C ) IAV infection following acid bypass is comparable between control and δWD MEFs . IAV was bound on ice for 1 h , and warmed at either neutral pH ( 6 . 8 ) or low pH ( pH 5 . 0 ) for 2 min , followed by incubation in STOP medium containing NH4Cl for 18 hours . Cells were fixed and stained for NP and Hoechst , imaged by automated confocal microscopy and maximum intensity projection images analysed to quantify viral infectivity . The results from N = 3 independent experiments ( 5 , 000 - 10 , 000 cells quantified per experiment ) , and the means are shown . Scale bar ; 500 µm .

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C) IAV infection following acid bypass is comparable between control and δWD MEFs. IAV was bound on ice for 1 h, and warmed at either neutral pH (6.8) or low pH (pH 5.0) for 2 min, followed by incubation in STOP medium containing NH4Cl for 18 hours. Cells were fixed and stained for NP and Hoechst, imaged by automated confocal microscopy and maximum intensity projection images analysed to quantify viral infectivity. The results from N=3 independent experiments (5,000-10,000 cells quantified per experiment), and the means are shown. Scale bar; 500 µm.


D ) . IAV fusion with endosomes is increased in δWD MEFs . Cells were infected with dual labeled ( R18 / SP - DiOC18 ) IAV for 1 h , fixed , counterstained with Hoechst ( nuclei blue ) and WGA - AF647 ( cell perimeter in white ) . Cells were imaged by automated confocal microscopy and the number of fusion events per cell ( represented by number of SP - DiOC18 puncta ) was quantified . Fused and non - fused viral particles are shown as green and red spots respectively . The results and means ( horizontal lines ) of n = 3 independent experiments ( 30 - 60 cells per experiment ) are shown . Representative cells overlayed with the cell boundary segmented from the WGA staining are shown on the right .

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D). IAV fusion with endosomes is increased in δWD MEFs. Cells were infected with dual labeled (R18/SP-DiOC18) IAV for 1 h, fixed, counterstained with Hoechst (nuclei blue) and WGA-AF647 (cell perimeter in white). Cells were imaged by automated confocal microscopy and the number of fusion events per cell (represented by number of SP-DiOC18 puncta) was quantified. Fused and non-fused viral particles are shown as green and red spots respectively. The results and means (horizontal lines) of n = 3 independent experiments (30-60 cells per experiment) are shown. Representative cells overlayed with the cell boundary segmented from the WGA staining are shown on the right.


E , F ) Numbers of fusion positive cells is increased for δWD MEFs . MEFs from δWD ( white circles ) or littermate control mice ( black circles ) were incubated with dual - labelled ( SP - DiOC18 / R18 ) IAV at 40C for 45 min and warmed to 37 ° C for 30 ad 60 minutes . Cells were harvested by trypsinisation , fixed in PFA and analysed by flow cytometry to determine to determine percentage of cells positive for fusion ( E ) and median fluorescence intensity ( MFI ) of dequenched SP - DiOC18 signa ( F ) . Graphs show individual replicates ( n = 3 ) with a horizontal line at the mean and were compared using two - way ANOVA with Bonferroni post - tests * * P < 0 . 01 , * * * P < 0 . 001 ) .

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E, F) Numbers of fusion positive cells is increased for δWD MEFs. MEFs from δWD (white circles) or littermate control mice (black circles) were incubated with dual-labelled (SP-DiOC18/R18) IAV at 40C for 45 min and warmed to 37°C for 30 ad 60 minutes. Cells were harvested by trypsinisation, fixed in PFA and analysed by flow cytometry to determine to determine percentage of cells positive for fusion (E) and median fluorescence intensity (MFI) of dequenched SP-DiOC18 signa (F). Graphs show individual replicates (n = 3) with a horizontal line at the mean and were compared using two-way ANOVA with Bonferroni post-tests **P<0.01, ***P<0.001).


( G , H ) Endocytosis of IAV increases in δWD MEFs . MEFs from δWD or littermate control mice were incubated with AlexFluor 488 - labelled IAV at 40C for 1hr and warmed to 37 ° C for 15 Cells were fixed and permeabilised and stained with antibody against HA ( red ) . The blue line indicates the plasma membrane of the cell . Green arrows indicate examples of viruses only labelled with green fluorescence . Double - labelled virus particles ( green - red ) represent non - endocytosed particles bound at the cell surface , whereas green particles represent endocytosed particles . The results and means of n = 3 independent experiments ( 50 - 100 cells per experiment ) are shown .

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(G, H) Endocytosis of IAV increases in δWD MEFs. MEFs from δWD or littermate control mice were incubated with AlexFluor 488-labelled IAV at 40C for 1hr and warmed to 37°C for 15 Cells were fixed and permeabilised and stained with antibody against HA (red). The blue line indicates the plasma membrane of the cell. Green arrows indicate examples of viruses only labelled with green fluorescence. Double-labelled virus particles (green-red) represent non-endocytosed particles bound at the cell surface, whereas green particles represent endocytosed particles. The results and means of n = 3 independent experiments (50-100 cells per experiment) are shown.


( I , J ) Interferon signalling increases in δWD MEFs . MEFs from δWD or littermate control mice were infected with IAV . At the indicated time points , mRNA transcripts were evaluated by qPCR for IFIT1 ( I ) and ISG15 ( J ) .

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(I, J) Interferon signalling increases in δWD MEFs. MEFs from δWD or littermate control mice were infected with IAV. At the indicated time points, mRNA transcripts were evaluated by qPCR for IFIT1 (I) and ISG15 (J).


( A ) Western blot analysis showing the expression of the WRNIP1 protein in wild - type cells ( shWRNIP1WT ) and WRNIP1 - deficient ( shWRNIP1 ) or mutant ( shWRNIP1T294A ) cells . MRC5SV fibroblasts were used as a positive control . The membrane was probed with an anti - FLAG or anti - WRNIP1 . GAPDH was used as a loading control . Below each lane of the blot the ratio of WRNIP1 protein to total protein , then normalised to MRC5SV , is reported .

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

(A) Western blot analysis showing the expression of the WRNIP1 protein in wild-type cells (shWRNIP1WT) and WRNIP1-deficient (shWRNIP1) or mutant (shWRNIP1T294A) cells. MRC5SV fibroblasts were used as a positive control. The membrane was probed with an anti-FLAG or anti-WRNIP1. GAPDH was used as a loading control. Below each lane of the blot the ratio of WRNIP1 protein to total protein, then normalised to MRC5SV, is reported.


( B ) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT , shWRNIP1 and shWRNIP1T294A cells . Cells were pulse - labelled with CldU , and then subjected to a pulse - labelling with IdU . ( C ) Analysis of replication fork velocity ( fork speed ) in the cells under unperturbed conditions . The length of the green tracks were measured . Mean values are represented as horizontal black lines ( ns , not significant ; Student ' s t - test ) .

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(B) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were pulse-labelled with CldU, and then subjected to a pulse-labelling with IdU.(C) Analysis of replication fork velocity (fork speed) in the cells under unperturbed conditions. The length of the green tracks were measured. Mean values are represented as horizontal black lines (ns, not significant; Student's t-test).


( D ) Cells were treated as in ( B ) . For each replication origin , the length of the right - fork signal was measured and plotted against the length of the left - fork signal . A schematic representation of symmetric and asymmetric forks is given . If the ratio between the left - fork length and the right - fork length deviated by more than 33 % from 1 ( that is , outside the violet dashed lines in the graphs ) , the fork was considered asymmetric . The percentage of asymmetric forks was calculated for all cell lines . N = number of forks counted for each cell line . R represents linear correlation coefficient .

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(D) Cells were treated as in (B). For each replication origin, the length of the right-fork signal was measured and plotted against the length of the left-fork signal. A schematic representation of symmetric and asymmetric forks is given. If the ratio between the left-fork length and the right-fork length deviated by more than 33% from 1 (that is, outside the violet dashed lines in the graphs), the fork was considered asymmetric. The percentage of asymmetric forks was calculated for all cell lines. N= number of forks counted for each cell line. R represents linear correlation coefficient.


( E ) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT , shWRNIP1 and shWRNIP1T294A cells . Cells were pulse - labelled with CldU , treated with 4mM HU and then subjected to a pulse - labelling with IdU . ( F ) Graphs show the percentage of red ( CldU ) tracts ( stalled forks ) or red - green ( CldU - IdU ) contiguous tracts ( restarting forks ) in the cells . Mean shown , n = 3 . Error bars represent standard error . ( * , p < 0 . 05 ; * * , p < 0 . 01 ; Student ' s t test ) . Representative DNA fiber images are shown . Scale bars , 10 µm .

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(E) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were pulse-labelled with CldU, treated with 4mM HU and then subjected to a pulse-labelling with IdU.(F) Graphs show the percentage of red (CldU) tracts (stalled forks) or red-green (CldU-IdU) contiguous tracts (restarting forks) in the cells. Mean shown, n = 3. Error bars represent standard error. (*, p < 0.05; **, p < 0.01; Student's t test). Representative DNA fiber images are shown. Scale bars, 10 µm.


( G ) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT , shWRNIP1 and shWRNIP1T294A cells . Cells were sequentially pulse - labelled with CldU and IdU as indicated , then treated or not with 4 mM HU . ( H ) Representative IdU tract length distributions in all cell lines under unperturbed conditions ( left graph ) or after HU treatment ( right graph ) . Median tract lengths are given in parentheses . See also Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are shown . Scale bars , 10 µm .

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(G) Experimental scheme of dual labelling of DNA fibers in shWRNIP1WT, shWRNIP1 and shWRNIP1T294A cells. Cells were sequentially pulse-labelled with CldU and IdU as indicated, then treated or not with 4 mM HU.(H) Representative IdU tract length distributions in all cell lines under unperturbed conditions (left graph) or after HU treatment (right graph). Median tract lengths are given in parentheses. See also Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are shown. Scale bars, 10 µm.


( A ) Experimental scheme of dual labelling of DNA fibers in wild - type cells ( shWRNIP1WT ) or WRNIP1 - deficient cells ( shWRNIP1 ) . Cells were sequentially pulse - labelled with CldU and IdU as indicated , then left untreated or treated with 4 mM HU in combination or not with 50 µM Mirin . ( B ) Representative IdU tract length distributions in shWRNIP1WT ( left graph ) or shWRNIP1 cells ( right graph ) after treatment . Median tract lengths are given in parentheses . See Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are shown . Scale bars , 10 µm .

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

(A) Experimental scheme of dual labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were sequentially pulse-labelled with CldU and IdU as indicated, then left untreated or treated with 4 mM HU in combination or not with 50 µM Mirin.(B) Representative IdU tract length distributions in shWRNIP1WT (left graph) or shWRNIP1 cells (right graph) after treatment. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are shown. Scale bars, 10 µm.


( A ) Evaluation of ssDNA accumulation at parental - strand by immunofluorescence microscopy analysis in wild - type ( shWRNIP1WT ) or WRNIP1 - deficient ( shWRNIP1 ) cells . Experimental design of ssDNA assay is shown . Cells were labelled with IdU for 24 h , as indicated , washed and left to recover for 2 h , then treated or not with 4 mM HU . In parallel samples , the MRE11 activity is chemically inhibited with 50 µM Mirin , alone or in combination with HU - induced replication stress . After treatment , cells were fixed and stained with an anti - IdU antibody without denaturing the DNA to specifically detect parental ssDNA . Horizontal black lines represent the mean SE ; n = 3 . Error bars represent standard error ( ns , not significant ; * * , p < 0 . 01 ; * * * * , p < 0 . 0001 ; two - tailed Student ' s t test ) . Representative images are shown . DNA was counterstained with DAPI ( blue ) .

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(A) Evaluation of ssDNA accumulation at parental-strand by immunofluorescence microscopy analysis in wild-type (shWRNIP1WT) or WRNIP1-deficient (shWRNIP1) cells. Experimental design of ssDNA assay is shown. Cells were labelled with IdU for 24 h, as indicated, washed and left to recover for 2 h, then treated or not with 4 mM HU. In parallel samples, the MRE11 activity is chemically inhibited with 50 µM Mirin, alone or in combination with HU-induced replication stress. After treatment, cells were fixed and stained with an anti-IdU antibody without denaturing the DNA to specifically detect parental ssDNA. Horizontal black lines represent the mean SE; n = 3. Error bars represent standard error (ns, not significant; **, p<0.01; ****, p<0.0001; two-tailed Student's t test). Representative images are shown. DNA was counterstained with DAPI (blue).


( B ) Analysis of chromatin binding of MRE11 and RAD51 in shWRNIP1WT and shWRNIP1 cells . Chromatin fractions of cells , treated or not with 4 mM HU , were analysed by immunoblotting . The membrane was probed with the anti - WRNIP1 , anti - MRE11 and anti - RAD51 antibodies . LAMIN B1 was used as a loading for the chromatin fraction . Total amount of RAD51 and MRE11 ( Input ) in the cells was determined with the relevant antibodies . LAMIN B1 was used as a loading control . In the graph , the fold increase respect to the wild - type untreated of the normalized ratio of the chromatin - bound RAD51 ( or MRE11 ) / total RAD51 ( or MRE11 ) is reported for each cell line .

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(B) Analysis of chromatin binding of MRE11 and RAD51 in shWRNIP1WT and shWRNIP1 cells. Chromatin fractions of cells, treated or not with 4 mM HU, were analysed by immunoblotting. The membrane was probed with the anti-WRNIP1, anti-MRE11 and anti-RAD51 antibodies. LAMIN B1 was used as a loading for the chromatin fraction. Total amount of RAD51 and MRE11 (Input) in the cells was determined with the relevant antibodies. LAMIN B1 was used as a loading control. In the graph, the fold increase respect to the wild-type untreated of the normalized ratio of the chromatin-bound RAD51 (or MRE11)/ total RAD51 (or MRE11) is reported for each cell line.


( C ) Analysis of DNA - protein interactions between ssDNA and endogenous RAD51 in shWRNIP1WT and shWRNIP1 cells by in situ PLA assay . Experimental designed used for the assay is given . Cells were labelled with IdU for 24 h , as indicated , washed and left to recover for 2 h , then treated or not with 4 mM HU for 4 h . Next , cells were fixed , stained with an anti - IdU antibody without denaturing the DNA to specifically detect parental - strand ssDNA , and subjected to PLA assay as described in the " Experimental procedures " section . Antibodies raised against IdU or RAD51 were used to reveal ssDNA or endogenous RAD51 respectively . Each red spot represents a single interaction between ssDNA and RAD51 . No spot has been revealed in cells stained with each single antibody ( negative control ) . DNA was counterstained with DAPI ( blue ) . Representative images of the PLA assay are given . Graph shows the number of PLA spots per cell . Horizontal black lines represent the mean value ( ns , not significant ; * * , p < 0 . 01 ; two - tailed Student ' s t test ) ; n = 3 .

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

(C) Analysis of DNA-protein interactions between ssDNA and endogenous RAD51 in shWRNIP1WT and shWRNIP1 cells by in situ PLA assay. Experimental designed used for the assay is given. Cells were labelled with IdU for 24 h, as indicated, washed and left to recover for 2 h, then treated or not with 4 mM HU for 4 h. Next, cells were fixed, stained with an anti-IdU antibody without denaturing the DNA to specifically detect parental-strand ssDNA, and subjected to PLA assay as described in the "Experimental procedures" section. Antibodies raised against IdU or RAD51 were used to reveal ssDNA or endogenous RAD51 respectively. Each red spot represents a single interaction between ssDNA and RAD51. No spot has been revealed in cells stained with each single antibody (negative control). DNA was counterstained with DAPI (blue). Representative images of the PLA assay are given. Graph shows the number of PLA spots per cell. Horizontal black lines represent the mean value (ns, not significant; **, p < 0.01; two-tailed Student's t test); n = 3.


( D ) Localization of WRNIP1 , MRE11 and RAD51 to stalled replication forks . Forks were isolated by CldU - co - immunoprecipitation ( CldU - IP ) . shWRNIP1WT or shWRNIP1 cells were pulse - labelled with CldU , then fixed or treated with HU . Cells were cross - linked , and the nuclear extracts were isolated ( Input ) and subjected to CldU - IP using an anti - CldU antibody ( CldU - IP ) . The membranes were probed with the anti - WRNIP1 or anti - RAD51 antibodies . After stripping , the membranes were probed with an anti - MRE11 antibody . LAMIN B1 and GAPDH were used as loading controls ( Input ) . Ponceau S was used as a loading control of CldU - IP . Dot blot analysis was performed to confirm that equal amounts of immunoprecipitatedDNA from each sample . 10 % of each IP was loaded on a nitrocellulose membrane . The membrane was probed with an anti - CldU antibody . The graph shows the normalized ratio of the proteins co - immunoprecipitated with CldU ( CldUCo - IP proteins ) / the total of labelled DNAimmunoprecipitated with CldU ( CldUIP ) for each cell line after replication stress from two independent experiments . The dots in the graph represent the individual data points from each single experiment . Horizontal black line represents the mean value from two replicates ; n = 2 .

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

(D) Localization of WRNIP1, MRE11 and RAD51 to stalled replication forks. Forks were isolated by CldU-co-immunoprecipitation (CldU-IP). shWRNIP1WT or shWRNIP1 cells were pulse-labelled with CldU, then fixed or treated with HU. Cells were cross-linked, and the nuclear extracts were isolated (Input) and subjected to CldU-IP using an anti-CldU antibody (CldU-IP). The membranes were probed with the anti-WRNIP1 or anti-RAD51 antibodies. After stripping, the membranes were probed with an anti-MRE11 antibody. LAMIN B1 and GAPDH were used as loading controls (Input). Ponceau S was used as a loading control of CldU-IP. Dot blot analysis was performed to confirm that equal amounts of immunoprecipitatedDNA from each sample. 10% of each IP was loaded on a nitrocellulose membrane. The membrane was probed with an anti-CldU antibody. The graph shows the normalized ratio of the proteins co-immunoprecipitated with CldU (CldUCo-IP proteins)/ the total of labelled DNAimmunoprecipitated with CldU (CldUIP) for each cell line after replication stress from two independent experiments. The dots in the graph represent the individual data points from each single experiment. Horizontal black line represents the mean value from two replicates; n = 2.


( A ) Experimental scheme of pulse - labelling of DNA fibers in wild - type cells ( shWRNIP1WT ) or WRNIP1 - deficient cells ( shWRNIP1 ) . Cells were labelled with IdU and exposed or not to 25 μM RAD51 inhibitor , then treated or not with 4 mM HU . ( B ) Representative IdU tract length distributions in shWRNIP1WT cells ( left graph ) or shWRNIP1 cells ( right graph ) . Median tract lengths are reported in parentheses . See Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are reported . Scale bars , 10 µm .

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(A) Experimental scheme of pulse-labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were labelled with IdU and exposed or not to 25 μM RAD51 inhibitor, then treated or not with 4 mM HU.(B) Representative IdU tract length distributions in shWRNIP1WT cells (left graph) or shWRNIP1 cells (right graph). Median tract lengths are reported in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are reported. Scale bars, 10 µm.


( C ) Scheme of DNA fiber tract analysis in shWRNIP1 cells . Cells were transfected with an empty vector or a plasmid expressing a wild - type human RAD51 , and 48 h thereafter labelled with IdU and treated or not with 4 mM HU . ( D ) Representative IdU tract length distributions in shWRNIP1 cells or shWRNIP1 cells expressing exogenous wild - type RAD51 after HU exposure . Median tract lengths are given in parentheses . See Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are given . Scale bars , 10 µm . Western blot shows the expression of the RAD51 protein in shWRNIP1 cells . The membrane was probed with an anti - RAD51 . LAMIN B1 was used as a loading control .

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

(C) Scheme of DNA fiber tract analysis in shWRNIP1 cells. Cells were transfected with an empty vector or a plasmid expressing a wild-type human RAD51, and 48 h thereafter labelled with IdU and treated or not with 4 mM HU.(D) Representative IdU tract length distributions in shWRNIP1 cells or shWRNIP1 cells expressing exogenous wild-type RAD51 after HU exposure. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are given. Scale bars, 10 µm. Western blot shows the expression of the RAD51 protein in shWRNIP1 cells. The membrane was probed with an anti-RAD51. LAMIN B1 was used as a loading control.


( A ) Co - immunoprecipitation experiments in HEK293T cells transfected with empty vector or FLAG - WRNIP1 plasmid . Cells were treated or not with HU . After treatment , cell lysates were immunoprecipitated ( FLAG - IP ) using anti - FLAG antibody . The presence of WRNIP1 , BRCA2 and RAD51 was assessed by immunoblotting using the anti - FLAG , anti - RAD51 and anti - BRCA2 antibodies , respectively . Whole cell extracts were analysed ( Input ) . The membrane was probed with the same antibodies used for IP . GAPDH was used as a loading control .

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

(A) Co-immunoprecipitation experiments in HEK293T cells transfected with empty vector or FLAG-WRNIP1 plasmid. Cells were treated or not with HU. After treatment, cell lysates were immunoprecipitated (FLAG-IP) using anti-FLAG antibody. The presence of WRNIP1, BRCA2 and RAD51 was assessed by immunoblotting using the anti-FLAG, anti-RAD51 and anti-BRCA2 antibodies, respectively. Whole cell extracts were analysed (Input). The membrane was probed with the same antibodies used for IP. GAPDH was used as a loading control.


( B ) Analysis of protein - protein interactions between WRNIP1 and endogenous RAD51 in wild - type ( shWRNIP1WT ) or WRNIP1 - mutant ( shWRNIP1T294A ) cells by in situ PLA assay . Cells were labelled with IdU for 24 h , washed and left to recover for 2 h , then treated or not with 4 mM HU . Antibodies raised against FLAG - Tag and RAD51 were used to reveal FLAG - WRNIP1 or endogenous RAD51 respectively . Each red spot represents a single interaction between WRNIP1 and RAD51 . No spot has been revealed in cells stained with each single antibody ( negative control ) . DNA was counterstained with DAPI ( blue ) . Representative images of the PLA assay are shown . Graph shows the mean number of PLA spots per cell SE . Error bars represent standard error ( ns , not significant ; two - tailed Student ' s t test ) ; n = 3 .

O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Analysis of protein-protein interactions between WRNIP1 and endogenous RAD51 in wild-type (shWRNIP1WT) or WRNIP1-mutant (shWRNIP1T294A) cells by in situ PLA assay. Cells were labelled with IdU for 24 h, washed and left to recover for 2 h, then treated or not with 4 mM HU. Antibodies raised against FLAG-Tag and RAD51 were used to reveal FLAG-WRNIP1 or endogenous RAD51 respectively. Each red spot represents a single interaction between WRNIP1 and RAD51. No spot has been revealed in cells stained with each single antibody (negative control). DNA was counterstained with DAPI (blue). Representative images of the PLA assay are shown. Graph shows the mean number of PLA spots per cell SE. Error bars represent standard error (ns, not significant; two-tailed Student's t test); n = 3.


( C ) Experimental scheme of pulse - labelling of DNA fibers in wild - type cells ( shWRNIP1WT ) or WRNIP1 - deficient cells ( shWRNIP1 ) . Cells were transfected with BRCA2 siRNA ( siBRCA2 ) , and 48 h thereafter labelled with IdU , then treated or not with 4 mM HU . ( D ) Representative IdU tract length distributions in shWRNIP1WT / siBRCA2 or shWRNIP1siBRCA2 cells treated or not with HU . Median tract lengths are given in parentheses . See Tables S1 and S2 for details on the data sets and statistical test . Representative DNA fiber images are reported . Scale bars , 10 µm . Western blot shows BRCA2 depletion in shWRNIP1WT and shWRNIP1 cells . The membrane was probed with an anti - BRCA2 or anti - WRNIP1 . GAPDH was used as a loading control .

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

(C) Experimental scheme of pulse-labelling of DNA fibers in wild-type cells (shWRNIP1WT) or WRNIP1-deficient cells (shWRNIP1). Cells were transfected with BRCA2 siRNA (siBRCA2), and 48 h thereafter labelled with IdU, then treated or not with 4 mM HU.(D) Representative IdU tract length distributions in shWRNIP1WT/siBRCA2 or shWRNIP1siBRCA2 cells treated or not with HU. Median tract lengths are given in parentheses. See Tables S1 and S2 for details on the data sets and statistical test. Representative DNA fiber images are reported. Scale bars, 10 µm. Western blot shows BRCA2 depletion in shWRNIP1WT and shWRNIP1 cells. The membrane was probed with an anti-BRCA2 or anti-WRNIP1. GAPDH was used as a loading control.


( 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 .

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

(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 ) 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 .

O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O 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 ) 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 .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(C) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(E) 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 ) .

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

(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 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 .

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

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.


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 .

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

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.


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 .

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

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 .

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

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 .

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

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

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

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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 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 .

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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 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 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 .

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

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 ) .

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

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 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 .

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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 and 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 , SEM .

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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 ) . The proteins were separated on a 10 % polyacrylamide gel and 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 and pCtIP ( mock - treated ) or λCtIP . Averages shown ; n = 3 ; error bars , SEM . Statistical denotations , * * ( p < 0 . 01 ) , two - tailed t - test .

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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 complex instead of MRN . Averages shown ; n = 3 ; error bars , SEM . Statistical denotations , ns ( p > 0 . 05 ) , 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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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 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 .

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

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 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 .

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

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 .

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

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

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 the top oligonucleotide ( 20 nt away from the 3 ' - end , see cartoon ) .

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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 .

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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 and without pCtIP . Averages shown ; n = 3 ; error bars , SEM .

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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 cells cultured with and without folate for 28 days .

Population 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 .

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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 .

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 ) .

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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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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 cells cultured at the indicated folate concentrations for 48 days .

A 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 .

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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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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 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 ) .

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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 .

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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 .

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

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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 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 .

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

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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 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 .

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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 intensity projection images 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 ) 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 .

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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. (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 .

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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. (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 .

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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. (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 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 .

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(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 .

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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 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 .

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(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 .

(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 proteins that accumulate upon ABA / NAA treatment and 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.


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 .

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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.


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 .

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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 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 )

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(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 images of root explants from WT , atg2 - 2 and 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 ) 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 .

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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 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

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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 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 ) .

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

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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 the 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 , 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 .

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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 and total brain volumes of Mpdz + / + and 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 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 .

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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 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 .

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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 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 .

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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 + / + 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 .

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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 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 .

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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 ) The images are representative of 2 Mpdz + / + mice and 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 ) . The images are representative of 2 Mpdz + / + mice and 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.