( D ) Lectin - Fc binding to full - SARS - CoV - 2 Spike with or without de - N - glycosylation by PNGase F . " PNGase F only " denotes wells that were not coated with Spike . Results are shown as mean OD values ± SD normalized to BSA controls ( technical replicates , N = 3 ) .

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(D) Lectin-Fc binding to full-length SARS-CoV-2 Spike with or without de-N-glycosylation by PNGase F. "PNGase F only" denotes wells that were not coated with the Spike protein. Results are shown as mean OD values ± SD normalized to BSA controls (technical replicates, N=3).


( E ) High speed AFM of single trimeric Spike visualizing real - time interaction dynamics with lectins . Top panel shows 5 frames of trimeric Spike alone imaged on mica . Middle bottom panels show 5 sequential frames of trimeric Spike / Clec4g trimeric Spike / CD209c complexes , acquired at a rate of 153 . 6 303 ms / frame , respectively . Association dissociation events between lectin Spike are indicated by red arrows , respectively . blue dotted ellipses display of complexes showing low conformational mobility . Color schemes indicate height of molecules in nanometers ( nm ) . Volumes of single trimeric Spike , trimeric Spike / Clec4g trimeric Spike / CD209c complexes are indicated , as well as numbers of lectins bound to trimeric Spike , averaged over experimental recording period .

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(E) High speed AFM of single trimeric Spike visualizing the real-time interaction dynamics with lectins. Top panel shows 5 frames of trimeric Spike alone imaged on mica. Middle and bottom panels show 5 sequential frames of trimeric Spike/Clec4g and trimeric Spike/CD209c complexes, acquired at a rate of 153.6 and 303 ms/frame, respectively. Association and dissociation events between lectin and Spike are indicated by white and red arrows, respectively. The blue dotted ellipses display the core of the complexes showing low conformational mobility. Color schemes indicate height of the molecules in nanometers (nm). Volumes of single trimeric Spike, trimeric Spike/Clec4g and trimeric Spike/CD209c complexes are indicated, as well as numbers of lectins bound to trimeric Spike, averaged over the experimental recording period.


( A ) ELISA analyses of human lectin - hIgG1 Fc - fusion ( hCLEC4g , hCD209 , hCD299 ) binding to untreated or heat - denatured full - SARS - CoV - 2 Spike . A human ACE2 - hIgG1 - Fc fusion ( hACE2 ) was used to control for complete denaturation of Spike . Results are shown as mean OD values ± SD normalized to a BSA control ( technical replicates , N = 3 ) .

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(A) ELISA analyses of human lectin-hIgG1 Fc-fusion protein (hCLEC4g, hCD209, hCD299) binding to untreated or heat-denatured full-length SARS-CoV-2 Spike. A human ACE2-hIgG1-Fc fusion protein (hACE2) was used to control for the complete denaturation of Spike. Results are shown as mean OD values ± SD normalized to a BSA control (technical replicates, N=3).


( B ) hCLEC4g , hCD209 , hCD299 binding to full - SARS - CoV - 2 Spike with or without de - N - glycosylation by PNGase F . " PNGase F only " denotes wells that were not coated with Spike . Results are shown as mean OD values ± SD normalized against BSA control ( technical replicates , N = 3 ) .

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(B) hCLEC4g, hCD209, and hCD299 binding to full-length SARS-CoV-2 Spike with or without de-N-glycosylation by PNGase F. "PNGase F only" denotes wells that were not coated with the Spike protein. Results are shown as mean OD values ± SD normalized against the BSA control (technical replicates, N=3).


( C , D ) SPR with immobilized full - trimeric Spike , probed with various concentrations of hCLEC4G ( C ) hCD209 ( D ) .

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(C, D) SPR analysis with immobilized full-length trimeric Spike, probed with various concentrations of hCLEC4G (C) and hCD209 (D).


( C ) Infectivity of mouse Clec4g , CD209c , Asgr1 hCLEC4G pre - treated SARS - CoV - 2 in Vero E6 . Data represent two to three independent experiments for Clec4g , CD209c hCLEC4G lectins ( N = 5 - 9 ) , one experiment for Asgr1 ( N = 2 - 4 ) . ( D ) Infectivity of mouse Clec4g , CD209c , Asgr1 hCLEC4G pre - treated SARS - CoV - 2 in human airway epithelial Calu3 measured in triplicates compared to non - treated SARS - CoV - 2 infected Calu3 measured in quadruplicates ( technical replicates ) . : ( C ) ( D ) Viral was measured with qRT - PCR 15 hours after infection with 0 . 02 MOI ( 103 PFUs ) of SARS - CoV - 2 . Data are presented as fold changes of viral loads over mock ( only SARS - CoV - 2 was added ) treated controls ( mean log10 values ± SD ) . no controls were negative for SARS - CoV - 2 .

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(C) Infectivity of mouse Clec4g, CD209c, Asgr1 and hCLEC4G pre-treated SARS-CoV-2 virus in Vero E6 cells. Data represent two to three independent experiments for Clec4g, CD209c and hCLEC4G lectins (N=5-9) and, one experiment for Asgr1 (N=2-4). (D) Infectivity of mouse Clec4g, CD209c, Asgr1 and hCLEC4G pre-treated SARS-CoV-2 virus in human airway epithelial Calu3 cells measured in triplicates and compared to non-treated SARS-CoV-2 infected Calu3 cells measured in quadruplicates (technical replicates). : (C) and (D) Viral RNA was measured with qRT-PCR 15 hours after infection with 0.02 MOI (103 PFUs) of SARS-CoV-2. Data are presented as fold changes of viral loads over mock (only SARS-CoV-2 was added) treated controls (mean log10 values ± SD). All no virus controls were negative for SARS-CoV-2.


A . Immunoblot of extracts prepared from synchronized at ring ( R ) , trophozoite ( T ) schizont ( S ) stages with anti - histone H3 C - terminus antibodies identifies three bands : full - histone H3 , an intermediate form ( H3 - int ) a fully processed form ( H3p ) . Anti - histone H4 anti - PfAldolase antibodies served as loading controls for fraction , respectively .

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A. Immunoblot analysis of nuclear and cytoplasmic extracts prepared from parasites synchronized at ring (R), trophozoite (T) and schizont (S) stages with anti-histone H3 C-terminus antibodies identifies three bands: full-length histone H3, an intermediate form (H3-int) and a fully processed form (H3p). Anti-histone H4 and anti-PfAldolase antibodies served as loading controls for the nuclear and cytoplasmic fraction, respectively.


B . Immunoblot assays of mononucleosomes with anti - histone H3 C - terminus or anti - histone H4 antibodies . right panel shows a mononoucleosome preparation separated on a denaturing polyacrylamide gel stained with Coomassie brilliant blue ( C . B . ) .

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B. Immunoblot assays of mononucleosomes with anti-histone H3 C-terminus or anti-histone H4 antibodies. The right panel shows a mononoucleosome preparation separated on a denaturing polyacrylamide gel and stained with Coomassie brilliant blue (C.B.).


C . Immunoblot of a schizont - stage histone preparation with antibodies against indicated post - translational modification ( PTM ) in N - terminal tail of histone H3 ( upper panel ) .

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C. Immunoblot analysis of a schizont-stage histone preparation with antibodies against the indicated post-translational modification (PTM) in the N-terminal tail of histone H3 (upper panel).


B . GST - PfH3 or GST - PfH2A were incubated with increasing amounts of extracts , reactions resolved using denaturing gel electrophoresis analyzed by immunoblotting with anti - GST antibodies .

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B. GST-PfH3 or GST-PfH2A were incubated with increasing amounts of nuclear extracts, the reactions resolved using denaturing gel electrophoresis and analyzed by immunoblotting with anti-GST antibodies.


C . GST - PfH3 was incubated with 25 μg of extracts indicated protease inhibitor . resulting products were analyzed by denaturing gel electrophoresis immunoblotting with anti - GST antibodies . ( - ) indicates a reaction without extract protease inhibitor .

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C. GST-PfH3 was incubated with 25 μg of nuclear extracts and the indicated protease inhibitor. The resulting products were analyzed by denaturing gel electrophoresis and immunoblotting with anti-GST antibodies. (-) indicates a reaction without nuclear extract and protease inhibitor.


A . Indirect immunofluorescence assays were performed to determine localization of ectopically expressed PfH3p - HA in ring ( R ) , trophozoite ( T ) schizont ( S ) stages of P . falciparum asexual growth . PfH3p - HA was detected using anti - HA antibodies ( green ) endogenous histone H3 with anti - histone H3 N - terminal antibodies ( red ) . DAPI ( blue ) was used to stain nucleus . Scale bar = 5 μm .

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A. Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p-HA in ring (R), trophozoite (T) and schizont (S) stages of P. falciparum asexual growth. PfH3p-HA was detected using anti-HA antibodies (green) and endogenous histone H3 with anti-histone H3 N-terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm.


B . Nuclei isolated from wild - type ( WT ) or PfH3p - HA - expressing ( WT + PfH3p - HA ) schizont stage were treated with 4 U / ml of micrococcal nuclease ( MNase ) for indicated amounts of time , purified migrated on a 2 % agarose gel , stained with ethidium bromide . Mononucleosomes purified after 10 minutes of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis either stained with Coomassie Brilliant blue ( C . B . ) or visualized by immunoblotting with anti - HA ( α - HA ) or anti - C - terminal histone H3 ( α - H3c ) antibodies .

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B. Nuclei isolated from wild-type (WT) or PfH3p-HA-expressing (WT + PfH3p-HA) schizont stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 minutes of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant blue (C.B.) or visualized by immunoblotting with anti-HA (α-HA) or anti-C-terminal histone H3 (α-H3c) antibodies.


C . Co - immunoprecipitation ( IP ) experiments of purified mononucleosomes obtained from wild - type ( WT ) or transfected ( WT + PfH3p - HA ) schizont stage were performed with either anti - HA antibodies or mouse IgG . Immunoprecipitated products ( right panel ) were analyzed by immunoblotting using anti - HA or anti - histone H4 antibodies .

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C. Co-immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild-type (WT) or transfected (WT + PfH3p-HA) schizont stage parasites were performed with either anti-HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti-HA or anti-histone H4 antibodies.


A . Genome - wide distribution of ectopically expressed PfH3p - HA in P . falciparum schizont stages is represented as fold - enrichment of PfH3p - HA ChIP - seq signal over Input ( PfH3p - HA / Input in blue ; y - axis scale 1 - 10 ) or over PfH3n ( PfH3p - HA / Input in teal ; y - axis scale 1 - 10 ) calculated using MACS2 . coverage of PfH3p - HA ( red ; y - axis scale 0 - 50 ) , Input ( black ; y - axis scale 0 - 30 ) PfH3n ( green ; y - axis scale 0 - 30 ) is also shown represents average reads per million over 1000 nt bins of genome . x - axis represents chromosome in Mb . Major MACS2 - derived peaks identified in replicates are indicated using an orange arrowhead . B . Principal Component of different ChIP - seq replicates ( bigwig files derived from deduplicated bam files ) was performed using plotPCA function of deepTools on a multibigwig summary file , over 150 nt bins . Eigen values of top two principal components PC1 PC2 are shown meaningful clustering of replicates highlighted . Rep = Biological Replicate . C . Summary of PfH3p - enriched peaks - corresponding genes - identified in PfH3p - HA versus Input or PfH3p - HA versus PfH3n comparisons using MACS2 peak - calling . overlap between two comparisons is indicated using a Venn diagram . FE = Fold Enrichment ; q - value represents Benjamini Hochberg false discovery rate . D . genomic context of six peaks identified in three PfH3p - HA ChIP - seq replicates is shown . fold enrichment of PfH3p - HA ChIP - seq signal over Input ( PfH3p - HA / Input ; blue ) or PfH3n ( PfH3p - HA / PfH3n ; teal ) is shown , as are coverage plots of PfH3p - HA ( red ) , PfH3n ( green ) Input ( black ) , which are represented as average reads per million over 1000 nt bins of genome . replication genes : Replication A 1 ( RPA1 ) , Proliferating Antigen 1 ( PCNA1 ) , Single Stranded binding ( SSB ) , Topoisomerase I ( TOPO - I ) , polymerase alpha subunit ( pol a ) , Topoisomerase II ( TOPO - II ) are highlighted their directionality indicated . Note that data in part D correspond to Biological Replicate 1 .

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A. Genome-wide distribution of ectopically expressed PfH3p-HA in P. falciparum schizont stages is represented as fold-enrichment of PfH3p-HA ChIP-seq signal over Input (PfH3p-HA / Input in blue; y-axis scale 1-10) or over PfH3n (PfH3p-HA / Input in teal; y-axis scale 1-10) calculated using MACS2. The coverage of PfH3p-HA (red; y-axis scale 0-50), Input (black; y-axis scale 0-30) and PfH3n (green; y-axis scale 0-30) is also shown and represents average reads per million over 1000 nt bins of the genome. The x-axis represents chromosome size in Mb. Major MACS2-derived peaks identified in all replicates are indicated using an orange arrowhead. B. Principal Component Analysis of the different ChIP-seq replicates (bigwig files derived from deduplicated bam files) was performed using the plotPCA function of deepTools on a multibigwig summary file, over 150 nt bins. The Eigen values of the top two principal components PC1 and PC2 are shown and meaningful clustering of replicates highlighted. Rep = Biological Replicate. C. Summary of the PfH3p-enriched peaks - and corresponding genes - identified in the PfH3p-HA versus Input or PfH3p-HA versus PfH3n comparisons using MACS2 peak-calling analysis. The overlap between the two comparisons is indicated using a Venn diagram. FE = Fold Enrichment; q-value represents the Benjamini and Hochberg false discovery rate. D. The genomic context of the six peaks identified in all three PfH3p-HA ChIP-seq replicates is shown. The fold enrichment of PfH3p-HA ChIP-seq signal over Input (PfH3p-HA / Input; blue) or PfH3n (PfH3p-HA / PfH3n; teal) is shown, as are the coverage plots of PfH3p-HA (red), PfH3n (green) and Input (black), which are represented as average reads per million over 1000 nt bins of the genome. DNA replication genes: Replication Protein A 1 (RPA1), Proliferating Cell Nuclear Antigen 1 (PCNA1), Single Stranded DNA binding protein (SSB), Topoisomerase I (TOPO-I), DNA polymerase alpha subunit (DNA pol a), and Topoisomerase II (TOPO-II) are highlighted and their directionality indicated. Note that the data in part D correspond to Biological Replicate 1.


( D ) smFISH showing LAT in Ntrk1 - positive neurons . SCG - derived cultures were infected with HSV - 1 in presence of ACV allowed to establish latency over 7 days before being processed for smFISH using probes for HSV - 1 LAT ( green ) neuronal marker Ntrk1 ( TrkA ) ( red ) or fibroblast marker Col3a1 ( red ) . Signal intensities for LAT probe were quantified in neurons fibroblasts plotted by ImageJ . of LAT - positive were quantified displayed as bar graphs with mean ± . Left : 3 biological replicates , signal intensities of 50 were quantified for each replicate . Middle : 3 biological replicates , signal intensities of 30 were quantified for each replicate . Right : blue red dots represent number of biological replicates done for of each type . Scale bar , 10 μm . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * * * * , p < 0 . 0001 ) . P values are calculated using two - unpaired Student ' s t . P values > 0 . 05 were not significant ( ns ) .

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(D) smFISH images showing LAT RNA in Ntrk1-positive neurons. SCG-derived cultures were infected with HSV-1 in the presence of ACV and allowed to establish latency over 7 days before being processed for smFISH using probes for HSV-1 LAT (green) and neuronal marker Ntrk1 (TrkA) (red) or fibroblast marker Col3a1 (red). Signal intensities for the LAT probe were quantified in neurons and fibroblasts and plotted by ImageJ. The percentage of LAT-positive cells were quantified and displayed as bar graphs with mean ± SEM. Left: 3 biological replicates, signal intensities of 50 cells were quantified for each replicate. Middle: 3 biological replicates, signal intensities of 30 cells were quantified for each replicate. Right: the blue and red dots represent the number of biological replicates done for quantification of each cell type. Scale bar, 10 μm. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( A ) smFISH showing selected viral mRNAs in reactivating neurons probed for ICP27 ( immediate - early ) , UL30 ( early ) , UL36 ( true - late ) or markers , Ntrk ( neurons ) , or Col3a1 ( fibroblasts ) . was visualized using DAPI ( blue ) . To induce reactivation , latently - infected SCG - derived cultures were simultaneously treated with LY294002 ( 20 μM ) WAY - 150138 ( 20 μg / mL ) cultured for 48 hrs prior to processing for smFISH . Scale bar , 10 μm .

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(A) smFISH images showing selected viral mRNAs in reactivating neurons probed for ICP27 (immediate-early), UL30 (early), UL36 (true-late) or cellular markers, Ntrk (neurons), or Col3a1 (fibroblasts). Nuclear DNA was visualized using DAPI (blue). To induce reactivation, latently-infected SCG-derived cultures were simultaneously treated with LY294002 (20 μM) and WAY-150138 (20 μg/mL) and cultured for 48 hrs prior to processing for smFISH. Scale bar, 10 μm.


( B ) of positive for a viral mRNA or for GFP was quantified at different time points displayed as bar graphs with mean ± . Latently - infected cultures were untreated ( latent ) or treated with LY294002 WAY - 150138 for 18 , 48 , or 72 hrs . Each dot corresponds to value from each biological replicate . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - unpaired Student ' s t .

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(B) The percentage of cells positive for a viral mRNA or for GFP fluorescence was quantified at different time points and displayed as bar graphs with mean ± SEM. Latently-infected cultures were untreated (latent) or treated with LY294002 and WAY-150138 for 18, 48, or 72 hrs. Each dot corresponds to the value from each biological replicate. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.


( C - D ) Reactivating neurons probed simultaneously for two separate viral productive cycle mRNAs . Signal overlap is evident as or yellow foci in merge . Bar , 10 μm . In ( D ) , of positive nuclei with overlapping UL30 UL36 smFISH signals were quantified as mean ± ( n = 3 biological replicates ) . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - unpaired Student ' s t .

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(C-D) Reactivating neurons probed simultaneously for two separate viral productive cycle mRNAs. Signal overlap is evident as white or yellow nuclear foci in the merge. Bar, 10 μm. In (D), the percentage of positive nuclei with overlapping UL30 and UL36 smFISH signals were quantified as the mean ± SEM (n=3 biological replicates). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.


( B ) Differential gene expression of transcripts showed a significant upregulation of at least 19 viral transcript units ( TU ) a subset of genes in neurons undergoing reactivation Three host transcripts were also downregulated . Gadd45b is highlighted with a green asterisk . Vertical hashed blue lines indicate Log2 fold changes > 1 . 5 < - 1 . 5 , while horizontal hashed blue indicates an adjusted p - value of 0 . 01 . Significantly regulated genes are named shown as magenta circles .

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(B) Differential gene expression analysis of cellular transcripts showed a significant upregulation of at least 19 viral transcript units (TU) and a subset of cellular genes in neurons undergoing reactivation Three host transcripts were also downregulated. Gadd45b is highlighted with a green asterisk. Vertical hashed blue lines indicate Log2 fold changes >1.5 and < -1.5, while the horizontal hashed blue line indicates an adjusted p-value of 0.01. Significantly regulated genes are named and shown as magenta circles.


( A ) smFISH showing Gadd45b mRNA in latent ( UL36 - negative ) neurons / Gadd45b mRNA in viral UL36 - positive neurons . Latently - infected SCG neurons treated with LY294002 WAY - 150138 for 48 hrs were processed using smFISH to detect Gadd45b ( green ) viral UL36 ( red ) mRNA colocalization . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 μm . ( B ) smFISH processed as in ( A ) showing Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with LY294002 . Scale bar , 10 μm .

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(A) smFISH images showing nuclear Gadd45b mRNA in latent (UL36-negative) neurons and nuclear/cytoplasmic Gadd45b mRNA in viral UL36-positive neurons. Latently-infected SCG neurons treated with LY294002 and WAY-150138 for 48 hrs were processed using smFISH to detect Gadd45b (green) and viral UL36 (red) mRNA levels and colocalization. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. (B) smFISH images processed as in (A) showing nuclear Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with LY294002. Scale bar, 10 μm.


( E ) of HSV - 1 latently - infected neurons or reactivating neurons at 48 hrs post treatment with LY294002 WAY - 150138 were scored for either predominantly or / Gadd45b mRNA localization displayed as mean ± . Each purple dot represents a biological replicate experiment . ( F ) of or / Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with either LY294002 or in DMSO . Each blue dot represents a biological replicate experiment . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * * , p < 0 . 0001 ) .

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(E) The percentage of HSV-1 latently-infected neurons or reactivating neurons at 48 hrs post treatment with LY294002 and WAY-150138 were scored for either predominantly nuclear or nuclear/cytoplasmic Gadd45b mRNA localization and displayed as mean ± SEM. Each purple dot represents a biological replicate experiment. (F) Quantification of nuclear or nuclear/cytoplasmic Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with either LY294002 or in DMSO. Each light blue dot represents a biological replicate experiment. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ****, p<0.0001).


( H ) Combined smFISH immunofluorescence ( IF ) showing high GFP - expressing neurons ( GFP - positive ) or low GFP - expressing ( GFP absent ) neurons simultaneously probed for Gadd45b mRNA . Reactivation was induced by treatment with LY294002 WAY - 150138 for 60 hrs processed for simultaneous smFISH IF . Right panel shows ( as in G ) of smFISH signal of Gadd45b mRNA IF signal of GFP at 48 hrs post treatment with LY294002 WAY - 150138 ( n = 3 biological replicates ) . signal was quantified , plotted , analysed by ImageJ . Bar , 10 μm . Data information : : R R square values were calculated using Correlation by Prism 8 .

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(H) Combined smFISH and immunofluorescence (IF) images showing high GFP-expressing neurons (GFP-positive) or low GFP-expressing (GFP absent) neurons simultaneously probed for Gadd45b mRNA. Reactivation was induced by treatment with LY294002 and WAY-150138 for 60 hrs and processed for simultaneous smFISH and IF staining. Right panel shows quantification (as in G) of the smFISH signal intensity of Gadd45b mRNA and IF signal intensity of GFP protein at 48 hrs post treatment with LY294002 and WAY-150138 (n=3 biological replicates). The signal intensity was quantified, plotted, and analysed by ImageJ. Bar, 10 μm. Data information: : R and R square values were calculated using Correlation analysis by Prism 8.


( A ) Viral polymerase inhibitor phosphonoacetic acid ( PAA , 300 μg / ml ) treatment suppressed Gadd45b mRNA expression . Latently - infected SCG neurons were treated with LY294002 for 18 hrs to induce reactivation then treated with or without PAA until termination time - points indicated . was then collected of Gadd45b mRNA quantified by RT - qPCR ( n = 3 biological replicates , bars error bars are mean ± SD ) . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - unpaired Student ' s t .

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(A) Viral DNA polymerase inhibitor phosphonoacetic acid (PAA, 300 μg/ml) treatment suppressed Gadd45b mRNA expression. Latently-infected SCG neurons were treated with LY294002 for 18 hrs to induce reactivation then treated with or without PAA until the termination time-points indicated. RNA was then collected and levels of Gadd45b mRNA quantified by RT-qPCR (n=3 biological replicates, the bars and error bars are mean ± SD). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.


( C ) Immunofluorescence showing - enriched Gadd45b in uninfected neurons at 72 hrs post treatment with LY294002 . Scale bar , 20 μm . ( D ) signal showing of / Gadd45b at 72 hrs post treatment with LY294002 WAY - 150138 neurons . signal was quantified by ImageJ . 3 biological replicates , 10 EdU positive 10 EdU negative were quantified for each replicate . horizonal error bars are mean ± SD . ( E ) of Gadd45b - localized neurons were quantified from latent neurons treated with LY294002 WAY - 150138 for 72 hrs . Each dot represents each biological replicate ; 20 were quantified for each replicate . bars error bars are mean ± SD . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - unpaired Student ' s t .

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(C) Immunofluorescence images showing nuclear-enriched Gadd45b in uninfected neurons at 72 hrs post treatment with LY294002. Scale bar, 20 μm. (D) The signal intensity quantification showing the ratio of nuclear/cytoplasmic Gadd45b at 72 hrs post treatment with LY294002 and WAY-150138 neurons. The signal intensity was quantified by ImageJ. 3 biological replicates, 10 EdU positive cells and 10 EdU negative cells were quantified for each replicate. The horizonal line and error bars are mean ± SD. (E) The percentage of Gadd45b nuclear puncta-localized neurons were quantified from latent neurons treated with LY294002 and WAY-150138 for 72 hrs. Each dot represents each biological replicate; 20 cells were quantified for each replicate. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.


( G ) Latent SCG neurons treated with LY294002 WAY - 150138 for 72 hrs , Gadd45b - presenting neurons were ICP4 negative ( none observed in over 60 Gadd45b - positive neurons ) , but a small population ( ~ 5 % ) in Gadd45b - enriched neurons were ICP4 positive . Scale bar , 20 μm .

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(G) Latent SCG neurons treated with LY294002 and WAY-150138 for 72 hrs, the Gadd45b nuclear puncta-presenting neurons were ICP4 negative (none observed in over 60 Gadd45b nuclear puncta-positive neurons), but a small population (~5%) in Gadd45b nuclear-enriched neurons were ICP4 positive. Scale bar, 20 μm.


( A ) Depletion of Gadd45b expression using lentiviral - transduced shRNAs in latently - infected SCG neurons case cause HSV - 1 reactivation . HSV1 - GFP - Us11 latently - infected cultures were infected with lentiviruses expressing either two different shRNAs against Gadd45b mRNA or a non - silencing shRNA ( NS ) . Reactivation was quantified by scoring of wells expressing GFP after 5 days . Knockdown efficiencies for shRNAs in latently - infected SCG neurons were confirmed by RT - qPCR using collected in parallel . Reactivation rates were quantified from 30 wells for each condition , 3 biological replicates . RT - qPCR were quantified from 3 biological replicates . bars error bars are mean ± SD . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - unpaired Student ' s t . P values > 0 . 05 were not significant ( ns ) .

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(A) Depletion of Gadd45b expression using lentiviral-transduced shRNAs in latently-infected SCG neurons case cause HSV-1 reactivation. HSV1-GFP-Us11 latently-infected cultures were infected with lentiviruses expressing either two different shRNAs against Gadd45b mRNA or a non-silencing shRNA (NS). Reactivation was quantified by scoring the percentage of wells expressing GFP after 5 days. Knockdown efficiencies for individual shRNAs in latently-infected SCG neurons were confirmed by RT-qPCR using RNA collected in parallel. Reactivation rates were quantified from 30 wells for each condition, 3 biological replicates. RT-qPCR were quantified from 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( B ) smFISH shows that depletion of Gadd45b leads to increased UL36 expression . smFISH was performed 3 days post - lentiviral transduction . smFISH signals were quantified plotted using ImageJ Prism . Left : 3 biological replicates , 30 were quantified for each replicate . horizonal lines are mean . Right : 3 biological replicates . horizonal lines error bars are mean ± SD .

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(B) smFISH shows that depletion of Gadd45b leads to increased UL36 expression. smFISH was performed 3 days post-lentiviral transduction. smFISH signals were quantified and plotted using ImageJ and Prism. Left: 3 biological replicates, 30 cells were quantified for each replicate. The horizonal lines are mean. Right: 3 biological replicates. The horizonal lines and error bars are mean ± SD.


( C ) Ectopic expression of Gadd45b - Myc - Flag inhibits viral mRNA expression . Lentivirus - delivered ectopic expression of Gadd45b - Myc - Flag in SCG neurons was confirmed by immunoblotting . Latently - infected SCG neurons were transduced with Gadd45b - Myc - Flag for 3 days then treated with LY294002 WAY - 150138 to induce reactivation . was collected at indicated time - points ( LY treatment for 18 or 72 hours ) of viral UL36 mRNA quantified by RT - qPCR . 3 biological replicates . bars error bars are mean ± SD . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - unpaired Student ' s t . P values > 0 . 05 were not significant ( ns ) .

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(C) Ectopic expression of Gadd45b-Myc-Flag inhibits viral mRNA expression. Lentivirus-delivered ectopic expression of Gadd45b-Myc-Flag in SCG neurons was confirmed by immunoblotting. Latently-infected SCG neurons were transduced with Gadd45b-Myc-Flag for 3 days and then treated with LY294002 and WAY-150138 to induce reactivation. RNA was collected at the indicated time-points (LY treatment for 18 or 72 hours) and levels of viral UL36 mRNA quantified by RT-qPCR. 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( D ) Ectopic expression of Gadd45b - Myc - Flag antagonizes LY294002 - induced HSV - 1 reactivation . Latently - infected SCG neurons were either transduced with vector control ( Ctrl ) or Gadd45b - Myc - Flag from lentivirus for 3 days prior to addition of LY294002 . Reactivation was quantified by scoring of wells expressing GFP at indicated days . of wells expressing GFP - positive was scored as mean ± ( n = 3 biological replicates ) . Reactivation rates were quantified from 30 wells for each condition , 3 biological replicates .

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(D) Ectopic expression of Gadd45b-Myc-Flag antagonizes LY294002-induced HSV-1 reactivation. Latently-infected SCG neurons were either transduced with vector control (Ctrl) or Gadd45b-Myc-Flag from lentivirus for 3 days prior to addition of LY294002. Reactivation was quantified by scoring the percentage of wells expressing GFP fluorescence at the indicated days. The percentage of wells expressing GFP-positive was scored as the mean ± SEM (n=3 biological replicates). Reactivation rates were quantified from 30 wells for each condition, 3 biological replicates.


( F ) Ectopic expression of Gadd45b - Myc - Flag suppressed viral ICP4 mRNA expression . Latently - infected SCG neurons were transduced with Gadd45b for 3 days then treated with LY294002 WAY - 150138 for 72 hrs . Viral ICP4 mRNA was quantified by qRT - PCR . 3 biological replicates . bars error bars are mean ± SD . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - unpaired Student ' s t . P values > 0 . 05 were not significant ( ns ) .

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(F) Ectopic expression of Gadd45b-Myc-Flag suppressed viral ICP4 mRNA expression. Latently-infected SCG neurons were transduced with Gadd45b for 3 days and then treated with LY294002 and WAY-150138 for 72 hrs. Viral ICP4 mRNA was quantified by qRT-PCR. 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( G ) Immunoblots for viral ICP4 in latent SCG neurons treated with LY294002 WAY - 150138 in presence or absence of Gadd45b - Myc - Flag was quantified by ImageJ ( n = 3 biological replicates ) . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - unpaired Student ' s t . P values > 0 . 05 were not significant ( ns ) .

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(G) Immunoblots for viral ICP4 protein in latent SCG neurons treated with LY294002 and WAY-150138 in the presence or absence of Gadd45b-Myc-Flag was quantified by ImageJ (n=3 biological replicates). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( A ) THP1 were infected with shown strains of HSV - 1 ( MOI 3 ) . Supernatants were harvested from untreated cultures or infected for 12 or 18 hours for measurement of type I IFN bioactivity .

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(A) THP1 cells were infected with the shown strains of HSV-1 (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 hours for measurement of type I IFN bioactivity.


( B ) THP1 were treated with 0 . 1 μg / ml of acyclovir ( ACV ) infected with KOS HSV - 1 F HSV - 1 strains ( MOI 3 ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity .

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(B) THP1 cells were treated with 0.1 μg/ml of acyclovir (ACV) and infected with the KOS HSV-1 and F HSV-1 strains (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.


( C ) THP1 were treated with infectious or UV - inactivated HSV - 1 ( KOS ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity .

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(C) THP1 cells were treated with infectious or UV-inactivated HSV-1 (KOS). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.


( D ) MDMs were infected with ICP0 - deficient or revertant HSV - 1 ( KOS ) , MOI 3 . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity .

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(D) MDMs were infected with ICP0-deficient or revertant HSV-1 (KOS), MOI 3. Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.


( E ) THP1 - derived deficient for cGAS or STING were infected with shown strains of HSV - 1 ( MOI 3 ) or stimulated with poly ( I : C ) ( 2 µg / ml ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity .

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(E) THP1-derived cells deficient for cGAS or STING were infected with the shown strains of HSV-1 (MOI 3) or stimulated with poly(I:C) (2 µg/ml). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.


( A ) MDMs were infected for 18 h with HSV - 1 ( KOS ) , MOI 1 . viral were detected by mass spectrometry using iTRAQ - labeling . Identified viral are shown .

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(A) MDMs were infected for 18 h with HSV-1 (KOS), MOI 1. Cytosolic viral proteins were detected by mass spectrometry using iTRAQ-labeling. Identified viral proteins are shown.


( B ) MDMs were infected with KOS or F strains of HSV - 1 ( MOI 3 ) . lysates were harvested 12 hpi , of ICP27 β - actin were determined by Western blotting .

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(B) MDMs were infected with the KOS or F strains of HSV-1 (MOI 3). Cell lysates were harvested 12 hpi, and levels of ICP27 and β-actin were determined by Western blotting.


( C ) HFFs MDMs were infected with HSV - 1 ( KOS ) MOI 0 . 1 1 . 0 . Culture medium was replaced 3 hpi , isolated 24 hpi for viral plaque . Data are presented as means of triplicates + / - st . dev .

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(C) HFFs and MDMs were infected with HSV-1 (KOS) MOI 0.1 and 1.0. Culture medium was replaced 3 hpi, and isolated 24 hpi for viral plaque assay. Data are presented as means of triplicates +/- st.dev.


( D ) THP1 were infected with indicated strains of HSV - 1 ( MOI 3 ) for 12 hrs . lysates were isolated of ICP27 β - actin were determined by Western blotting .

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(D) THP1 cells were infected with the indicated strains of HSV-1 (MOI 3) for 12 hrs. Cell lysates were isolated and levels of ICP27 and β-actin were determined by Western blotting.


( A ) THP1 were infected with HSV - 1 KOS or an ICP27 - deficient mutant ( ΔICP27 ) on KOS genetic background ( MOI 3 ) . Supernatants were harvested from untreated cultures or infected for 12 or 18 hours for measurement of type I IFN bioactivity .

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(A) THP1 cells were infected with HSV-1 KOS or an ICP27-deficient mutant (ΔICP27) on KOS genetic background (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 hours for measurement of type I IFN bioactivity.


( B ) MDMs were infected with HSV - 1 KOS or ΔICP27 ( MOI 3 ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity .

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(B) MDMs were infected with HSV-1 KOS or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.


( C ) THP1 - derived deficient for cGAS or STING were infected with HSV - 1 KOS or ΔICP27 ( MOI 3 ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity .

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(C) THP1-derived cells deficient for cGAS or STING were infected with HSV-1 KOS or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.


( D ) HEK293T were transfected with 12 ng STING plasmid , 5 or 25 ng ICP27 plasmid , reporter gene constructs as indicated ( 2x104 per well ) . Reporter gene activity was measured in lysates isolated 24 h post transfection .

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(D) HEK293T cells were transfected with 12 ng STING plasmid DNA, 5 or 25 ng ICP27 plasmid DNA, and reporter gene constructs as indicated (2x104 cells per well). Reporter gene activity was measured in lysates isolated 24 h post transfection.


( E ) THP1 were infected with HSV - 1 KOS or ΔICP27 ( MOI 3 ) in presence or absence of acyclovir ( ACV ) . Supernatants were isolated 18 hpi for measurement of type I IFN

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(E) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3) in the presence or absence of acyclovir (ACV). Supernatants were isolated 18 hpi for measurement of type I IFN bioactivity


( A ) THP1 were infected with HSV - 1 KOS or ΔICP27 ( MOI 3 ) . extracts were isolated at indicated time points post infection , of ICP27 , RCC1 , GAPDH were determined by Western blotting .

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(A) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3). Cytoplasmic and nuclear extracts were isolated at the indicated time points post infection, and levels of ICP27, RCC1, and GAPDH were determined by Western blotting.


( B ) THP1 were infected with HSV - 1 KOS or ΔICP27 ( MOI 10 ) . were fixed at indicated time points post infection , stained with DAPI an antibody against ICP27 , visualized by confocal microscopy . Scale bar 10 μm .

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(B) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 10). The cells were fixed at the indicated time points post infection, and stained with DAPI and an antibody against ICP27, and visualized by confocal microscopy. Scale bar 10 μm.


( C , D ) THP1 were infected with HSV - 1 KOS , ΔICP27 or HSV - 1 ΔNLS . localization of major NLS is illustrated in panel C . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity .

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(C, D) THP1 cells were infected with HSV-1 KOS, ΔICP27 or HSV-1 ΔNLS. The localization of the major NLS is illustrated in panel C. Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.


( E , F ) THP1 were infected with HSV - 1 KOS or ΔICP27 ( MOI 3 ) . ( E ) extracts ( F ) were isolated at indicated time points post infection . Western blotting was used to determine of phospho - total TBK1 were determined in fractions of phospho - IRF3 RCC1 in fractions .

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(E, F) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3). Cytoplasmic (E) and nuclear extracts (F) were isolated at the indicated time points post infection. Western blotting was used to determine levels of phospho- and total TBK1 were determined in the cytoplasmic fractions and levels of phospho-IRF3 and RCC1 in the nuclear fractions.


( G - I ) HEK293T were ( G ) transfected with STING , TBK1 , IRF3 - 5D , ICP27 , together with reporter gene constructs indicated . Reporter gene activity was measured 24 h post transfection .

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(G-I) HEK293T cells were (G) transfected with STING, TBK1, IRF3-5D, and ICP27, together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post transfection.


( G - I ) HEK293T were ( ( H ) transfected with STING stimulated for 16 h with cGAMP ( 4 μg / ml ) together with reporter gene constructs indicated . Reporter gene activity was measured 24 h post transfection .

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(G-I) HEK293T cells were ((H) transfected with STING and stimulated for 16 h with cGAMP (4 μg/ml) together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post transfection.


( G - I ) HEK293T were ( I ) transfected with TRIF , MAVS , STING , ICP27 , together with reporter gene constructs indicated . Reporter gene activity was measured 24 h post transfection .

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(G-I) HEK293T cells were (I) transfected with TRIF, MAVS, STING, and ICP27, together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post transfection.


( A ) Lysates from THP1 infected with HSV - 1 KOS ( MOI 10 ) were subjected to immunoprecipitation using an anti - STING antibody , presence of TBK1 , STING , ICP27 in precipitates was detected by Western blotting .

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(A) Lysates from THP1 cells infected with HSV-1 KOS (MOI 10) were subjected to immunoprecipitation using an anti-STING antibody, and the presence of TBK1, STING, and ICP27 in the precipitates was detected by Western blotting.


( B , C ) THP1 were infected with HSV - 1 KOS ( MOI 10 ) for 6 8 h . were fixed stained with DAPI anti - ICP27 together with anti - pTBK1 ( B ) . stainings were visualized by confocal microscopy . Arrowheads , colocalizations between ICP27 pTBK1 . Scale bar 10 μm .

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(B, C) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h. The cells were fixed and stained with DAPI and anti-ICP27 together with anti-pTBK1 (B). The stainings were visualized by confocal microscopy. Arrowheads, colocalizations between ICP27 and pTBK1. Scale bar 10 μm.


( B , C ) THP1 were infected with HSV - 1 KOS ( MOI 10 ) for 6 8 h . were fixed stained with DAPI anti - ICP27 together with anti - STING ( C ) . stainings were visualized by confocal microscopy . Arrowheads , colocalizations between ICP27 STING . Scale bar 10 μm .

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(B, C) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h. The cells were fixed and stained with DAPI and anti-ICP27 together with anti-STING (C). The stainings were visualized by confocal microscopy. Arrowheads, colocalizations between ICP27 and STING. Scale bar 10 μm.


( D ) TBK1 was immunoprecipitated from whole lysates from HEK293T transfected with ICP27 each of adaptor TRIF , MAVS , STING . precipitates were immunoblotted for TBK1 , ICP27 , STING .

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(D) TBK1 was immunoprecipitated from whole cell lysates from HEK293T cells transfected with ICP27 and each of the adaptor proteins TRIF, MAVS, and STING. The precipitates were immunoblotted for TBK1, ICP27, and STING.


( E ) THP1 control STING KO were infected with HSV - 1 KOS ( MOI 10 ) for 6 8 h total lysates were generated . TBK1 was immunoprecipitated , subjected to Western blotting using antibodies against ICP27 , phospho - TBK1 ( pTBK1 ) , TBK1 .

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(E) THP1 control and STING KO cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h and total lysates were generated. TBK1 was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27, phospho-TBK1 (pTBK1), and TBK1.


( F ) THP1 control TBK1 KO were infected with HSV - 1 KOS ( MOI 10 ) for 8 h total lysates were generated . STING was immunoprecipitated , subjected to Western blotting using antibodies against ICP27 STING .

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(F) THP1 control and TBK1 KO cells were infected with HSV-1 KOS (MOI 10) for 8 h and total lysates were generated. STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING.


( G ) THP1 were infected with HSV - 1 KOS ( MOI 10 ) for 8 h in presence or absence of TBK1 inhibitor BX795 ( BX , 200 nM ) . Total lysates were generated STING was immunoprecipitated , subjected to Western blotting using antibodies against ICP27 STING .

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(G) THP1 cells were infected with HSV-1 KOS (MOI 10) for 8 h in the presence or absence of the TBK1 inhibitor BX795 (BX, 200 nM). Total lysates were generated and STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING.


( H ) THP1 were infected with HSV - 1 KOS ( MOI 10 ) for 6 8 h total lysates were generated . Phospho - TBK1 was immunoprecipitated , subjected to Western blotting using antibodies against ICP27 total TBK1 .

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(H) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h and total lysates were generated. Phospho-TBK1 was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and total TBK1.


( I ) TBK1 was immunoprecipitated from whole lysates from HEK293T transfected with ICP27 STING ( either WT or S366A ) . precipitates were immunoblotted for TBK1 , ICP27 , STING .

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(I) TBK1 was immunoprecipitated from whole cell lysates from HEK293T cells transfected with ICP27 and STING (either WT or S366A). The precipitates were immunoblotted for TBK1, ICP27, and STING.


( C ) THP1 were infected with HSV - 1 ( KOS ) , HSV - 2 ( HG52 ) , HSV - 1 ΔICP27 , or HSV - 1 ΔICP27 rescued with HSV - 2 ICP27 ( MOI 3 ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity .

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(C) THP1 cells were infected with HSV-1 (KOS), HSV-2 (HG52), HSV-1 ΔICP27, or HSV-1 ΔICP27 rescued with HSV-2 ICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.


( D ) RAW264 . 7 were infected with HSV - 1 ( KOS ) , PRV ( vBecker3 ) corresponding mutants lacking ICP27 ( UL54 in case of PRV ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity .

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(D) RAW264.7 cells were infected with HSV-1 (KOS), PRV (vBecker3) and the corresponding mutants lacking ICP27 (UL54 in the case of PRV). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.


( F ) THP1 were infected with HSV - 1 ( KOS ) , ΔRGG , or ΔICP27 ( MOI 3 ) . Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity . ICP27 expression in infected was determined by Western blotting .

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(F) THP1 cells were infected with HSV-1 (KOS), ΔRGG, or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. ICP27 expression in the infected cells was determined by Western blotting.


( G ) THP1 were infected with HSV - 1 KOS or HSV - 1 ΔRGG ( MOI 10 ) for 8 h total lysates were generated . STING was immunoprecipitated , subjected to Western blotting using antibodies against ICP27 STING .

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(G) THP1 cells were infected with HSV-1 KOS or HSV-1 ΔRGG (MOI 10) for 8 h and total lysates were generated. STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING.


A . Results of CRISPR screening analyzed by MAGeCK algorithm .

A. Results of CRISPR screening analyzed by the MAGeCK algorithm.


B . Wild type ( WT ) NCOA4 KO MEFs were pretreated with 25 μg / ml ferric ammonium citrate ( FAC ) for 16 h then chased with 20 μg / ml cycloheximide ( CHX ) for indicated times . Soluble ferritin expression were determined by immunoblotting with indicated antibodies .

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B. Wild type (WT) and NCOA4 KO MEFs were pretreated with 25 μg/ml ferric ammonium citrate (FAC) for 16 h and then chased with 20 μg/ml cycloheximide (CHX) for the indicated times. Soluble ferritin expression levels were determined by immunoblotting with the indicated antibodies.


C . WT MEFs untreated or treated with 10 μg / ml FAC for 12 h were lysed with SDS - containing sample buffer or Triton buffer , analyzed by immunoblotting with indicated antibodies .

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C. WT MEFs untreated or treated with 10 μg/ml FAC for 12 h were lysed with SDS-containing sample buffer or Triton buffer, and analyzed by immunoblotting with the indicated antibodies.


E . Subcellular fractions of MEFs treated with 20 μM Dfo for 12 h , 10 μg / ml FAC for 12 h , or 100 μM Deferoxamine ( Dfo ) for 6 h after treatment with 10 μg / ml FAC for 12 h were prepared by differential centrifugation as described in ( D ) . Each fraction was subjected to SDS - PAGE analyzed by immunoblotting with indicated antibodies .

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E. Subcellular fractions of MEFs treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12 h, or 100 μM Deferoxamine (Dfo) for 6 h after treatment with 10 μg/ml FAC for 12 h were prepared by differential centrifugation as described in (D). Each fraction was subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.


F . WT MEFs were untreated , treated with 10 μg / ml FAC for 12h or treated with 10 μg / ml FAC , 10 μM E64d 10 μg / ml pepstatin A ( pep A ) for 12h . were immunostained with anti - NCOA4 anti - LAMP1 . Scale bar , 10 μm . Quantitative data are shown as means ± SD of three biological replicates . At least 30 were quantified in each replicate . P = 0 . 033 ( Welch Two Sample t - ) .

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F. WT MEFs were untreated, treated with 10 μg/ml FAC for 12h or treated with 10 μg/ml FAC, 10 μM E64d and 10 μg/ml pepstatin A (pep A) for 12h. The cells were immunostained with anti-NCOA4 and anti-LAMP1. Scale bar, 10 μm. Quantitative data are shown as the means ± SD of three biological replicates. At least 30 cells were quantified in each replicate. P=0.033 (Welch Two Sample t-test).


G . NCOA4 were photobleached in MEFs stably expressing NCOA4 - GFP after treatment with 10 μg / ml FAC for 12 h , then recovery was monitored . p62 were photobleached in MEFs stably expressing GFP - human p62 , then recovery was monitored . Representative are shown . Time 0 indicates start of recovery after photobleaching . Scale bars , 2 μm . H . Quantitative data of recovery in ( G ) shown as means ± SD . 26 dots ( NCOA4 ) or 25 dots ( p62 ) were quantified from three biological replicates .

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G. NCOA4 puncta were photobleached in MEFs stably expressing NCOA4-GFP after treatment with 10 μg/ml FAC for 12 h, and then fluorescent recovery was monitored. p62 puncta were photobleached in MEFs stably expressing GFP-human p62, and then fluorescent recovery was monitored. Representative images are shown. Time 0 indicates the start of recovery after photobleaching. Scale bars, 2 μm. H. Quantitative data of fluorescence recovery in (G) shown as means ± SD. 26 dots (NCOA4) or 25 dots (p62) were quantified from three biological replicates.


I . Time - lapse imaging of MEFs stably expressing NCOA4 - GFP . Representative fusion fission are shown . Red arrowheads indicate fission event . Scale bars , 5 μm .

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I. Time-lapse imaging of MEFs stably expressing NCOA4-GFP. Representative fusion and fission images are shown. Red arrowheads indicate the fission event. Scale bars, 5 μm.


B . NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg / ml FAC for 12 h then fixed for imaging . were immunostained with anti - myc antibody . Representative are shown . Scale bar , 10 μm .

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B. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h and then fixed for imaging. Cells were immunostained with anti-myc antibody. Representative images are shown. Scale bar, 10 μm.


C . Number of per in ( B ) are shown as means ± of at least 40 in each condition from two biological replicates .

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C. Number of puncta per cell in (B) are shown as the means ± SEM of at least 40 cells in each condition from two biological replicates.


D . NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg / ml FAC for 12 h , fractionated , analyzed by immunoblotting with indicated antibodies .

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D. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h, fractionated, and analyzed by immunoblotting with the indicated antibodies.


E . DISOPRED3 disorder score of human NCOA4 .

E. DISOPRED3 disorder score of human NCOA4.


F . WT MEFs stably expressing NCOA4 IDR ( aa 167 - 334 ) fused with tandem FKBP were treated with 10 μg / ml FAC or 20 μM Dfo for 12 h then cultured with 0 . 1 nM FKBP ligand ( AP20187 ) for 6h . were fixed for imaging immunostained with anti - myc to analyze of fusion . Scale bar , 10μm . G . Number of in ( F ) are shown as means ± of at least 35 in each condition from two biological replicates . P < 0 . 0001 ( Dfo + ligand vs FAC + ligand ) P = 0 . 048 ( FAC vs FAC + ligand ) ( Kruskal - Wallis ANOVA with Dunn ' s multiple comparison ) . P values were adjusted using Bonferroni method .

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F. WT MEFs stably expressing NCOA4 IDR (aa 167-334) fused with tandem FKBP were treated with 10 μg/ml FAC or 20 μM Dfo for 12 h and then cultured with 0.1 nM FKBP ligand (AP20187) for 6h. The cells were fixed for imaging and immunostained with anti-myc to analyze puncta of the fusion protein. Scale bar, 10μm. G. Number of puncta in (F) are shown as means ± SEM of at least 35 cells in each condition from two biological replicates. P<0.0001 (Dfo+ligand vs FAC+ligand) and P=0.048 (FAC vs FAC+ligand) (Kruskal-Wallis ANOVA with Dunn's multiple comparison test). P values were adjusted using the Bonferroni method.


H . Coomassie - stained SDS - PAGE gel of purified . I . amount of co - purifying iron in purified in ( H ) was measured by inductively coupled plasma mass spectrometry ( ICP - MS ) . Data are shown as means ± SD of three biological replicates . P = 0 . 032 ( Welch Two Sample t - ) .

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H. Coomassie-stained SDS-PAGE gel of purified proteins. I. The amount of co-purifying iron in purified proteins in (H) was measured by inductively coupled plasma mass spectrometry (ICP-MS). Data are shown as means ± SD of three biological replicates. P=0.032 (Welch Two Sample t-test).


A . Purified NCOA4 were incubated with FeSO4 for 1 h under aerobic conditions fractionated . Samples were subjected to SDS - PAGE stained with Coomassie Brilliant Blue . B . Quantitative results in ( A ) are shown as means ± of three biological replicates .

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A. Purified NCOA4 proteins were incubated with FeSO4 for 1 h under aerobic conditions and fractionated. Samples were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. B. Quantitative results in (A) are shown as means ± SEM of three biological replicates.


F . Electron micrograph of NCOA4 KO MEFs reconstituted with myc - hNCOA4 . Cryosections were labeled with anti - myc antibody . Scale bar , 200nm . G . of diameter of NCOA4 condensates . Red shows mean . dot plot represents diameter of 36 condensates from two biological replicates .

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F. Electron micrograph of NCOA4 KO MEFs reconstituted with myc-hNCOA4. Cryosections were labeled with anti-myc antibody. Scale bar, 200nm. G. Quantification of the diameter of NCOA4 condensates. Red line shows the mean. The dot plot represents the diameter of 36 condensates from two biological replicates.


A . MEFs were treated with 10 μg / ml FAC for indicated times harvested . lysates were fractionated analyzed by SDS - PAGE with indicated antibodies . Asterisks indicate a non - specific band . TfR1 ; Transferrin receptor

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A. MEFs were treated with 10 μg/ml FAC for the indicated times and harvested. Cell lysates were fractionated and analyzed by SDS-PAGE with the indicated antibodies. Asterisks indicate a non-specific band. TfR1; Transferrin receptor 1


B . MEFs were cultured for 12 h in medium containing 10 μg / ml FAC supplemented with DMSO or 10 μM E64d 10 μg / ml pepstatin A ( pepA ) . Samples were analyzed by immunoblotting with indicated antibodies . black arrowhead indicates an intact ferritin band , arrowhead indicates a partial degradation band of ferritin .

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B. MEFs were cultured for 12 h in medium containing 10 μg/ml FAC supplemented with DMSO or 10 μM E64d and 10 μg/ml pepstatin A (pepA). Samples were analyzed by immunoblotting with the indicated antibodies. The black arrowhead indicates an intact ferritin band, and the white arrowhead indicates a partial degradation band of ferritin.


C . NCOA4 KO MEFs reconstituted with hNCOA4 - myc were cultured with 10 μg / ml FAC for indicated times fixed for imaging . were immunostained with ferritin myc antibodies . Scale bar , 10μm .

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C. NCOA4 KO MEFs reconstituted with hNCOA4-myc were cultured with 10 μg/ml FAC for the indicated times and fixed for imaging. Cells were immunostained with ferritin and myc antibodies. Scale bar, 10μm.


D . Myc ( NCOA4 ) were extracted , average signal intensities of myc ferritin in in ( C ) were plotted .

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D. Myc (NCOA4) puncta were extracted, and average signal intensities of myc and ferritin in the puncta in (C) were plotted.


E . MEFs expressing hNCOA4 - TurboID were treated with 10 μg / ml FAC for 6 h or 24 h then cultured with DMSO or 50 μM biotin for 30 min . were lysed with Triton buffer , lysates were pulled down by streptavidin beads . Inputs pulldown samples were analyzed with immunoblotting by indicated antibodies .

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E. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 6 h or 24 h and then cultured with DMSO or 50 μM biotin for 30 min. Cells were lysed with Triton buffer, and lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed with immunoblotting by indicated antibodies.


F . WT MEFs pretreated with 10 μg / ml FAC for 6 h or 24 h were chased with 20 μg / ml CHX analyzed by immunoblotting with indicated antibodies .

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F. WT MEFs pretreated with 10 μg/ml FAC for 6 h or 24 h were chased with 20 μg/ml CHX and analyzed by immunoblotting with the indicated antibodies.


G . NCOA4 KO MEFs reconstituted with myc - hNCOA4 were pretreated with 10 μg / ml FAC then cultured with 20 μg / ml CHX or 200 nM bafilomycin A1 ( BafA ) . were fixed for imaging immunostained using myc LAMP1 antibodies . Scale bar , 10μm .

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G. NCOA4 KO MEFs reconstituted with myc-hNCOA4 were pretreated with 10 μg/ml FAC and then cultured with 20 μg/ml CHX or 200 nM bafilomycin A1 (BafA). Cells were fixed for imaging and immunostained using myc and LAMP1 antibodies. Scale bar, 10μm.


A . WT , ATG7 KO , TAX1BP1 KO , FIP200KO MEFs were treated or not treated with 10 μg / ml FAC for 12 h . lysates were fractionated analyzed by immunoblotting with indicated antibodies .

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A. WT, ATG7 KO, TAX1BP1 KO, and FIP200KO MEFs were treated or not treated with 10 μg/ml FAC for 12 h. Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies.


C . HEK293T were transfected with GFP - NCOA4 indicated FLAG - His - TAX1BP1 variants . Soluble lysates extracted from transfected were immunoprecipitated with anti - FLAG antibody . Inputs immunoprecipitated samples were analyzed by immunoblotting with indicated antibodies .

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C. HEK293T cells were transfected with GFP-NCOA4 and the indicated FLAG-His-TAX1BP1 variants. Soluble lysates extracted from transfected cells were immunoprecipitated with anti-FLAG antibody. Inputs and immunoprecipitated samples were analyzed by immunoblotting with the indicated antibodies.


WT MEFs TAX1BP1 KO MEFs reconstituted with indicated TAX1BP1 variants were untreated or treated with 10 μg / ml FAC for 12 h . ( D , F ) lysates were fractionated analyzed by immunoblotting with indicated antibodies .

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WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were untreated or treated with 10 μg/ml FAC for 12 h. (D, F) Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies.


WT MEFs TAX1BP1 KO MEFs reconstituted with indicated TAX1BP1 variants were treated with 10 μg / ml FAC for 12 h . ( E ) were fixed for imaging immunostained to analyze ferritin localization . Scale bar , 10μm .

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WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were treated with 10 μg/ml FAC for 12 h. (E) Cells were fixed for imaging and immunostained to analyze ferritin localization. Scale bar, 10μm.


A . MEFs were pretreated with 10 μg / ml FAC for 12 h then cultured with 100 μM Dfo for indicated time . were lysed with SDS - containing sample buffer or Triton buffer . Lysates were analyzed by immunoblotting with indicated antibodies .

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A. MEFs were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo for the indicated time. Cells were lysed with SDS-containing sample buffer or Triton buffer. Lysates were analyzed by immunoblotting with the indicated antibodies.


B . MEFs were treated with 20 μM Dfo for 12 h , 10 μg / ml FAC for 12h , or 100 μM Dfo for 6 h after treatment with 10 μg / ml FAC for 12 h . Triton - soluble lysates were subjected to glycerol gradient ultracentrifugation .

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B. MEFs were treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12h, or 100 μM Dfo for 6 h after treatment with 10 μg/ml FAC for 12 h. Triton-soluble lysates were subjected to glycerol gradient ultracentrifugation analysis.


C . WT TAX1BP1 KO MEFs were pretreated with 25 μg / ml FAC for 16 h then cultured with 100 μM Dfo . Lysates were fractionated then analyzed by immunoblotting with indicated antibodies . D . Soluble FTL bands in ( C ) were quantified by densitometry . Data are shown as means ± SD of three biological replicates .

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C. WT and TAX1BP1 KO MEFs were pretreated with 25 μg/ml FAC for 16 h and then cultured with 100 μM Dfo. Lysates were fractionated and then analyzed by immunoblotting with the indicated antibodies. D. Soluble FTL bands in (C) were quantified by densitometry. Data are shown as the means ± SD of three biological replicates.


E . TAX1BP1 KO MEFs ( sg1 clone ) stably expressing FLAG - OsTIR1 ( F74G ) FLAG - mAID - mTAX1BP1 were pretreated with 10 μg / ml FAC for 12 h , cultured with DMSO or 1 μM 5 - Ph - IAA for 2 h , then treated with 100 μM Dfo . Soluble lysates were analyzed by immunoblotting with indicated antibodies . 5 - Ph - IAA , a derivative of Auxin ; mAID , mini auxin - inducible

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E. TAX1BP1 KO MEFs (sg1 clone) stably expressing FLAG-OsTIR1 (F74G) and FLAG-mAID-mTAX1BP1 were pretreated with 10 μg/ml FAC for 12 h, cultured with DMSO or 1 μM 5-Ph-IAA for 2 h, and then treated with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies. 5-Ph-IAA, a derivative of Auxin; mAID, mini auxin-inducible degron


F . TAX1BP1 KO MEFs ( clone sg1 ) in which ATG7 was knocked down were pretreated with 10 μg / ml FAC for 12 h then cultured with 100 μM Dfo . Soluble lysates were analyzed by immunoblotting with indicated antibodies .

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F. TAX1BP1 KO MEFs (clone sg1) in which ATG7 was knocked down were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies.


G . TAX1BP1 KO MEFs ( clone sg1 ) stably expressing myc - hNCOA4 pretreated with 25 μg / ml FAC for 16 h were cultured with 100 μM Dfo , soluble lysates were analyzed by immunoblotting with indicated antibodies .

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G. TAX1BP1 KO MEFs (clone sg1) stably expressing myc-hNCOA4 pretreated with 25 μg/ml FAC for 16 h were cultured with 100 μM Dfo, and soluble lysates were analyzed by immunoblotting with the indicated antibodies.


H , I . NCOA4 KO MEFs reconstituted with NCOA4 variants were pretreated with 10 μg / ml FAC for 12 h then chased with ( H ) 20 μg / ml CHX or ( I ) 100 μM Dfo for indicated times . Soluble lysates were analyzed by immunoblotting with indicated antibodies .

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H, I. NCOA4 KO MEFs reconstituted with NCOA4 variants were pretreated with 10 μg/ml FAC for 12 h and then chased with (H) 20 μg/ml CHX or (I) 100 μM Dfo for the indicated times. Soluble lysates were analyzed by immunoblotting with the indicated antibodies.


J . MEFs expressing hNCOA4 - TurboID were treated with 10 μg / ml FAC for 3 h ( indicated as F ) or 100 μM DFO for 3 h after treatment with 10 μg / ml FAC for 12 h ( indicated as D ) , then cultured with DMSO or 50 μM biotin for 30 min . Triton - soluble lysates were pulled down by streptavidin beads . Inputs pulldown samples were analyzed by immunoblotting with indicated antibodies . black arrowheads indicate hNCOA4 - TurboID endogenous NCOA4 , respectively .

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J. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 3 h (indicated as F) or 100 μM DFO for 3 h after treatment with 10 μg/ml FAC for 12 h (indicated as D), and then cultured with DMSO or 50 μM biotin for 30 min. Triton-soluble cell lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed by immunoblotting with the indicated antibodies. The black and white arrowheads indicate hNCOA4-TurboID and endogenous NCOA4, respectively.


A . U2OS were either mock or treated with 10 μM XL413 for 30 minutes , at which point 4 mM HU was added further incubated for indicated times . Whole extracts were then analysed by western blotting with indicated antibodies . Reduction of phosphorylation of Ser40 / 41 on MCM2 is indicative of CDC7 inhibition by XL413 . Representative of two independent experiments .

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A. U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes, at which point 4 mM HU was added and cells further incubated for the indicated times. Whole cell extracts were then analysed by western blotting with the indicated antibodies. Reduction of phosphorylation of Ser40/41 on MCM2 is indicative of CDC7 inhibition by XL413. Representative of two independent experiments.


B . U2OS were either mock treated or treated with 10 μM XL413 , 4 mM HU or both for 24 hours before performing neutral comet assays . Representative of comets are shown . Scale bar = 100 μM . In dot plots ~ 800 comets per each condition were analysed , means are indicated with a red their value shown above plot . Data are from two independent experiments .

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B. U2OS cells were either mock treated or treated with 10 μM XL413, 4 mM HU or both for 24 hours before performing neutral comet assays. Representative images of comets are shown. Scale bar = 100 μM. In the dot plots ~800 comets per each condition were analysed, means are indicated with a red line and their value shown above the plot. Data are from two independent experiments.


C . U2OS were either untreated or treated with 10 μM XL413 or with 5 µM AZD6738 alone or in combination , where indicated , after 30 minutes 4 mM HU was added for a further 2 hours . Whole extracts were analysed by western blotting with indicated antibodies . Total stain ( TPS ) as a loading control . Representative of two independent experiments .

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C. U2OS cells were either untreated or treated with 10 μM XL413 or with 5 µM AZD6738 alone or in combination and, where indicated, after 30 minutes 4 mM HU was added for a further 2 hours. Whole cell extracts were analysed by western blotting with the indicated antibodies. Total protein stain (TPS) as a loading control. Representative of two independent experiments.


D . U2OS were either untreated or treated with 4 mM HU for 5 hours in presence or absence of 10 μM XL413 which had either been added to 30 minutes prior ( pre - treatment ) or at same time ( co - treatment ) to addition of HU . Whole extracts were analysed by western blotting with indicated antibodies total stain ( TPS ) is displayed as a loading control . Representative of two independent experiments .

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D. U2OS cells were either untreated or treated with 4 mM HU for 5 hours in the presence or absence of 10 μM XL413 which had either been added to cells 30 minutes prior (pre-treatment) or at the same time (co-treatment) to the addition of HU. Whole cell extracts were analysed by western blotting with the indicated antibodies and total protein stain (TPS) is displayed as a loading control. Representative of two independent experiments.


B . CSK soluble - enriched fractions were analysed by western blotting with indicated antibodies . Representative of two independent experiments .

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B. CSK soluble and chromatin-enriched protein fractions were analysed by western blotting with the indicated antibodies. Representative of two independent experiments.


C - D . Flow cytometry to assess of pS139 Histone H2AX ( c ) . Histograms show mono - parametric of against pS139 Histone H2AX . Histograms are overlaid to appreciate changes in pS139 Histone H2AX upon treatment ( Red lines ) relative to appropriate experimental baseline controls ( Grey lines ) . Data are representative of two independent experiments .

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C-D. Flow cytometry analysis to assess levels of pS139 Histone H2AX (c). Histograms show the mono-parametric analysis of cell count against pS139 Histone H2AX intensity. Histograms are overlaid to appreciate changes in pS139 Histone H2AX intensity upon treatment (Red lines) relative to appropriate experimental baseline controls (Grey lines). Data are representative of two independent experiments.


A . U2OS were labelled with EdU for 30 minutes , then EdU was washed were further incubated for either 1 or 2 hours in presence of thymidine . At indicated time points were fixed binding to EdU - labelled captured by mediated Pull down technique ( DmChP ) . Graphical experimental outline is shown above by western blot of relevant in both input captured material .

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A. U2OS cells were labelled with EdU for 30 minutes, then EdU was washed and cells were further incubated for either 1 or 2 hours in presence of thymidine. At the indicated time points cells were fixed and proteins binding to EdU-labelled DNA captured by DNA mediated chromatin Pull down technique (DmChP). Graphical experimental outline is shown above the analysis by western blot of relevant proteins in both input and captured material.


B . U2OS were labelled with EdU for 30 minutes then treated with 4 mM HU for 2 hours in presence or absence of 10 μM XL413 . binding to EdU - labelled captured by DmChP were then analysed by western blot as above .

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B. U2OS cells were labelled with EdU for 30 minutes and then treated with 4 mM HU for 2 hours in the presence or absence of 10 μM XL413. Proteins binding to EdU-labelled DNA captured by DmChP were then analysed by western blot as above.


C . As in panel B but incubation with HU was extended to 24 hours . Black arrow indicates MRE11 electrophoretic - mobility shift .

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C. As in panel B but incubation with HU was extended to 24 hours. Black arrow indicates MRE11 electrophoretic-mobility shift.


D . U2OS were either mock or treated with 10 μM XL413 for 30 minutes , at which point 4 mM HU was added incubated for further 24 hours . Extracts prepared from HU treated were then incubated in presence or absence of λ - phosphatase . were analysed by western blotting with anti - MRE11 antibodies . Total stain ( TPS ) is used as a loading control . Black arrow indicates MRE11 electrophoretic - mobility shift .

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D. U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes, at which point 4 mM HU was added and cells incubated for further 24 hours. Extracts prepared from HU treated cells were then incubated in presence or absence of λ-phosphatase. Proteins were analysed by western blotting with anti-MRE11 antibodies. Total protein stain (TPS) is used as a loading control. Black arrow indicates MRE11 electrophoretic-mobility shift.


E . U2OS were treated with 4 mM HU in presence or absence of 10 μM XL413 for 24 hours . PCNA ( green ) MRE11 ( red ) were detected by immunofluorescence . Inset I - II represent enlargements of selected region of merged .

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E. U2OS cells were treated with 4 mM HU in the presence or absence of 10 μM XL413 for 24 hours. PCNA (green) and MRE11 (red) were detected by immunofluorescence. Inset I-II represent enlargements of selected region of the merged images.


F . of PCNA MRE11 colocalization was assessed with ImageJ in ~ 70 randomly selected for each condition from four biological replicates expressed as Pearson ' s correlation coefficient . Error bars represent . Statistical significance was assessed by student t ( * p ˂ 0 .

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F. Quantification of PCNA and MRE11 colocalization was assessed with ImageJ in ~70 randomly selected cells for each condition from four biological replicates and expressed as Pearson's correlation coefficient. Error bars represent SEM. Statistical significance was assessed by student t test (*p˂0.05


A . MCF10A were either mock or treated with 10 μM XL413 labelled with IdU ( red ) for 30 minutes at which point 4 mM HU was added for 2 hours . HU was then washed off labelled with CldU ( green ) in continued presence or absence of XL413 . A set of representative fibres from each condition is shown of IdU ( red ) only tracts is plotted . At least 200 replication forks were analysed for each condition . Error bars represent from three biological repeats statistical significance was assessed by student t ( * * p ˂ 0 .

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A. MCF10A cells were either mock or treated with 10 μM XL413 and labelled with IdU (red) for 30 minutes at which point 4 mM HU was added for 2 hours. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413. A set of representative DNA fibres from each condition is shown and the percentage of IdU (red) only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent the SEM from three biological repeats and statistical significance was assessed by the student t test (** p˂0.01