( 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 ) .

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

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

(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 .

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

(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 .

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


( G ) 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 .

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(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 .

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


( B ) 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 .

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


( C ) Evaluation of 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 .

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


( D ) Experimental scheme for evaluation of 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 .

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


( E ) Experimental scheme of 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 .

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


( F ) Experimental design of 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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(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).


SILAC - based proteomics data identify known ( red ) & novel ( green ) hypoxia - induced proteins in SW620 cells . One sample T - test was performed .

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SILAC-based proteomics data identify known (red) & novel (green) hypoxia-induced proteins in SW620 cells. One sample T-test was performed.


Western blotting confirmed GPRC5A as a hypoxia - induced protein in SILAC lysates .

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Western blotting confirmed GPRC5A as a hypoxia-induced protein in SILAC lysates.


Validation of GPRC5A western blot data using siRNA . * Non - specific band of ~ 60kDa not depleted by GPRC5A siRNA .

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Validation of GPRC5A western blot data using siRNA. *Non-specific band of ~60kDa not depleted by GPRC5A siRNA.


Confocal microscopy showing plasma membrane GPRC5A expression in hypoxic SW620 cells ( scale bars : 75um ) .

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Confocal microscopy showing plasma membrane GPRC5A expression in hypoxic SW620 cells (scale bars: 75um).


Western blotting showing GPRC5A upregulation by hypoxia in a panel of colorectal tumour cell lines .

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Western blotting showing GPRC5A upregulation by hypoxia in a panel of colorectal tumour cell lines.


Basal & hypoxia - induced GPRC5A protein expression was decreased by HIF - 1 / 2α depletion .

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Basal & hypoxia-induced GPRC5A protein expression was decreased by HIF-1/2α depletion.


Depletion of HIF - 1β decreased GPRC5A protein upregulation in hypoxia .

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Depletion of HIF-1β decreased GPRC5A protein upregulation in hypoxia.


Hypoxia mimetic DMOG induced HIF - 1 / 2α , CA9 & GPRC5A protein expression . Dual HIF - 1 / 2α depletion reduced GPRC5A induction by DMOG .

Hypoxia mimetic DMOG induced HIF-1/2α, CA9 & GPRC5A protein expression. Dual HIF-1/2α depletion reduced GPRC5A induction by DMOG.


qRT - PCR demonstrating that GPRC5A mRNA was upregulated by hypoxia . GPRC5A was normalised to HPRT ( error bars ± SD ) .

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qRT-PCR demonstrating that GPRC5A mRNA was upregulated by hypoxia. GPRC5A was normalised to HPRT (error bars ± SD).


qRT - PCR demonstrating that HIF - 1 / 2α depletion decreased GPRC5A induction during hypoxia . GPRC5A was normalised to HPRT ( error bars ± SD ) .

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qRT-PCR demonstrating that HIF-1/2α depletion decreased GPRC5A induction during hypoxia. GPRC5A was normalised to HPRT (error bars ± SD).


ChIP / PCR analyses identify HIF - 1α a binding to the GPRC5A promoter region containing a putative optimal HRE ( error bars ± SD ) .

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ChIP/PCR analyses identify HIF-1α a binding to the GPRC5A promoter region containing a putative optimal HRE (error bars ± SD).


Expression of CA9 in formalin fixed paraffin embedded hypoxic SW620 cells by IHC . Reduced CA9 expression with siRNA confirms antibody specificity ( scale bars : 200um ) .

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Expression of CA9 in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced CA9 expression with siRNA confirms antibody specificity (scale bars: 200um).


Expression of ; GPRC5A in formalin fixed paraffin embedded hypoxic SW620 cells by IHC . Reduced ; GPRC5A expression with siRNA confirms antibody specificity ( scale bars : 200um ) .

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Expression of ; GPRC5A in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced ; GPRC5A expression with siRNA confirms antibody specificity (scale bars: 200um).


IHC analysis of serial sections from human colorectal tissue from patients with mesenteric ischaemia ( strangulated colon ) . GPRC5A is co - expressed with CA9 in the colonic epithelial cells ( scale bars : 50um ) .

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IHC analysis of serial sections from human colorectal tissue from patients with mesenteric ischaemia (strangulated colon). GPRC5A is co-expressed with CA9 in the colonic epithelial cells (scale bars: 50um).


Quantitative RT - PCR analysis of mouse intestinal tissue . Gene expression was normalised to housekeeping gene Tbp . Raw data from three independent experiments ( n = 3 mice ) are shown ( error bars ± SEM ) .

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Quantitative RT-PCR analysis of mouse intestinal tissue. Gene expression was normalised to housekeeping gene Tbp. Raw data from three independent experiments (n=3 mice) are shown (error bars ± SEM).


Tg [ fli1 : eGFP ; vhl - / - ] & Tg [ fli1 : eGFP ] zebrafish embryos ( 5 days post - fertilisation ) demonstrate excessive angiogenesis & increased expression of HIF target genes ( scale bars : 100um ) .

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Tg[fli1:eGFP; vhl-/-] & Tg[fli1:eGFP] zebrafish embryos (5 days post-fertilisation) demonstrate excessive angiogenesis & increased expression of HIF target genes (scale bars: 100um).


gprc5ba was induced in vhl mutant zebrafish embryos & fli1 : eGFP zebrafish embryos exposed to 5 % O2 ( vs normoxia ) for 24 hours ( RT - PCR ) .

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gprc5ba was induced in vhl mutant zebrafish embryos & fli1:eGFP zebrafish embryos exposed to 5% O2 (vs normoxia) for 24 hours (RT-PCR).


Kaplan - Meier curve following analysis of transcriptomics dataset GSE24551 . Event - free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA .

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Kaplan-Meier curve following analysis of transcriptomics dataset GSE24551. Event-free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA.


GPRC5A depletion markedly increases caspase - 3 activation / PARP cleavage during hypoxia .

GPRC5A depletion markedly increases caspase-3 activation/PARP cleavage during hypoxia.


Three independent siRNA sequences targeting GPRC5A induce caspase - 3 activation / PARP cleavage during hypoxia .

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Three independent siRNA sequences targeting GPRC5A induce caspase-3 activation/PARP cleavage during hypoxia.


GPRC5A depletion reduces hypoxic cell growth / survival . Crystal violet cell assays show reduced cell growth / survival in GPRC5A depleted cells during hypoxia [ n = 3 ] .

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GPRC5A depletion reduces hypoxic cell growth/survival. Crystal violet cell assays show reduced cell growth/survival in GPRC5A depleted cells during hypoxia [n=3].


Expression of an siRNA - resistant GPRC5A cDNA rescues hypoxic GPRC5A - depleted cells from apoptosis . Upper : doxycycline - induced expression GPRC5Asi1R rescues increases caspase - 3 / PARP cleavage induced by GPRC5A depletion in hypoxia . Lower : generation of an siRNA1 resistant GPRC5A cDNA by synonymous mutations .

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Expression of an siRNA-resistant GPRC5A cDNA rescues hypoxic GPRC5A-depleted cells from apoptosis. Upper: doxycycline-induced expression GPRC5Asi1R rescues increases caspase-3/PARP cleavage induced by GPRC5A depletion in hypoxia. Lower: generation of an siRNA1 resistant GPRC5A cDNA by synonymous mutations.


Caspase inhibitor QVD prevents caspase - 3 activation / PARP cleavage by GPRC5A depletion in hypoxia .

Caspase inhibitor QVD prevents caspase-3 activation/PARP cleavage by GPRC5A depletion in hypoxia.


GPRC5A depletion in hypoxia induces apoptosis as determined by the violet ratiometric membrane asymmetry probe / dead cell apoptosis assay and flow

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GPRC5A depletion in hypoxia induces apoptosis as determined by the violet ratiometric membrane asymmetry probe/dead cell apoptosis assay and flow cytometry


Hypoxia induced YAP stabilisation via Ser397 dephosphorylated was abrogated in GPRC5A depleted cells .

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Hypoxia induced YAP stabilisation via Ser397 dephosphorylated was abrogated in GPRC5A depleted cells.


Hypoxia - induced nuclear localisation of YAP was attenuated in GPRC5A depleted cells .

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Hypoxia-induced nuclear localisation of YAP was attenuated in GPRC5A depleted cells.


Hypoxia activated TEAD activity ( 8x GTIIC - luc reporter ) but this was reduced by GPRC5A depletion . A representative triplicate experiment is shown ( n = 3 ) .

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Hypoxia activated TEAD activity (8x GTIIC-luc reporter) but this was reduced by GPRC5A depletion. A representative triplicate experiment is shown (n=3).


Hypoxia reduced LATS activity and expression but this was prevented by GPRC5A depletion .

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Hypoxia reduced LATS activity and expression but this was prevented by GPRC5A depletion.


Constitutively active RhoA ( G14V ) expression restored YAP stabilisation ( Ser397 dephosphorylation ) by hypoxia in GPRC5A depleted cells .

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Constitutively active RhoA (G14V) expression restored YAP stabilisation (Ser397 dephosphorylation) by hypoxia in GPRC5A depleted cells.


The YAP / TEAD inhibitor verteporfin selectively inhibited cancer cell survival in hypoxia by crystal violet assay ( n = 3 independent experiments ) .

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The YAP/TEAD inhibitor verteporfin selectively inhibited cancer cell survival in hypoxia by crystal violet assay (n=3 independent experiments).


YAP knockdown was sufficient to induce caspase - 3 activation / PARP cleavage in hypoxia and was not further enhanced by GPRC5A depletion .

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YAP knockdown was sufficient to induce caspase-3 activation/PARP cleavage in hypoxia and was not further enhanced by GPRC5A depletion.


Crystal violet assays show that YAP was required downstream of GPRC5A to promote cell survival . GPRC5A depleted cells were rescued by expression of an siRNA - resistant GPRC5A cDNA ( GPRC5Asi1R ) but this was prevented by co - depletion of YAP ( n = 3 independent experiments ) .

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Crystal violet assays show that YAP was required downstream of GPRC5A to promote cell survival. GPRC5A depleted cells were rescued by expression of an siRNA-resistant GPRC5A cDNA (GPRC5Asi1R) but this was prevented by co-depletion of YAP (n=3 independent experiments).


Expression of an siRNA - resistant GPRC5A rescued the critical phenotypes of GPRC5A depletion . GPRC5Asi1R expression prevented PARP cleavage in hypoxia as well as restoring hypoxia - induced YAP stabilisation ( Ser397 dephosphorylation ) ; these phenotypes were reversed by YAP depletion .

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Expression of an siRNA-resistant GPRC5A rescued the critical phenotypes of GPRC5A depletion. GPRC5Asi1R expression prevented PARP cleavage in hypoxia as well as restoring hypoxia-induced YAP stabilisation (Ser397 dephosphorylation); these phenotypes were reversed by YAP depletion.


GPRC5A depletion attenuated hypoxia - induced BCL - XL expression .

GPRC5A depletion attenuated hypoxia-induced BCL-XL expression.


Constitutively active YAP ( S127A ) expression induced BCL - XL expression and prevents caspase - 3 activation / PARP cleavage by GPRC5A depletion in hypoxia .

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Constitutively active YAP (S127A) expression induced BCL-XL expression and prevents caspase-3 activation/PARP cleavage by GPRC5A depletion in hypoxia.


GPRC5Asi1R expression restored BCL - XL expression and prevents the appearance of cleaved caspase - 3 induced by GPRC5A depletion in hypoxia .

GPRC5Asi1R expression restored BCL-XL expression and prevents the appearance of cleaved caspase-3 induced by GPRC5A depletion in hypoxia.


( A ) Immunoblot analysis of NRF1 , NRF2 - α and PGC1 - α in mouse PMs infected with VSV or HSV - 1 or treated with poly ( I : C ) or ISD for the indicated time points . ACTIN was used as a loading control . Ratios of target proteins versus loading control normalized to the 0 - h time point of each condition .

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(A) Immunoblot analysis of NRF1, NRF2-α and PGC1-α in mouse PMs infected with VSV or HSV-1 or treated with poly(I:C) or ISD for the indicated time points. ACTIN was used as a loading control. Ratios of target proteins versus loading control normalized to the 0-h time point of each condition.


( B ) qPCR analysis of Nrf1 , Gabpa and Ppargc1a mRNA in mouse PMs infected with VSV or HSV - 1 or treated with poly ( I : C ) or ISD for the indicated time points ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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(B) qPCR analysis of Nrf1, Gabpa and Ppargc1a mRNA in mouse PMs infected with VSV or HSV-1 or treated with poly(I:C) or ISD for the indicated time points (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( C ) Immunoblot analysis of NRF1 expression in mouse PMs transfected with p65 - specific siRNA ( si - p65 ) or control siRNA ( si - NC ) for 48 h , followed by infection with VSV or HSV - 1 for 8 h . GAPDH was used as a loading control . Ratios of target proteins versus loading control normalized to the untreated sample of each condition .

O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of NRF1 expression in mouse PMs transfected with p65-specific siRNA (si-p65) or control siRNA (si-NC) for 48 h, followed by infection with VSV or HSV-1 for 8 h. GAPDH was used as a loading control. Ratios of target proteins versus loading control normalized to the untreated sample of each condition.


( E ) Immunoblot analysis of mitochondrial proteins and autophagy marker LC3 in mouse PMs transfected with NRF1 - specific siRNA ( si - NRF1 ) or control siRNA ( si - NC ) for 48 h , followed by infection with VSV or HSV - 1 for the indicated time points . ACTIN was used as a loading control . Ratios of target proteins versus loading control normalized to the untreated sample of each condition .

O O O 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 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

(E) Immunoblot analysis of mitochondrial proteins and autophagy marker LC3 in mouse PMs transfected with NRF1-specific siRNA (si-NRF1) or control siRNA (si-NC) for 48 h, followed by infection with VSV or HSV-1 for the indicated time points. ACTIN was used as a loading control. Ratios of target proteins versus loading control normalized to the untreated sample of each condition.


( F ) Mitochondrial mass determined by mtDNA / nuclear DNA ( nDNA ) ratio in mouse PMs transfected with NRF1 - specific siRNA ( si - NRF1 ) or control siRNA ( si - NC ) for 48 h , followed by infection with VSV or HSV - 1 for the indicated time points ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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

(F) Mitochondrial mass determined by mtDNA/nuclear DNA (nDNA) ratio in mouse PMs transfected with NRF1-specific siRNA (si-NRF1) or control siRNA (si-NC) for 48 h, followed by infection with VSV or HSV-1 for the indicated time points (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


RNA - sequencing ( RNA - seq ) analysis of gene expression in wild - type and NRF1 - KO mouse PMs ( n = 3 biological replicates per group ) infected with VSV for 8 h . ( C ) Pheatmap analysis showing downregulated DEGs encoding mitochondrial inner membrane proteins . TPM , transcripts per kilobase of exon model per million mapped reads .

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

RNA-sequencing (RNA-seq) analysis of gene expression in wild-type and NRF1-KO mouse PMs (n=3 biological replicates per group) infected with VSV for 8 h. (C) Pheatmap analysis showing downregulated DEGs encoding mitochondrial inner membrane proteins. TPM, transcripts per kilobase of exon model per million mapped reads.


( D ) Mitochondrial mass determined by mtDNA / nuclear DNA ( nDNA ) ratio in wild - type and NRF1 - KO mouse PMs infected with VSV or HSV - 1 for the indicated time points ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments ( D ) and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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

(D) Mitochondrial mass determined by mtDNA/nuclear DNA (nDNA) ratio in wild-type and NRF1-KO mouse PMs infected with VSV or HSV-1 for the indicated time points (n=3 biological replicates per group). Data information: Data are from three independent experiments (D) and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( F and G ) The representative curves of oxygen consumption rate ( OCR ) in wild - type and NRF1 - KO mouse PMs infected with VSV ( F ) or HSV - 1 ( G ) for 8 h ( n = 5 biological replicates per group ) . Oligomycin : 1 µM , FCCP : 4 µM , Rotenone : 1 µM . Quantification of basal and maximal OCR . Data information : Data are from at least three biological replicates and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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

(F and G) The representative curves of oxygen consumption rate (OCR) in wild-type and NRF1-KO mouse PMs infected with VSV (F) or HSV-1 (G) for 8 h (n=5 biological replicates per group). Oligomycin: 1 µM, FCCP: 4 µM, Rotenone: 1 µM. Quantification of basal and maximal OCR. Data information: Data are from at least three biological replicates and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( H and I ) Electron micrographs of mitochondria in wild - type and NRF1 - KO mouse PMs infected with VSV or HSV - 1 for 8 h . Scale bars , 500 nm ( H ) . ( I ) Quantitative analysis of mitochondrial cristae ( the ratio of cristae number to mitochondrial area ) in ( H ) ( 20 - 30 mitochondria per group , biological replicates ) . Data information : Data are from at least three biological replicates and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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

(H and I) Electron micrographs of mitochondria in wild-type and NRF1-KO mouse PMs infected with VSV or HSV-1 for 8 h. Scale bars, 500 nm (H). (I) Quantitative analysis of mitochondrial cristae (the ratio of cristae number to mitochondrial area) in (H) (20-30 mitochondria per group, biological replicates). Data information: Data are from at least three biological replicates and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( A ) Luciferase activity of IFN - β , NF - κB and ISRE promoter reporter in HEK293T cells transfected with empty vector or plasmids encoding incremental NRF1 - Myc for 24 h followed by infection with VSV for 8 h ( n = 4 biological replicates per group ) .

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

(A) Luciferase activity of IFN-β, NF-κB and ISRE promoter reporter in HEK293T cells transfected with empty vector or plasmids encoding incremental NRF1-Myc for 24 h followed by infection with VSV for 8 h (n=4 biological replicates per group).


( B and C ) Immunoblot analysis of phosphorylated and total TBK1 , p65 and STAT1 in wild - type and NRF1 - KO mouse PMs infected with VSV or HSV - 1 ( B ) or stimulated with poly ( I : C ) or ISD ( C ) for the indicated time points . GAPDH was used as a loading control . Ratios of phosphorylated proteins versus total proteins normalized to the 0 - h or 4 - h time point of each wild - type sample .

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 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

(B and C) Immunoblot analysis of phosphorylated and total TBK1, p65 and STAT1 in wild-type and NRF1-KO mouse PMs infected with VSV or HSV-1 (B) or stimulated with poly(I:C) or ISD (C) for the indicated time points. GAPDH was used as a loading control. Ratios of phosphorylated proteins versus total proteins normalized to the 0-h or 4-h time point of each wild-type sample.


( D ) qPCR analysis of Ifnb1 , Il6 , Tnfa and Cxcl10 mRNA in wild - type and NRF1 - KO mouse PMs infected with VSV or HSV - 1 or stimulated with poly ( I : C ) or ISD for the indicated time points ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ND , not detectable , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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

(D) qPCR analysis of Ifnb1, Il6, Tnfa and Cxcl10 mRNA in wild-type and NRF1-KO mouse PMs infected with VSV or HSV-1 or stimulated with poly(I:C) or ISD for the indicated time points (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ND, not detectable, *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( E ) ELISA quantification of IFN - β , IL6 and TNF - α secretion in wild - type and NRF1 - KO mouse PMs treated as in ( D ) ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ND , not detectable , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ELISA quantification of IFN-β, IL6 and TNF-α secretion in wild-type and NRF1-KO mouse PMs treated as in (D) (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ND, not detectable, *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( A ) 8 - week - old Nrf1F / F and Nrf1MyeKO mice ( n = 3 for PBS treatment and n = 6 for virus treatment ) were intravenously infected with VSV ( 4 × 107 PFU / mouse ) or HSV - 1 ( 1 × 107 PFU / mouse ) . After 24 h , ELISA quantification of IFN - β , IL6 and TNF - α in sera . Data information : Data are presented as mean ± s . e . m . Two - tailed , unpaired Student ' s t test were used to measure significance . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 8-week-old Nrf1F/F and Nrf1MyeKO mice (n=3 for PBS treatment and n=6 for virus treatment) were intravenously infected with VSV (4×107 PFU/mouse) or HSV-1 (1×107 PFU/mouse). After 24 h, ELISA quantification of IFN-β, IL6 and TNF-α in sera. Data information: Data are presented as mean ± s.e.m. Two-tailed, unpaired Student's t test were used to measure significance. *p < 0.05, **p < 0.01, ***p < 0.001.


( B ) qPCR analysis of Ifnb1 , Il6 and Tnfa mRNA in the lung of Nrf1F / F and Nrf1MyeKO mice infected with VSV as in A ( n = 6 per group ) . Data information : Data are presented as mean ± s . e . m . Two - tailed , unpaired Student ' s t test were used to measure significance . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR analysis of Ifnb1, Il6 and Tnfa mRNA in the lung of Nrf1F/F and Nrf1MyeKO mice infected with VSV as in A (n=6 per group). Data information: Data are presented as mean ± s.e.m. Two-tailed, unpaired Student's t test were used to measure significance. *p < 0.05, **p < 0.01, ***p < 0.001.


( C ) qPCR analysis of VSV mRNA and plaque assay of VSV titers in the lungs of Nrf1F / F and Nrf1MyeKO mice infected with VSV as in A ( n = 6 per group ) . Data information : Data are presented as mean ± s . e . m . Two - tailed , unpaired Student ' s t test were used to measure significance . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR analysis of VSV mRNA and plaque assay of VSV titers in the lungs of Nrf1F/F and Nrf1MyeKO mice infected with VSV as in A (n=6 per group). Data information: Data are presented as mean ± s.e.m. Two-tailed, unpaired Student's t test were used to measure significance. *p < 0.05, **p < 0.01, ***p < 0.001.


( E ) qPCR analysis of Ifnb1 , Il6 and Tnfa mRNA in the brains of Nrf1F / F and Nrf1MyeKO mice infected with HSV - 1 as in ( A ) ( n = 6 per group ) . ( F ) qPCR analysis of HSV - 1 UL30 mRNA and plaque assay of HSV - 1 titers in the brain of Nrf1F / F and Nrf1MyeKO mice infected with HSV - 1 as in ( A ) ( n = 6 per group ) . Data information : Data are presented as mean ± s . e . m . Two - tailed , unpaired Student ' s t test were used to measure significance . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR analysis of Ifnb1, Il6 and Tnfa mRNA in the brains of Nrf1F/F and Nrf1MyeKO mice infected with HSV-1 as in (A) (n=6 per group). (F) qPCR analysis of HSV-1 UL30 mRNA and plaque assay of HSV-1 titers in the brain of Nrf1F/F and Nrf1MyeKO mice infected with HSV-1 as in (A) (n=6 per group). Data information: Data are presented as mean ± s.e.m. Two-tailed, unpaired Student's t test were used to measure significance. *p < 0.05, **p < 0.01, ***p < 0.001.


( G ) Survival of Nrf1F / F and Nrf1MyeKO mice ( n = 10 per group ) at various times after intraperitoneal ( i . p . ) infection with HSV - 1 ( 2 × 107 PFU per mouse ) . Data information : Data are presented as mean ± s . e . m . log - rank ( Mantel - Cox ) test ( G ) were used to measure significance . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(G) Survival of Nrf1F/F and Nrf1MyeKO mice (n=10 per group) at various times after intraperitoneal (i.p.) infection with HSV-1 (2×107 PFU per mouse). Data information: Data are presented as mean ± s.e.m. log-rank (Mantel-Cox) test (G) were used to measure significance. *p < 0.05, **p < 0.01, ***p < 0.001.


( A ) mtROS levels measured by MitoSOX staining and flow cytometry in wild - type and NRF1 - KO mouse PMs infected with VSV ( left ) or HSV - 1 ( right ) for the indicated time points . MFI , mean fluorescence intensity ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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

(A) mtROS levels measured by MitoSOX staining and flow cytometry in wild-type and NRF1-KO mouse PMs infected with VSV (left) or HSV-1 (right) for the indicated time points. MFI, mean fluorescence intensity (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( B ) DNA was isolated from digitonin extracts of wild - type and NRF1 - KO mouse PMs infected with VSV ( left ) or HSV - 1 ( right ) for the indicated time points . Cytosolic translocation of mtDNA was quantified via qPCR using the indicated primer sets . 18S rRNA served as a control ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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

(B) DNA was isolated from digitonin extracts of wild-type and NRF1-KO mouse PMs infected with VSV (left) or HSV-1 (right) for the indicated time points. Cytosolic translocation of mtDNA was quantified via qPCR using the indicated primer sets. 18S rRNA served as a control (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( C and D ) Wild - type and NRF1 - KO mouse PMs infected with VSV ( 8 h ) or HSV - 1 ( 4 h ) were imaged by confocal microscope after staining with anti - DNA ( DNA ) and anti - Prohibitin ( Mito ) antibodies . Scale bars , 5 µm ( C ) . ( D ) The number of cytosolic mtDNA puncta per cell was quantitated by ImageJ ( n = 30 cells per group ) . Data information : Data are from three independent experiments or representative data ( C ) and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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

(C and D) Wild-type and NRF1-KO mouse PMs infected with VSV (8 h) or HSV-1 (4 h) were imaged by confocal microscope after staining with anti-DNA (DNA) and anti-Prohibitin (Mito) antibodies. Scale bars, 5 µm (C). (D) The number of cytosolic mtDNA puncta per cell was quantitated by ImageJ (n=30 cells per group). Data information: Data are from three independent experiments or representative data (C) and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( E ) ELISA quantification of IFN - β ( left ) and IL6 ( right ) in wild - type and NRF1 - KO mouse PMs pretreated with or without EtBr ( 450 ng / ml , 72 h ) , followed by infection with VSV or HSV - 1 for 8 h ( n = 3 biological replicates per group ) . ( F ) ELISA quantification of IFN - β ( left ) and IL6 ( right ) in wild - type and NRF1 - KO mouse PMs pretreated with or without MitoQ ( 100 nM , 2 h ) , followed by infection with VSV or HSV - 1 for 8 h ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ELISA quantification of IFN-β (left) and IL6 (right) in wild-type and NRF1-KO mouse PMs pretreated with or without EtBr (450 ng/ml, 72 h), followed by infection with VSV or HSV-1 for 8 h (n=3 biological replicates per group). (F) ELISA quantification of IFN-β (left) and IL6 (right) in wild-type and NRF1-KO mouse PMs pretreated with or without MitoQ (100 nM, 2 h), followed by infection with VSV or HSV-1 for 8 h (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( H ) qPCR analysis of Ifnb1 and Il6 mRNA in the lungs of Nrf1F / F and Nrf1MyeKO mice pretreated with MitoQ as in ( G ) and 1 h later infected i . p . with VSV ( 5 × 108 PFU / mouse ) for 24 h ( n = 6 per group ) . ( I ) qPCR analysis of Ifnb1 and Il6 mRNA in the brains of Nrf1F / F and Nrf1MyeKO mice pretreated with MitoQ as in ( G ) and 1 h later infected i . p . with HSV - 1 ( 1 × 107 PFU / mouse ) for 24 h ( n = 6 per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , using a two - tailed , unpaired Student ' s t test .

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

(H) qPCR analysis of Ifnb1 and Il6 mRNA in the lungs of Nrf1F/F and Nrf1MyeKO mice pretreated with MitoQ as in (G) and 1 h later infected i.p. with VSV (5×108 PFU/mouse) for 24 h (n=6 per group). (I) qPCR analysis of Ifnb1 and Il6 mRNA in the brains of Nrf1F/F and Nrf1MyeKO mice pretreated with MitoQ as in (G) and 1 h later infected i.p. with HSV-1 (1×107 PFU/mouse) for 24 h (n=6 per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, using a two-tailed, unpaired Student's t test.


( A ) Immunoblot analysis and Phos - tag SDS - PAGE analysis of mouse PMs infected with VSV or HSV - 1 for the indicated time points .

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(A) Immunoblot analysis and Phos-tag SDS-PAGE analysis of mouse PMs infected with VSV or HSV-1 for the indicated time points.


( C ) Interaction between TBK1 and NRF1 in HEK293T cells was detected by co - immunoprecipitation of differentially tagged proteins .

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

(C) Interaction between TBK1 and NRF1 in HEK293T cells was detected by co-immunoprecipitation of differentially tagged proteins.


( G ) Endogenous phospho - NRF1 S318 signal was detected in wild - type and NRF1 S318A mouse PMs after the infection with different viruses for 8 h . The red arrows indicate phosphorylated signals for NRF1 .

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(G) Endogenous phospho-NRF1 S318 signal was detected in wild-type and NRF1 S318A mouse PMs after the infection with different viruses for 8 h. The red arrows indicate phosphorylated signals for NRF1.


( J ) ELISA quantification of IFN - β and IL6 secretion in wild - type and NRF1 S318A mouse PMs treated as in ( I ) ( n = 3 biological replicates per group ) . Data information : Data are from three independent experiments and presented as mean ± s . e . m . ns , not significant ( p > 0 . 05 ) , * p < 0 . 05 , * * p < 0 . 01 . Two - tailed , unpaired Student ' s t test were used to measure significance .

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

(J) ELISA quantification of IFN-β and IL6 secretion in wild-type and NRF1 S318A mouse PMs treated as in (I) (n=3 biological replicates per group). Data information: Data are from three independent experiments and presented as mean ± s.e.m. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01. Two-tailed, unpaired Student's t test were used to measure significance.


( L ) Survival of wild - type and NRF1 S318A mice ( n = 10 per group ) at various times after intraperitoneal infection with HSV - 1 ( 2 × 107 PFU per mouse ) . Data information : log - rank ( Mantel - Cox ) test ( L ) were used to measure significance .

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(L) Survival of wild-type and NRF1 S318A mice (n=10 per group) at various times after intraperitoneal infection with HSV-1 (2×107 PFU per mouse). Data information: log-rank (Mantel-Cox) test (L) were used to measure significance.


Confocal images demonstrate overlap of GFP tagged mtON with MitoTrackerTM Red CMXRos stained C . elegans hypodermal mitochondria . Scale bar 10 µm .

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Confocal images demonstrate overlap of GFP tagged mtON with MitoTrackerTM Red CMXRos stained C. elegans hypodermal mitochondria. Scale bar 10 µm.


Immunoblot comparing the cytosolic supernatant and the mitochondria - enriched pellet of isolation fractions . GFP - tagged mtON migrates at the predicted molecular weight of 82 kDa accounting for the mitochondria - target sequence , the proton pump , and GFP . mtON is observed only in the mitochondrial fraction compared to marker proteins HSP60 ( mitochondria ) and actin ( cytosol ) . All blots are from the same lanes on one membrane .

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 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

Immunoblot comparing the cytosolic supernatant and the mitochondria-enriched pellet of isolation fractions. GFP-tagged mtON migrates at the predicted molecular weight of 82 kDa accounting for the mitochondria-target sequence, the proton pump, and GFP. mtON is observed only in the mitochondrial fraction compared to marker proteins HSP60 (mitochondria) and actin (cytosol). All blots are from the same lanes on one membrane.


Representative TMRE fluorescence traces in arbitrary units ( a . u . ) before and after Δψm dissipation with FCCP . Dashed lines indicate where FCCP was added . + / - ATR traces were performed in the absence of succinate . mtON activation was continuous throughout the traces . Light green trace is from mitochondria with ATR , gray traces are without . Quantification of change in fluorescence ( ΔF ) normalized to the maximum change in fluorescence given by succinate respiration ( ΔFmax ) for each mitochondrial preparation . Data are from the maximum light dose in Fig . EV 3A . Two - way ANOVA with Sidak ' s test for multiple comparisons , * p = 0 . 0469 , + ATR succinate vs . + ATR + light p = 0 . 9978 , n = 6 mitochondrial isolations . Data show mean ± SEM .

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

Representative TMRE fluorescence traces in arbitrary units (a.u.) before and after Δψm dissipation with FCCP. Dashed lines indicate where FCCP was added. +/- ATR traces were performed in the absence of succinate. mtON activation was continuous throughout the traces. Light green trace is from mitochondria with ATR, gray traces are without. Quantification of change in fluorescence (ΔF) normalized to the maximum change in fluorescence given by succinate respiration (ΔFmax) for each mitochondrial preparation. Data are from the maximum light dose in Fig. EV 3A. Two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0469, +ATR succinate vs. +ATR +light p = 0.9978, n = 6 mitochondrial isolations. Data show mean ± SEM.


Representative BCECF - AM 490 / 440 nm ratio trace . Ratio of 545 nm fluorescence intensity at either 440 or 490 nm excitation . Dashed lines are where light or FCCP treatment occurred . Light green trace is from mitochondria with ATR , gray traces are without . Quantification of change in BCECF - AM fluorescence ratio normalized to maximum change given by succinate matrix alkalization . Two - way ANOVA with Sidak ' s test for multiple comparisons , - ATR succinate vs . - ATR , + light * p = 0 . 0212 , + ATR succinate vs . + ATR light p = 0 . 999 , + ATR succinate vs . + ATR , - light p = 0 . 0237 , + ATR , + light vs , + ATR , - light p = 0 . 0474 , n = 3 mitochondrial isolations . Some individual points overlap . Data show mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 BCECF-AM 490/440 nm ratio trace. Ratio of 545 nm fluorescence intensity at either 440 or 490 nm excitation. Dashed lines are where light or FCCP treatment occurred. Light green trace is from mitochondria with ATR, gray traces are without. Quantification of change in BCECF-AM fluorescence ratio normalized to maximum change given by succinate matrix alkalization. Two-way ANOVA with Sidak's test for multiple comparisons, -ATR succinate vs. -ATR, +light *p = 0.0212, +ATR succinate vs. +ATR light p = 0.999, +ATR succinate vs. +ATR,-light p = 0.0237, +ATR, +light vs, +ATR, -light p = 0.0474, n = 3 mitochondrial isolations. Some individual points overlap. Data show mean ± SEM.


ATP levels normalized to total ATP synthesis given by succinate respiration . Data from Fig . EV 3B . Succinate data shown for comparison after normalization . Two - way ANOVA with Sidak ' s test for multiple comparisons , * p = 0 . 0011 , + ATR succinate vs + ATR light p = 0 . 5680 , n = 3 , 7 , 3 , 7 , biological replicates for each bar from left to right . Some individual points overlap . Data show mean + SEM .

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ATP levels normalized to total ATP synthesis given by succinate respiration. Data from Fig. EV 3B. Succinate data shown for comparison after normalization. Two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0011, +ATR succinate vs +ATR light p = 0.5680, n = 3,7,3,7, biological replicates for each bar from left to right. Some individual points overlap. Data show mean + SEM.


O2 required to consume 50 nmoles of ADP after mtON activation . Data are the maximum illumination from Fig . EV 4D using time - matched dark conditions , One - way ANOVA * p = 0 . 013 ( - ATR , - light vs . + ATR , + light p = 0 . 010 . - ATR , + light vs . + ATR , + light p = 0 . 013 . ) - ATR , - light n = 5 , rest n = 6 mitochondrial preparations . Data show mean + SEM .

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O2 required to consume 50 nmoles of ADP after mtON activation. Data are the maximum illumination from Fig. EV 4D using time-matched dark conditions, One-way ANOVA *p = 0.013 (-ATR, -light vs. +ATR, +light p = 0.010. -ATR, +light vs. +ATR, +light p = 0.013.) -ATR, -light n = 5, rest n = 6 mitochondrial preparations. Data show mean + SEM.


Day 1 adult animals expressing mtON were exposed to 50 µM rotenone ( ETC complex I inhibitor ) for 5 hours and survival was scored . Illumination was continuous throughout toxin exposure ( see methods ) . ATR alone was protective ( - ATR , - light vs . + ATR , - light p = 0 . 016 . ) The effect of mtON activation was greater than the ATR alone effect , one - way ANOVA with Tukey ' s post hoc test * p = 0 . 01 . ( - ATR , - light vs + ATR , + light p = 0 . 0002 . - ATR , + light vs . + ATR , + light p = 0 . 0009 . n = 3 plates each condition with at least 15 animals per plate ) . Data show mean ± SEM . mtON - expressing animals were exposed to 50 µM antimycin A ( complex III inhibitor ) and survival was scored 18 hours later . One - way ANOVA with Tukey ' s post hoc test * p = 0 . 02 ( - ATR , - light vs + ATR , + light p = 0 . 03 , - ATR , + light vs . + ATR , + light p = 0 . 01 . n = 5 plates each condition with at least 15 animals per plate ) . Data show mean ± SEM . mtON - expressing animals were exposed to 0 . 25 M azide ( complex IV inhibitor ) for 1 hour and scored for survival 1 hour after recovering , one - way ANOVA with Tukey ' s post hoc test * p = 0 . 0002 . ( - ATR , - light vs + ATR , + light p < 0 . 0001 , - ATR , + light vs . + ATR , + light p < 0 . 0001 . n = 3 plates each condition with at least 15 animals per plate ) . Data show mean ± SEM . mtON - expressing animals were exposed to 31 µM oligomycin A ( ATP synthase inhibitor ) and survival was scored 18 hours later . No significant differences were found by one - way ANOVA ( n = 3 plates each condition with at least 15 animals per plate ) . Data show mean ± SEM .

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

Day 1 adult animals expressing mtON were exposed to 50 µM rotenone (ETC complex I inhibitor) for 5 hours and survival was scored. Illumination was continuous throughout toxin exposure (see methods). ATR alone was protective (-ATR, -light vs. +ATR, -light p = 0.016.) The effect of mtON activation was greater than the ATR alone effect, one-way ANOVA with Tukey's post hoc test *p = 0.01. (-ATR, -light vs +ATR, +light p = 0.0002. -ATR, +light vs. +ATR, +light p = 0.0009. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 50 µM antimycin A (complex III inhibitor) and survival was scored 18 hours later. One-way ANOVA with Tukey's post hoc test *p = 0.02 (-ATR, -light vs +ATR, +light p = 0.03, -ATR, +light vs. +ATR, +light p = 0.01. n = 5 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 0.25 M azide (complex IV inhibitor) for 1 hour and scored for survival 1 hour after recovering, one-way ANOVA with Tukey's post hoc test *p = 0.0002. (-ATR, -light vs +ATR, +light p < 0.0001, -ATR, +light vs. +ATR, +light p <0.0001. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 31 µM oligomycin A (ATP synthase inhibitor) and survival was scored 18 hours later. No significant differences were found by one-way ANOVA (n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM.


mtON - expressing animals in a complex I mutant background ( gas - 1 ) were scored for locomotion speed in the presence of food by counting body bends per minute . mtON activation rescued the decreased locomotion of the gas - 1 mutant background , one - way ANOVA with Tukey ' s post hoc test , - ATR , + light vs . + ATR , + light * p = 0 . 0145 , + ATR , + light vs . wildtype ( WT ) * p = 0 . 0008 ( - ATR , - light vs + ATR , + light p = 0 . 0272 , + ATR , - light vs . + ATR , + light p = 0 . 0332 , all conditions vs WT , p < 0 . 001 , n = 30 , 30 , 30 , 41 , 41 animals each bar from left to right ) . Data show mean ± standard deviation .

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

mtON-expressing animals in a complex I mutant background (gas-1) were scored for locomotion speed in the presence of food by counting body bends per minute. mtON activation rescued the decreased locomotion of the gas-1 mutant background, one-way ANOVA with Tukey's post hoc test, -ATR, +light vs. +ATR, +light *p = 0.0145, +ATR, +light vs. wildtype (WT) *p = 0.0008 (-ATR, -light vs +ATR, +light p = 0.0272, +ATR, -light vs. +ATR, +light p = 0.0332, all conditions vs WT, p < 0.001, n = 30, 30,30,41,41 animals each bar from left to right). Data show mean ± standard deviation.


Top : Immunoblot assessing the effect of mtON activation on AMPK phosphorylation status . Top bands ( ~ 62 kDa ) are phosphorylated AMPK signal , and bottom bands ( ~ 43 kDa ) are actin signal . Image is from the same membrane cut to separately probe for phosphorylated AMPK ( pAMPK ) and actin . Bottom : Densitometry analysis showing decreased pAMPK to actin ratio , as there is no known antibody directed against total AMPK in C . elegans . Phosphorylation increases in the absence of food , but low phosphorylation is preserved when mtON is activated , two - way ANOVA with Sidak ' s test for multiple comparisons , * p = 0 . 0203 , n = 3 , 3 , 3 , 4 , 4 biological replicates each bar from left to right . Data show mean + standard deviation .

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

Top: Immunoblot assessing the effect of mtON activation on AMPK phosphorylation status. Top bands (~62 kDa) are phosphorylated AMPK signal, and bottom bands (~43 kDa) are actin signal. Image is from the same membrane cut to separately probe for phosphorylated AMPK (pAMPK) and actin. Bottom: Densitometry analysis showing decreased pAMPK to actin ratio, as there is no known antibody directed against total AMPK in C. elegans. Phosphorylation increases in the absence of food, but low phosphorylation is preserved when mtON is activated, two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0203, n = 3,3,3,4,4 biological replicates each bar from left to right. Data show mean + standard deviation.


Locomotion was assessed by counting body bends per minute . Wild type animals were compared to aak - 2 ( ok524 ) mutant animals . 2 - sample , 2 - tailed unpaired t - test * p < 0 . 0001 , wild type n = 35 , aak - 2 n = 39 animals across at least 3 days . Data show mean ± standard deviation .

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Locomotion was assessed by counting body bends per minute. Wild type animals were compared to aak-2(ok524) mutant animals. 2-sample, 2-tailed unpaired t-test *p < 0.0001, wild type n = 35, aak-2 n = 39 animals across at least 3 days. Data show mean ± standard deviation.


Locomotion in response to mtON activation . Illumination was continuous throughout body bends measurement ( see methods ) . For AAK - 2 activation , animals were exposed to 1 mM AICAR for 4 hours before body bends measurement . One - way ANOVA with Tukey ' s post hoc test , - ATR , + light vs + ATR , + light * p < 0 . 0001 , + ATR , + light vs . AICAR * p < 0 . 0001 , + ATR , + light vs AICAR + light * p < 0 . 0001 . n = 36 , 39 , 37 , 46 , 36 , 36 animals each bar from left to right . Data show mean ± standard deviation .

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Locomotion in response to mtON activation. Illumination was continuous throughout body bends measurement (see methods). For AAK-2 activation, animals were exposed to 1 mM AICAR for 4 hours before body bends measurement. One-way ANOVA with Tukey's post hoc test, -ATR, +light vs +ATR, +light *p < 0.0001, +ATR, +light vs. AICAR *p < 0.0001, +ATR,+light vs AICAR +light *p < 0.0001. n = 36, 39, 37, 46, 36, 36 animals each bar from left to right. Data show mean ± standard deviation.


Survival after HR in day 1 adult animals is shown with hypoxic preconditioning ( PC , represented by diagonal stripes ) 2 - sample 2 - tailed unpaired t - test , ctrl vs . PC * p = 0 . 0035 , n = 4 , each averaged from 3 technical replicates with at least 15 animals per replicate . Data show mean ± SEM .

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Survival after HR in day 1 adult animals is shown with hypoxic preconditioning (PC, represented by diagonal stripes) 2-sample 2-tailed unpaired t-test, ctrl vs. PC *p = 0.0035, n = 4, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


Protection ( % ) is percent survival minus percent survival of control condition . PC data calculated from panel B . FCCP final concentrations 0 . 001 , 0 . 01 , 0 . 1 , 1 nM . One - way ANOVA with Tukey ' s post hoc test , PC vs . 0 . 001 nM FCCP , * p = 0 . 0012 , 0 . 001 nM FCCP vs 0 . 01 FCCP , * p = 0 . 0142 , n = 4 , 3 , 3 , 3 , 3 independent experiments each bar from left to right , each averaged from 3 technical replicates with at least 15 animals per replicate . Data show mean ± SEM .

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Protection (%) is percent survival minus percent survival of control condition. PC data calculated from panel B. FCCP final concentrations 0.001, 0.01, 0.1, 1 nM. One-way ANOVA with Tukey's post hoc test, PC vs. 0.001 nM FCCP, *p = 0.0012, 0.001 nM FCCP vs 0.01 FCCP, *p = 0.0142, n = 4,3,3,3,3 independent experiments each bar from left to right, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.