure 5A, 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 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.


Figure 1GFP - 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 ) 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 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 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 .

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

Figure 1GFP-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) 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 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 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.


Figure 2 ( 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 . 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 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 . The ratio of free GFP to loading control , normalized to the 16h pre - treated sample ( set to 1 ) is provided below each band .

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

Figure 2(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. 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 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. The ratio of free GFP to loading control, normalized to the 16h pre-treated sample (set to 1) is provided below each band.


Figure 3 ( 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 . 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 . 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 .

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

Figure 3(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.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.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.


Figure 4 ( 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 )

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

Figure 4(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)


Figure 5 ( 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 ) 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 .

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

Figure 5(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) 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.


Figure 6 ( 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 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 ) .

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

Figure 6(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 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).


Figure 1A . 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 + / + and Mpdz - / - mice ( n = 8 ÷ 12 , mean ± SD ) . 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 the indicated brain sections ( n = 4 , mean ± SD ) .

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

Figure 1A. 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+/+ and Mpdz-/- mice (n=8÷12, mean ± SD).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 the indicated brain sections (n=4, mean ± SD).


Figure 2A . 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 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 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 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 .

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

Figure 2A. 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 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 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 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.


Figure 3A . 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 + / + 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 .

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

Figure 3A. 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+/+ 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.


Figure 4A . 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 ) 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 ) . 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 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 ) .

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

Figure 4A. 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) 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). 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 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).


Figure 5A . 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 images of tight junctions proximal to the apical faces of CPECs in Mpdz + / + and Mpdz - / - mice . Scale bar , 100 nmD . 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 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 .

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

Figure 5A. 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 images of tight junctions proximal to the apical faces of CPECs in Mpdz+/+ and Mpdz-/- mice. Scale bar, 100 nmD. 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 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.


Figure 6A . 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 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 ) .

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

Figure 6A. 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 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).


Figure 7A . 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 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 . 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 ) .

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

Figure 7A. 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 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. 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).


Figure 8A . Mean protein concentration in the CSF of Mpdz + / + and Mpdz - / - P14 - P21 mice ( mean ± SD , n = 2 ÷ 3 ) . 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 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 . 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 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 ) .

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

Figure 8A. Mean protein concentration in the CSF of Mpdz+/+ and Mpdz-/- P14-P21 mice (mean ± SD, n=2÷3).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 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. 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 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).


Figure 1 ( A ) Immunoblots of mitochondrial fractions from siRNA ‐ transfected HEK293T cells ± CCCP . ( B ) Immunoblots against indicated proteases in siRNA ‐ transfected cells . ( 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 .

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Figure 1(A) Immunoblots of mitochondrial fractions from siRNA‐transfected HEK293T cells±CCCP.(B) Immunoblots against indicated proteases in siRNA‐transfected cells.(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.


Figure 2 ( 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 ratio from images as in A . N = 3 ; * P0 . 05 compared with NT siRNA . ( C ) Mitochondrial interconnectivity from images as in A , N = 3 . ( 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 .

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Figure 2(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 ratio from images as in A. N=3; *P0.05 compared with NT siRNA.(C) Mitochondrial interconnectivity from images as in A, N=3.(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.


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

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


Figure 4 ( 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 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 .

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Figure 4(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 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.


Figure 5 ( 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 ) 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 cells ± CCCP . ( 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 .

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Figure 5(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) 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 cells ±CCCP.(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.


Figure 1 ( 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 .

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Figure 1(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.


Figure 2 ( 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 ) .

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Figure 2(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).


Figure 4 ( 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 .

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Figure 4(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.


Figure 7 ( 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 .

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Figure 7(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.


Figure 8 ( 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 ) .

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Figure 8(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).


Figure 1A 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 and DNA FISH analysis of RAD54 ( IF ) , PML ( IF ) and telomeres ( FISH ) in ALT cell lines SaOS2 and HuO9 . Scale bars = 10 µmWestern 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 hrsCombined 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 .

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Figure 1A 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 and DNA FISH analysis of RAD54 (IF), PML (IF) and telomeres (FISH) in ALT cell lines SaOS2 and HuO9. Scale bars = 10 µmWestern 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 hrsCombined 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.


Figure 2Combined 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 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 . Combined IF and DNA FISH for GFP ( IF ) and telomeres ( FISH ) in SaOS2 and HuO9 cells . Cells were forward transfected with Polη - GFP , and then after 24 hours transfected with 20nM siRAD54 # 2 . Cells were stained for GFP and telomeres 48 hours after siRNA transfection . Scale bars = 10 µm Quantification of data in F . GFP negative cells were excluded from analysis . Cells were considered positive if they showed 1 or more colocalization events between GFP ( Polη ) and telomeres . At least 50 GFP positive cells were counted per condition per repeat , n = 3 . Values shown are mean ± sd . Values were compared using standard two - way ANOVA followed by Sidak ' s multiple comparison test .

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Figure 2Combined 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 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.Combined IF and DNA FISH for GFP (IF) and telomeres (FISH) in SaOS2 and HuO9 cells. Cells were forward transfected with Polη-GFP, and then after 24 hours transfected with 20nM siRAD54#2. Cells were stained for GFP and telomeres 48 hours after siRNA transfection. Scale bars = 10 µm Quantification of data in F. GFP negative cells were excluded from analysis. Cells were considered positive if they showed 1 or more colocalization events between GFP (Polη) and telomeres. At least 50 GFP positive cells were counted per condition per repeat, n=3. Values shown are mean±sd. Values were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.