Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , A flow chart for BMDM isolation and RNA-seq analysis, created through BioRender. b , Volcano plot showing the upregulated and downregulated genes in BMDM infected with VSV (12 hpi, MOI = 1.5) compared to non-infected control. Blue dot indicates downregulated genes, red dot indicates upregulated genes, green dot indicates upregulated interferon-stimulated genes (ISGs). c , KEGG analysis of upregulated genes in ( b ). d , Amino acid sequence comparison between human and mouse PARP10. e , Schematic diagram showing the KO of the mouse Parp10 gene. f , g , Western blot showing the PARP10 protein expression in multiple tissues from Parp10 +/+ and Parp10 -/- mice. Normalization to GAPDH is shown in ( g ). h , Relative expression of PARP10 from human single-cell RNA-seq data extracted from https://www.proteinatlas.org/ . i , Western blot validating the MAVS KO in HEK293T cells. j , Normalized mRNA levels of Ifnb1 / Ccl5 / Isg56 in WT/BX795-treated/MAVS KO HEK293T cells transfected with GFP vector or GFP-PARP10, followed by VSV infection (8 hpi, MOI = 1.5). n = 3. k , Western blot to determine the levels of p-IRF3, total IRF3, and PARP10 in Parp10 +/+ and Parp10 -/- BMDM at different time points post-VSV (MOI = 1.5) infection. l , Schematic diagram showing the EGFP KI strategy for the generation of the Parp10 mEGFP/mEGFP mice.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Isolation, RNA Sequencing, Infection, Control, Sequencing, Comparison, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Fold changes of differentially expressed ARTD genes in BMDM with VSV (12 hpi, MOI = 1.5) infection compared to non-infected control. n = 3. b , Normalized VSV mRNA levels in HEK293T cells transfected with different ARTD genes and infected with VSV (8 hpi, MOI = 1.5). n = 3. c , Normalized PARP10 mRNA levels in Parp10 +/+ and Parp10 -/- BMDM at different time points post-VSV (MOI = 1.5) infection. n = 3. d , Normalized VSV mRNA levels in Parp10 +/+ and Parp10 -/- BMDM infected with VSV (12 hpi, MOI = 1.5). n = 3. e , f , Normalized VSV mRNA levels in spleen ( e ) and liver ( f ) samples isolated from VSV (1x10 8 PFU/mouse, 24 hpi) infected Parp10 +/+ ( n = 4) and Parp10 -/- ( n = 5) mice. g , Normalized SFV mRNA levels in Parp10 +/+ and Parp10 -/- BMDM infected with SFV (12 hpi, MOI = 0.5). n = 3. h , Normalized Ifna1 and Ifnb1 mRNA levels in Parp10 +/+ and Parp10 -/- BMDM infected with VSV (12 hpi, MOI = 1.5). n = 3. i , Normalized VSV mRNA levels in BHK-21 cells transfected with GFP or GFP-PARP10 and infected with VSV (8 hpi, MOI = 1.5). n = 3. j , Normalized VSV mRNA levels in WT/BX795-treated/MAVS KO HEK293T cells transfected with GFP or GFP-PARP10, followed by VSV infection (8 hpi, MOI = 1.5). n = 3. k , Normalized VSV mRNA levels in HEK293T cells transfected with GFP or GFP-PARP10 during viral binding, entry, and endocytosis steps. The endocytosis steps were blocked with 10 μM Chloroquine. l , Western blot showing the expression of mEGFP-PARP10 in Parp10 +/+ and Parp10 mEGFP/mEGFP BMDM with or without VSV infection (MOI = 1.5) for the indicated times. m , Representative images of colocalization between endogenous PARP10 and VSV inclusion labeled by N protein in Parp10 mEGFP/mEGFP BMDM infected with VSV (6 hpi, MOI = 1.5) or in non-infection control. n , Control IF experiment as ( m ) except without primary antibody staining for PARP10, thus excluding the leaky signal from VSV-N channel. Data are shown as mean ± SD. in ( b )-( k ). Scale bar: 10 μm for ( m ) and ( n ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Infection, Control, Transfection, Isolation, Binding Assay, Western Blot, Expressing, Labeling, Staining

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images to show the colocalization between GFP-PARP10 foci and VSV inclusions. b , Representative images of GFP-PARP10 inducible U2OS cells stimulated with different doxycycline doses. Higher expression induced more foci formation. c , Western blot showing the protein expression of GFP-PARP10 with different doxycycline doses. d , Calculation of the cellular threshold concentration of GFP-PARP10 to form cytoplasmic foci. The absolute concentration in cells is converted by a standard curve created by measuring the intensity of recombinant PARP10 protein. Each black dot indicates one cell with condensates, the red dot indicates the cell without condensates, and the line marks the threshold concentration for condensate formation. At least 100 cells were measured and analyzed. e , f , FRAP experiments for GFP-PARP10 foci in cells. The fluorescent intensity in the region of interest (ROI) in ( e ) is quantified in ( f ). g , Representative images for the GFP-PARP10 droplets fusion process. h , Representative images of GFP-PARP10 foci with LysoTracker, lipid dye, RAB7 staining, and mCherry-RAB5 co-expression. i , Representative images for GFP-PARP10 foci in cells stained with G3BP1 and LSM14A. j , Correlative light electron microscopy (CLEM) images of GFP-PARP10 foci. GFP-PARP10 foci were identified by the high electron density and correlation with GFP fluorescence. MitoTracker in red indicates the location of mitochondria. k , Representative images of recombinant PARP10 protein condensation induced by 10% Ficoll under the indicated salt and protein concentrations. l , Western blot showing the ADP-ribosylation of recombinant PARP10 with the indicated concentrations of NAD + in vitro . The ADPr-modified PARP10 was blotted with eAf1521. m , Representative images of Ficoll-induced PARP10 condensation in vitro with the treatment of 1,6-Hexanediol or 2,5-Hexanediol. Data are shown as mean ± SD. in ( d ) and ( f ). Scale bar: 10 μm for ( a ), ( b ), ( e ), ( g )-( i ). 2 μm for ( j ) and 500 nm for the inset. 20 μm for ( k ) and ( m ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Expressing, Western Blot, Concentration Assay, Recombinant, Staining, Electron Microscopy, Fluorescence, In Vitro, Modification

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of endogenous PARP10 staining in WT and PARP10 KO U2OS cells. b , c , Western blot showing the expression of endogenous PARP10 in U2OS cells and verification of PARP10 KO. PARP10 was stained with commercial antibody from Proteintech ( b ) and homemade antibody ( c ). d , Western blot showing the induction of endogenous PARP10 in U2OS with IFNβ, IFNγ, and TNFα stimulation. Scale bar: 10 μm for ( a ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Staining, Western Blot, Expressing

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , The schematic diagram for PARP10 protein and predicted intrinsically disordered region (IDR) and net charge per residue (NCPR) pattern are shown below. b , The schematic diagram for PARP10 mutants used. c , Representative images of cells stably expressing PARP10 mutants in ( b ). d , Quantification of the percentage of cells with foci in ( c ). e , Western blot showing the relative protein expression of each construct used in ( c ). f , Representative images of oligo-dT FISH staining in GFP-PARP10 condensate. g , Representative images of GFP-PARP10 condensates with poly(I:C) treatment. Cells with G3BP1 foci were indicative of successful poly(I:C) transfection. Arrow indicates cells transfected with poly(I:C) and arrowhead indicates cells absent of poly(I:C). h , Representative images of GFP-PARP10 condensates with RNase A (100 μg/mL) treatment for 20 min. PBS was used as control buffer. i , Representative images of PARP10 WT condensation in the presence of indicated concentrations of polyA RNA or total RNA with or without 10% Ficoll. Data are shown as mean ± SD. in ( d ). Scale bar: 10 μm for ( c ), ( f )-( h ). 20 μm for ( i ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Residue, Stable Transfection, Expressing, Western Blot, Construct, Staining, Transfection, Control

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of ADPr staining with eAf1521 and mitochondria staining with Tom20. b , RRM sequence alignment of PARP10 with TDP43 and hnRNPA1. c , Diagram showing the constructs used in the assay, and the representative images are shown below for each construct. Scale bar: 10 μm. d , Quantification of percentage of cells with foci formation in ( c ). e , Western blot showing the interaction between PARP10 domains (RRM2/3, KH1, or KH2) and different fragments in ( c ), determined by co-immunoprecipitation assay.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Staining, Sequencing, Construct, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative 2D classes of the recombinant, full-length PARP10 protein under cryo-EM. Scale bar: 200 Å. b , Cryo-EM map of PARP10 RRM1-3 pentamer obtained from 3D reconstruction and refinement. c , Structural model of PARP10 RRM1-3 pentamer, which is generated by docking AlphaFold-predicted models of PARP10 RRM1-3 domains into the cryo-EM map shown in ( b ). The PARP10 RRM1-3 oligomer adopts an open ring conformation with an angle of 140 degrees. d , Detailed illustration of the RRM1-3 dimer interface at two orientations. One dimeric interface of RRM1-3 is zoomed in, and residues in the interface between two RRM1-3 protomers are shown in sticks. e , Two protomers of PARP10 RRM1-3 contact with each other by electrostatic interactions. One protomer is represented with electrostatic potential surface, and the other is shown in cartoon. f,g , Representative images of cells ( f ) stably expressing PARP10 mutants depicted in ( g ). Scale bar: 10 μm. h , Quantification of the percentage of cells with foci in ( f ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Recombinant, Cryo-EM Sample Prep, Generated, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , A representative cryo-EM micrograph of full-length PARP10 protein. Scale bar: 500 Å. b , 2D class averages were selected for 3D reconstruction. Scale bar: 180 Å. c , Flow chart for cryo-EM data analysis. d , Maps of two classes of PARP10 RRM rings with different twisted angles. e , 3D variability analysis and non-uniform refinement of RRM oligomers. f - h , Map local resolution ( f ), Fourier shell correlation (FSC) curves ( g ), and particle distribution used in 3D reconstruction ( h ) of PARP10 RRM1-3 pentamer. i , j , 2D classes and corresponding models to show the two types of higher-order oligomerization of RRM1-3. Scale bar: 200 Å.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , b , Representative images for GFP-PARP10 condensate in cells stained with ADPr recognition antibodies (E6F6A and 10H) ( a ) and reagent (hsPARP14 macro2-3-rFC) ( b ). The catalytic deficient mutant GFP-PARP10 G888W served as a negative control. c , d , FRAP experiments for GFP-PARP10 and mCherry-eAf1521 co-expression cells. The fluorescent intensity in the region of interest (ROI) in ( c ) is quantified in ( d ). e , f , Representative images ( e ) and line quantification ( f ) of fluorescein-NAD + enrichment in PARP10-WT but not G888W condensates induced by PEG-8000 in vitro . g-i , Representative images of the reactions to show the PEG-8000 induced PARP10 condensation ( g ). Western blot showing ADP-ribosylation of 10 μM PARP10 with or without 10% PEG-8000 addition ( h ). The quantification of the ADP-ribosylation activity was shown in ( i ). n = 2. j , k , Western blot showing the ADP-ribosylation activity of PARP10 WT and condensation-deficient mutants 3RE and 3EA in cells ( j ). ADP-ribosylation levels were blotted with eAf1521. The quantification of the ADP-ribosylation activity was shown in ( k ). n = 3. Data are shown as mean ± SD. in ( d ), ( i ), ( k ). Scale bar: 10 μm for ( a )-( c ); 20 μm for ( e ) and ( g ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Staining, Mutagenesis, Negative Control, Expressing, In Vitro, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of cells stably expressing GFP-PARP10 WT and catalytic deficient mutants (G888W and ΔCD). ADPr signals stained by eAf1521. b , Western blot showing the relative protein expression of each construct used in ( a ). c , Box plot for the partition coefficient (PC) of PARP10 WT and mutants in ( a ). Each symbol represents one condensate. At least 25 condensates were measured and analyzed. d , Phase separation threshold concentration was measured for PARP10 WT and mutants in ( a ). The absolute concentration in cells is converted by a standard curve created by measuring the intensity of recombinant PARP10 protein. Each symbol represents one cell. At least 100 cells were measured and analyzed. Each black dot indicates one cell with condensates, the red dot indicates the cell without condensates, and the line marks the threshold concentration for condensate formation. e , f , Representative images ( e ) and quantification ( f ) for FRAP experiments on PARP10 WT ( n = 25), G888W ( n = 11), and H887E ( n = 17). The arrow indicated the bleached foci. Data are shown as mean ± SD. g , h , Representative images of GFP-PARP10 WT ( g ) and G888W ( h ) mutant treated with PARP10 inhibitor OUL-35. i , Box plot quantification of the diameter of PARP10 condensate in ( g ) and ( h ). j , Representative images of GFP-PARP10 co-expression with mCherry-MACROD2/TARG1/ARH3/PARG103. k , Box plot quantification of the diameter of PARP10 condensate in ( j ). Data are shown as mean ± SD. l , Representative images of PARP10 WT/H887E condensation induced by 10% Ficoll with or without NAD + addition. m , Western blot showing the ADP-ribosylation and protein levels of PARP10 WT and H887E in ( l ). n , Western blot showing CHX chase assay for IFNβ-induced endogenous PARP10 protein level in U2OS cells coupled with MG132 or Bafilomycin A1 treatments. o , Western blot showing ADP-ribosylation of GFP-PARP10 WT in cells coupled with MG132 treatment for the indicated times. The accumulation of ubiquitin signal was detected with P4D1 ubiquitin antibody. The catalytic deficient mutant GFP-PARP10 G888W serves as a negative control. p , Western blot showing the accumulation of GFP-PARP10 protein in cells treated with different concentrations of PARP10 activity inhibitor OUL35. q , Quantification of the GFP-PARP10 protein levels in ( p ). Data are shown as mean ± SD. in ( q ). Scale bar: 10 μm for ( a ), ( e ), ( g ), ( h ), ( j ). 20 μm for ( l ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Stable Transfection, Expressing, Staining, Western Blot, Construct, Concentration Assay, Recombinant, Mutagenesis, Ubiquitin Proteomics, Negative Control, Activity Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , List of ADPr hydrolases used in the study. b , Western blot showing the effect of mCherry-tagged ADPr hydrolase and its catalytic deficient (CD) mutants on the GFP-PARP10 auto-ADP-ribosylation in HEK293T cells. The auto-ADP-ribosylation was probed by eAf1521. c , Representative images of GFP-PARP10 condensates stained for FK2. d , TUBE pulldown assay showing the ubiquitination of PARP10 in response to MG132 and IFNβ treatment. GST bead only and PARP10 KO cells were used as controls. e , Western blot showing the ubiquitination of PARP10 in cells. f , Representative images of GFP-PARP10 WT/G888W stable cells treated with CHX for different times. g , Box plot quantification of the diameter of PARP10 condensate in ( f ). Scale bar: 10 μm for ( c ) and ( f ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Western Blot, Staining, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a - d , Representative images of cells co-transfected with GFP/GFP-PARP10 WT/G888W and mCherry-DTX2 ( a ), DTX3L ( b ), DTX3 ( c ), DTX4 ( d ). e , Representative images of cells transfected with GFP-PARP10 WT/G888W and stained with DTX1 antibody. f , Representative images of cells co-transfected with GFP-PARP10 WT and mCherry-DTX2 WT or 5 Mut which disrupts the ADPr binding. g , Western blot showing the interaction between GFP-PARP10 WT and mCherry/mCherry-DTX2 WT/5 Mut, determined by co-immunoprecipitation assay. h , Representative images and summary of GFP-PARP10 condensates colocalized with FK2-positive TRIMs, but not with the FK2-negative catalytic mutants. Scale bar: 10 μm for ( a )-( f ) and ( h ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Transfection, Staining, Binding Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Normalized VSV mRNA levels in HEK293T cells transfected with GFP/GFP-PARP10 WT/G888W/3RE and infected with VSV (8 hpi, MOI = 1.5). n = 6. b , Normalized VSV mRNA levels in Parp10 +/+ , Parp10 -/- , Parp10 G837W/G837W and Parp10 3E/3E BMDM with or without VSV infection (12 hpi, MOI = 1.5). n = 5. c , NAD + levels in HEK293T cells transfected with GFP/GFP-PARP10 WT/G888W, with or without VSV infection (8 hpi, MOI = 1.5), measured by MS. n = 3. d , NAD + levels in WT U2OS, HEK293T and BMDM cells infected with VSV (MOI = 1.5) for the indicated times, measured by MS. n = 3. e , Heatmap of gene expression in BMDM infected with VSV (12 hpi, MOI = 1.5) or not. n = 3. Genes related to NAD + metabolic process were shown. f , Fold changes of NAD + consuming genes in BMDM with VSV (12 hpi, MOI = 1.5) infection relative to non-infected control. n = 3. g , NAD + levels in Parp10 +/+ (WT), Parp10 -/- , and Parp10 3E/3E BMDM with or without VSV infection (12 hpi, MOI = 1.5), measured by MS. n = 3. h , Schematic diagram of the NAD + salvage pathway and the effect of NAMPT inhibitor FK866. i , Normalized VSV mRNA levels in HEK293T cells transfected with GFP/GFP-PARP10 WT, followed by the supplement of the indicated concentrations of NR and VSV infection (8 hpi, MOI = 1.5). n = 3. j , NAD + levels of cells in ( i ), determined by bioluminescent assays. n = 3. k , Normalized VSV mRNA levels in HEK293T cells treated with FK866 or DMSO (control), followed by VSV infection (8 hpi, MOI = 1.5). n = 3. l , NAD + levels of cells in ( k ), determined by bioluminescent assays. n = 3. m , Normalized VSV mRNA levels in HEK293T cells transfected with GFP vector/GFP-SARM1-TIR WT/E642A, followed by VSV infection (8 hpi, MOI = 1.5). n = 3. n , NAD + levels of cells in ( m ), determined by bioluminescent assays. n = 3. o , NAD + consumption by recombinant PARP10 with or without the addition of PEG-8000 for condensation induction in vitro over time, determined by bioluminescent assays. p , Representative images of GFP-PARP10 condensate with or without VSV infection (24 hpi, MOI = 1.5). Scale bar: 10 μm. q , Box plot quantification of the diameter of PARP10 condensate in ( p ). r , Schematic diagram showing that PARP10 inhibits VSV infection by regulating NAD + homeostasis. Data are shown as mean ± SD. in ( a )-( d ), ( g ), ( i )-( n ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Transfection, Infection, Gene Expression, Control, Plasmid Preparation, Recombinant, In Vitro

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of cells transfected with mouse PARP10 WT and mutants. ADPr signals were detected using eAf1521. Scale bar: 10 μm. b , Schematic diagram showing the generation of Parp10 G837W/G837W and Parp10 3E/3E mice with verification by Sanger sequencing. c , Normalized Parp10 mRNA levels in Parp10 +/+ , Parp10 -/- , Parp10 G837W/G837W and Parp10 3E/3E BMDM infected with VSV (12 hpi, MOI = 1.5) or not. n = 3. Data are shown as mean ± SD. d , Western blot showing the PARP10 protein expression in Parp10 +/+ , Parp10 -/- , Parp10 G837W/G837W and Parp10 3E/3E BMDM infected with VSV (12 hpi, MOI = 1.5) or not. e , KEGG analysis of metabolism pathways affected by PARP10 expression in HEK293T cells. f , Heatmap of metabolite levels in HEK293T cells transfected with GFP/GFP-PARP10 WT/G888W/3RE, with or without VSV infection (8 hpi, MOI = 1.5), measured by MS. n = 3. Metabolites of particular interest are colored by category. Metabolites with significant changes ( p < 0.05) are shown. g , Western blot showing the ADP-ribosylation of GFP-PARP10 in cells infected with VSV (MOI = 1.5) for the indicated times. Cells transfected with GFP vector and catalytic-deficient mutant (G888W) served as negative controls. PARP10 ADP-ribosylation was probed with eAf1521. h , GSEA showing the enriched gene set in HEK293T cells expressing GFP-PARP10 relative to GFP vector. i , Heat map of gene expression in ( h ) gene set. The expression profile for GFP, GFP-PARP10 WT, G888W, and 3RE mutants was shown. j , k , Heat map of gene expression related to NAD + catalytic process ( j ) and NAD + consumption ( k ) from the whole blood samples in healthy ( n = 17) and COVID-19 ( n = 20) populations. l , Comparison of PARP10 expression between healthy ( n = 17) and COVID-19 ( n = 20) populations. Data are shown as mean ± SD.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Transfection, Sequencing, Infection, Western Blot, Expressing, Plasmid Preparation, Mutagenesis, Gene Expression, Comparison

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , A flow chart for BMDM isolation and RNA-seq analysis, created through BioRender. b , Volcano plot showing the upregulated and downregulated genes in BMDM infected with VSV (12 hpi, MOI = 1.5) compared to non-infected control. Blue dot indicates downregulated genes, red dot indicates upregulated genes, green dot indicates upregulated interferon-stimulated genes (ISGs). c , KEGG analysis of upregulated genes in ( b ). d , Amino acid sequence comparison between human and mouse PARP10. e , Schematic diagram showing the KO of the mouse Parp10 gene. f , g , Western blot showing the PARP10 protein expression in multiple tissues from Parp10 +/+ and Parp10 -/- mice. Normalization to GAPDH is shown in ( g ). h , Relative expression of PARP10 from human single-cell RNA-seq data extracted from https://www.proteinatlas.org/ . i , Western blot validating the MAVS KO in HEK293T cells. j , Normalized mRNA levels of Ifnb1 / Ccl5 / Isg56 in WT/BX795-treated/MAVS KO HEK293T cells transfected with GFP vector or GFP-PARP10, followed by VSV infection (8 hpi, MOI = 1.5). n = 3. k , Western blot to determine the levels of p-IRF3, total IRF3, and PARP10 in Parp10 +/+ and Parp10 -/- BMDM at different time points post-VSV (MOI = 1.5) infection. l , Schematic diagram showing the EGFP KI strategy for the generation of the Parp10 mEGFP/mEGFP mice.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Isolation, RNA Sequencing, Infection, Control, Sequencing, Comparison, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Fold changes of differentially expressed ARTD genes in BMDM with VSV (12 hpi, MOI = 1.5) infection compared to non-infected control. n = 3. b , Normalized VSV mRNA levels in HEK293T cells transfected with different ARTD genes and infected with VSV (8 hpi, MOI = 1.5). n = 3. c , Normalized PARP10 mRNA levels in Parp10 +/+ and Parp10 -/- BMDM at different time points post-VSV (MOI = 1.5) infection. n = 3. d , Normalized VSV mRNA levels in Parp10 +/+ and Parp10 -/- BMDM infected with VSV (12 hpi, MOI = 1.5). n = 3. e , f , Normalized VSV mRNA levels in spleen ( e ) and liver ( f ) samples isolated from VSV (1x10 8 PFU/mouse, 24 hpi) infected Parp10 +/+ ( n = 4) and Parp10 -/- ( n = 5) mice. g , Normalized SFV mRNA levels in Parp10 +/+ and Parp10 -/- BMDM infected with SFV (12 hpi, MOI = 0.5). n = 3. h , Normalized Ifna1 and Ifnb1 mRNA levels in Parp10 +/+ and Parp10 -/- BMDM infected with VSV (12 hpi, MOI = 1.5). n = 3. i , Normalized VSV mRNA levels in BHK-21 cells transfected with GFP or GFP-PARP10 and infected with VSV (8 hpi, MOI = 1.5). n = 3. j , Normalized VSV mRNA levels in WT/BX795-treated/MAVS KO HEK293T cells transfected with GFP or GFP-PARP10, followed by VSV infection (8 hpi, MOI = 1.5). n = 3. k , Normalized VSV mRNA levels in HEK293T cells transfected with GFP or GFP-PARP10 during viral binding, entry, and endocytosis steps. The endocytosis steps were blocked with 10 μM Chloroquine. l , Western blot showing the expression of mEGFP-PARP10 in Parp10 +/+ and Parp10 mEGFP/mEGFP BMDM with or without VSV infection (MOI = 1.5) for the indicated times. m , Representative images of colocalization between endogenous PARP10 and VSV inclusion labeled by N protein in Parp10 mEGFP/mEGFP BMDM infected with VSV (6 hpi, MOI = 1.5) or in non-infection control. n , Control IF experiment as ( m ) except without primary antibody staining for PARP10, thus excluding the leaky signal from VSV-N channel. Data are shown as mean ± SD. in ( b )-( k ). Scale bar: 10 μm for ( m ) and ( n ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Infection, Control, Transfection, Isolation, Binding Assay, Western Blot, Expressing, Labeling, Staining

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images to show the colocalization between GFP-PARP10 foci and VSV inclusions. b , Representative images of GFP-PARP10 inducible U2OS cells stimulated with different doxycycline doses. Higher expression induced more foci formation. c , Western blot showing the protein expression of GFP-PARP10 with different doxycycline doses. d , Calculation of the cellular threshold concentration of GFP-PARP10 to form cytoplasmic foci. The absolute concentration in cells is converted by a standard curve created by measuring the intensity of recombinant PARP10 protein. Each black dot indicates one cell with condensates, the red dot indicates the cell without condensates, and the line marks the threshold concentration for condensate formation. At least 100 cells were measured and analyzed. e , f , FRAP experiments for GFP-PARP10 foci in cells. The fluorescent intensity in the region of interest (ROI) in ( e ) is quantified in ( f ). g , Representative images for the GFP-PARP10 droplets fusion process. h , Representative images of GFP-PARP10 foci with LysoTracker, lipid dye, RAB7 staining, and mCherry-RAB5 co-expression. i , Representative images for GFP-PARP10 foci in cells stained with G3BP1 and LSM14A. j , Correlative light electron microscopy (CLEM) images of GFP-PARP10 foci. GFP-PARP10 foci were identified by the high electron density and correlation with GFP fluorescence. MitoTracker in red indicates the location of mitochondria. k , Representative images of recombinant PARP10 protein condensation induced by 10% Ficoll under the indicated salt and protein concentrations. l , Western blot showing the ADP-ribosylation of recombinant PARP10 with the indicated concentrations of NAD + in vitro . The ADPr-modified PARP10 was blotted with eAf1521. m , Representative images of Ficoll-induced PARP10 condensation in vitro with the treatment of 1,6-Hexanediol or 2,5-Hexanediol. Data are shown as mean ± SD. in ( d ) and ( f ). Scale bar: 10 μm for ( a ), ( b ), ( e ), ( g )-( i ). 2 μm for ( j ) and 500 nm for the inset. 20 μm for ( k ) and ( m ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Expressing, Western Blot, Concentration Assay, Recombinant, Staining, Electron Microscopy, Fluorescence, In Vitro, Modification

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of endogenous PARP10 staining in WT and PARP10 KO U2OS cells. b , c , Western blot showing the expression of endogenous PARP10 in U2OS cells and verification of PARP10 KO. PARP10 was stained with commercial antibody from Proteintech ( b ) and homemade antibody ( c ). d , Western blot showing the induction of endogenous PARP10 in U2OS with IFNβ, IFNγ, and TNFα stimulation. Scale bar: 10 μm for ( a ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Staining, Western Blot, Expressing

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , The schematic diagram for PARP10 protein and predicted intrinsically disordered region (IDR) and net charge per residue (NCPR) pattern are shown below. b , The schematic diagram for PARP10 mutants used. c , Representative images of cells stably expressing PARP10 mutants in ( b ). d , Quantification of the percentage of cells with foci in ( c ). e , Western blot showing the relative protein expression of each construct used in ( c ). f , Representative images of oligo-dT FISH staining in GFP-PARP10 condensate. g , Representative images of GFP-PARP10 condensates with poly(I:C) treatment. Cells with G3BP1 foci were indicative of successful poly(I:C) transfection. Arrow indicates cells transfected with poly(I:C) and arrowhead indicates cells absent of poly(I:C). h , Representative images of GFP-PARP10 condensates with RNase A (100 μg/mL) treatment for 20 min. PBS was used as control buffer. i , Representative images of PARP10 WT condensation in the presence of indicated concentrations of polyA RNA or total RNA with or without 10% Ficoll. Data are shown as mean ± SD. in ( d ). Scale bar: 10 μm for ( c ), ( f )-( h ). 20 μm for ( i ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Residue, Stable Transfection, Expressing, Western Blot, Construct, Staining, Transfection, Control

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of ADPr staining with eAf1521 and mitochondria staining with Tom20. b , RRM sequence alignment of PARP10 with TDP43 and hnRNPA1. c , Diagram showing the constructs used in the assay, and the representative images are shown below for each construct. Scale bar: 10 μm. d , Quantification of percentage of cells with foci formation in ( c ). e , Western blot showing the interaction between PARP10 domains (RRM2/3, KH1, or KH2) and different fragments in ( c ), determined by co-immunoprecipitation assay.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Staining, Sequencing, Construct, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative 2D classes of the recombinant, full-length PARP10 protein under cryo-EM. Scale bar: 200 Å. b , Cryo-EM map of PARP10 RRM1-3 pentamer obtained from 3D reconstruction and refinement. c , Structural model of PARP10 RRM1-3 pentamer, which is generated by docking AlphaFold-predicted models of PARP10 RRM1-3 domains into the cryo-EM map shown in ( b ). The PARP10 RRM1-3 oligomer adopts an open ring conformation with an angle of 140 degrees. d , Detailed illustration of the RRM1-3 dimer interface at two orientations. One dimeric interface of RRM1-3 is zoomed in, and residues in the interface between two RRM1-3 protomers are shown in sticks. e , Two protomers of PARP10 RRM1-3 contact with each other by electrostatic interactions. One protomer is represented with electrostatic potential surface, and the other is shown in cartoon. f,g , Representative images of cells ( f ) stably expressing PARP10 mutants depicted in ( g ). Scale bar: 10 μm. h , Quantification of the percentage of cells with foci in ( f ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Recombinant, Cryo-EM Sample Prep, Generated, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , A representative cryo-EM micrograph of full-length PARP10 protein. Scale bar: 500 Å. b , 2D class averages were selected for 3D reconstruction. Scale bar: 180 Å. c , Flow chart for cryo-EM data analysis. d , Maps of two classes of PARP10 RRM rings with different twisted angles. e , 3D variability analysis and non-uniform refinement of RRM oligomers. f - h , Map local resolution ( f ), Fourier shell correlation (FSC) curves ( g ), and particle distribution used in 3D reconstruction ( h ) of PARP10 RRM1-3 pentamer. i , j , 2D classes and corresponding models to show the two types of higher-order oligomerization of RRM1-3. Scale bar: 200 Å.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , b , Representative images for GFP-PARP10 condensate in cells stained with ADPr recognition antibodies (E6F6A and 10H) ( a ) and reagent (hsPARP14 macro2-3-rFC) ( b ). The catalytic deficient mutant GFP-PARP10 G888W served as a negative control. c , d , FRAP experiments for GFP-PARP10 and mCherry-eAf1521 co-expression cells. The fluorescent intensity in the region of interest (ROI) in ( c ) is quantified in ( d ). e , f , Representative images ( e ) and line quantification ( f ) of fluorescein-NAD + enrichment in PARP10-WT but not G888W condensates induced by PEG-8000 in vitro . g-i , Representative images of the reactions to show the PEG-8000 induced PARP10 condensation ( g ). Western blot showing ADP-ribosylation of 10 μM PARP10 with or without 10% PEG-8000 addition ( h ). The quantification of the ADP-ribosylation activity was shown in ( i ). n = 2. j , k , Western blot showing the ADP-ribosylation activity of PARP10 WT and condensation-deficient mutants 3RE and 3EA in cells ( j ). ADP-ribosylation levels were blotted with eAf1521. The quantification of the ADP-ribosylation activity was shown in ( k ). n = 3. Data are shown as mean ± SD. in ( d ), ( i ), ( k ). Scale bar: 10 μm for ( a )-( c ); 20 μm for ( e ) and ( g ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Staining, Mutagenesis, Negative Control, Expressing, In Vitro, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of cells stably expressing GFP-PARP10 WT and catalytic deficient mutants (G888W and ΔCD). ADPr signals stained by eAf1521. b , Western blot showing the relative protein expression of each construct used in ( a ). c , Box plot for the partition coefficient (PC) of PARP10 WT and mutants in ( a ). Each symbol represents one condensate. At least 25 condensates were measured and analyzed. d , Phase separation threshold concentration was measured for PARP10 WT and mutants in ( a ). The absolute concentration in cells is converted by a standard curve created by measuring the intensity of recombinant PARP10 protein. Each symbol represents one cell. At least 100 cells were measured and analyzed. Each black dot indicates one cell with condensates, the red dot indicates the cell without condensates, and the line marks the threshold concentration for condensate formation. e , f , Representative images ( e ) and quantification ( f ) for FRAP experiments on PARP10 WT ( n = 25), G888W ( n = 11), and H887E ( n = 17). The arrow indicated the bleached foci. Data are shown as mean ± SD. g , h , Representative images of GFP-PARP10 WT ( g ) and G888W ( h ) mutant treated with PARP10 inhibitor OUL-35. i , Box plot quantification of the diameter of PARP10 condensate in ( g ) and ( h ). j , Representative images of GFP-PARP10 co-expression with mCherry-MACROD2/TARG1/ARH3/PARG103. k , Box plot quantification of the diameter of PARP10 condensate in ( j ). Data are shown as mean ± SD. l , Representative images of PARP10 WT/H887E condensation induced by 10% Ficoll with or without NAD + addition. m , Western blot showing the ADP-ribosylation and protein levels of PARP10 WT and H887E in ( l ). n , Western blot showing CHX chase assay for IFNβ-induced endogenous PARP10 protein level in U2OS cells coupled with MG132 or Bafilomycin A1 treatments. o , Western blot showing ADP-ribosylation of GFP-PARP10 WT in cells coupled with MG132 treatment for the indicated times. The accumulation of ubiquitin signal was detected with P4D1 ubiquitin antibody. The catalytic deficient mutant GFP-PARP10 G888W serves as a negative control. p , Western blot showing the accumulation of GFP-PARP10 protein in cells treated with different concentrations of PARP10 activity inhibitor OUL35. q , Quantification of the GFP-PARP10 protein levels in ( p ). Data are shown as mean ± SD. in ( q ). Scale bar: 10 μm for ( a ), ( e ), ( g ), ( h ), ( j ). 20 μm for ( l ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Stable Transfection, Expressing, Staining, Western Blot, Construct, Concentration Assay, Recombinant, Mutagenesis, Ubiquitin Proteomics, Negative Control, Activity Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , List of ADPr hydrolases used in the study. b , Western blot showing the effect of mCherry-tagged ADPr hydrolase and its catalytic deficient (CD) mutants on the GFP-PARP10 auto-ADP-ribosylation in HEK293T cells. The auto-ADP-ribosylation was probed by eAf1521. c , Representative images of GFP-PARP10 condensates stained for FK2. d , TUBE pulldown assay showing the ubiquitination of PARP10 in response to MG132 and IFNβ treatment. GST bead only and PARP10 KO cells were used as controls. e , Western blot showing the ubiquitination of PARP10 in cells. f , Representative images of GFP-PARP10 WT/G888W stable cells treated with CHX for different times. g , Box plot quantification of the diameter of PARP10 condensate in ( f ). Scale bar: 10 μm for ( c ) and ( f ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Western Blot, Staining, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a - d , Representative images of cells co-transfected with GFP/GFP-PARP10 WT/G888W and mCherry-DTX2 ( a ), DTX3L ( b ), DTX3 ( c ), DTX4 ( d ). e , Representative images of cells transfected with GFP-PARP10 WT/G888W and stained with DTX1 antibody. f , Representative images of cells co-transfected with GFP-PARP10 WT and mCherry-DTX2 WT or 5 Mut which disrupts the ADPr binding. g , Western blot showing the interaction between GFP-PARP10 WT and mCherry/mCherry-DTX2 WT/5 Mut, determined by co-immunoprecipitation assay. h , Representative images and summary of GFP-PARP10 condensates colocalized with FK2-positive TRIMs, but not with the FK2-negative catalytic mutants. Scale bar: 10 μm for ( a )-( f ) and ( h ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Transfection, Staining, Binding Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Normalized VSV mRNA levels in HEK293T cells transfected with GFP/GFP-PARP10 WT/G888W/3RE and infected with VSV (8 hpi, MOI = 1.5). n = 6. b , Normalized VSV mRNA levels in Parp10 +/+ , Parp10 -/- , Parp10 G837W/G837W and Parp10 3E/3E BMDM with or without VSV infection (12 hpi, MOI = 1.5). n = 5. c , NAD + levels in HEK293T cells transfected with GFP/GFP-PARP10 WT/G888W, with or without VSV infection (8 hpi, MOI = 1.5), measured by MS. n = 3. d , NAD + levels in WT U2OS, HEK293T and BMDM cells infected with VSV (MOI = 1.5) for the indicated times, measured by MS. n = 3. e , Heatmap of gene expression in BMDM infected with VSV (12 hpi, MOI = 1.5) or not. n = 3. Genes related to NAD + metabolic process were shown. f , Fold changes of NAD + consuming genes in BMDM with VSV (12 hpi, MOI = 1.5) infection relative to non-infected control. n = 3. g , NAD + levels in Parp10 +/+ (WT), Parp10 -/- , and Parp10 3E/3E BMDM with or without VSV infection (12 hpi, MOI = 1.5), measured by MS. n = 3. h , Schematic diagram of the NAD + salvage pathway and the effect of NAMPT inhibitor FK866. i , Normalized VSV mRNA levels in HEK293T cells transfected with GFP/GFP-PARP10 WT, followed by the supplement of the indicated concentrations of NR and VSV infection (8 hpi, MOI = 1.5). n = 3. j , NAD + levels of cells in ( i ), determined by bioluminescent assays. n = 3. k , Normalized VSV mRNA levels in HEK293T cells treated with FK866 or DMSO (control), followed by VSV infection (8 hpi, MOI = 1.5). n = 3. l , NAD + levels of cells in ( k ), determined by bioluminescent assays. n = 3. m , Normalized VSV mRNA levels in HEK293T cells transfected with GFP vector/GFP-SARM1-TIR WT/E642A, followed by VSV infection (8 hpi, MOI = 1.5). n = 3. n , NAD + levels of cells in ( m ), determined by bioluminescent assays. n = 3. o , NAD + consumption by recombinant PARP10 with or without the addition of PEG-8000 for condensation induction in vitro over time, determined by bioluminescent assays. p , Representative images of GFP-PARP10 condensate with or without VSV infection (24 hpi, MOI = 1.5). Scale bar: 10 μm. q , Box plot quantification of the diameter of PARP10 condensate in ( p ). r , Schematic diagram showing that PARP10 inhibits VSV infection by regulating NAD + homeostasis. Data are shown as mean ± SD. in ( a )-( d ), ( g ), ( i )-( n ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Transfection, Infection, Gene Expression, Control, Plasmid Preparation, Recombinant, In Vitro

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of cells transfected with mouse PARP10 WT and mutants. ADPr signals were detected using eAf1521. Scale bar: 10 μm. b , Schematic diagram showing the generation of Parp10 G837W/G837W and Parp10 3E/3E mice with verification by Sanger sequencing. c , Normalized Parp10 mRNA levels in Parp10 +/+ , Parp10 -/- , Parp10 G837W/G837W and Parp10 3E/3E BMDM infected with VSV (12 hpi, MOI = 1.5) or not. n = 3. Data are shown as mean ± SD. d , Western blot showing the PARP10 protein expression in Parp10 +/+ , Parp10 -/- , Parp10 G837W/G837W and Parp10 3E/3E BMDM infected with VSV (12 hpi, MOI = 1.5) or not. e , KEGG analysis of metabolism pathways affected by PARP10 expression in HEK293T cells. f , Heatmap of metabolite levels in HEK293T cells transfected with GFP/GFP-PARP10 WT/G888W/3RE, with or without VSV infection (8 hpi, MOI = 1.5), measured by MS. n = 3. Metabolites of particular interest are colored by category. Metabolites with significant changes ( p < 0.05) are shown. g , Western blot showing the ADP-ribosylation of GFP-PARP10 in cells infected with VSV (MOI = 1.5) for the indicated times. Cells transfected with GFP vector and catalytic-deficient mutant (G888W) served as negative controls. PARP10 ADP-ribosylation was probed with eAf1521. h , GSEA showing the enriched gene set in HEK293T cells expressing GFP-PARP10 relative to GFP vector. i , Heat map of gene expression in ( h ) gene set. The expression profile for GFP, GFP-PARP10 WT, G888W, and 3RE mutants was shown. j , k , Heat map of gene expression related to NAD + catalytic process ( j ) and NAD + consumption ( k ) from the whole blood samples in healthy ( n = 17) and COVID-19 ( n = 20) populations. l , Comparison of PARP10 expression between healthy ( n = 17) and COVID-19 ( n = 20) populations. Data are shown as mean ± SD.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Transfection, Sequencing, Infection, Western Blot, Expressing, Plasmid Preparation, Mutagenesis, Gene Expression, Comparison

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , A flow chart for BMDM isolation and RNA-seq analysis, created through BioRender. b , Volcano plot showing the upregulated and downregulated genes in BMDM infected with VSV (12 hpi, MOI = 1.5) compared to non-infected control. Blue dot indicates downregulated genes, red dot indicates upregulated genes, green dot indicates upregulated interferon-stimulated genes (ISGs). c , KEGG analysis of upregulated genes in ( b ). d , Amino acid sequence comparison between human and mouse PARP10. e , Schematic diagram showing the KO of the mouse Parp10 gene. f , g , Western blot showing the PARP10 protein expression in multiple tissues from Parp10 +/+ and Parp10 -/- mice. Normalization to GAPDH is shown in ( g ). h , Relative expression of PARP10 from human single-cell RNA-seq data extracted from https://www.proteinatlas.org/ . i , Western blot validating the MAVS KO in HEK293T cells. j , Normalized mRNA levels of Ifnb1 / Ccl5 / Isg56 in WT/BX795-treated/MAVS KO HEK293T cells transfected with GFP vector or GFP-PARP10, followed by VSV infection (8 hpi, MOI = 1.5). n = 3. k , Western blot to determine the levels of p-IRF3, total IRF3, and PARP10 in Parp10 +/+ and Parp10 -/- BMDM at different time points post-VSV (MOI = 1.5) infection. l , Schematic diagram showing the EGFP KI strategy for the generation of the Parp10 mEGFP/mEGFP mice.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Isolation, RNA Sequencing, Infection, Control, Sequencing, Comparison, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Fold changes of differentially expressed ARTD genes in BMDM with VSV (12 hpi, MOI = 1.5) infection compared to non-infected control. n = 3. b , Normalized VSV mRNA levels in HEK293T cells transfected with different ARTD genes and infected with VSV (8 hpi, MOI = 1.5). n = 3. c , Normalized PARP10 mRNA levels in Parp10 +/+ and Parp10 -/- BMDM at different time points post-VSV (MOI = 1.5) infection. n = 3. d , Normalized VSV mRNA levels in Parp10 +/+ and Parp10 -/- BMDM infected with VSV (12 hpi, MOI = 1.5). n = 3. e , f , Normalized VSV mRNA levels in spleen ( e ) and liver ( f ) samples isolated from VSV (1x10 8 PFU/mouse, 24 hpi) infected Parp10 +/+ ( n = 4) and Parp10 -/- ( n = 5) mice. g , Normalized SFV mRNA levels in Parp10 +/+ and Parp10 -/- BMDM infected with SFV (12 hpi, MOI = 0.5). n = 3. h , Normalized Ifna1 and Ifnb1 mRNA levels in Parp10 +/+ and Parp10 -/- BMDM infected with VSV (12 hpi, MOI = 1.5). n = 3. i , Normalized VSV mRNA levels in BHK-21 cells transfected with GFP or GFP-PARP10 and infected with VSV (8 hpi, MOI = 1.5). n = 3. j , Normalized VSV mRNA levels in WT/BX795-treated/MAVS KO HEK293T cells transfected with GFP or GFP-PARP10, followed by VSV infection (8 hpi, MOI = 1.5). n = 3. k , Normalized VSV mRNA levels in HEK293T cells transfected with GFP or GFP-PARP10 during viral binding, entry, and endocytosis steps. The endocytosis steps were blocked with 10 μM Chloroquine. l , Western blot showing the expression of mEGFP-PARP10 in Parp10 +/+ and Parp10 mEGFP/mEGFP BMDM with or without VSV infection (MOI = 1.5) for the indicated times. m , Representative images of colocalization between endogenous PARP10 and VSV inclusion labeled by N protein in Parp10 mEGFP/mEGFP BMDM infected with VSV (6 hpi, MOI = 1.5) or in non-infection control. n , Control IF experiment as ( m ) except without primary antibody staining for PARP10, thus excluding the leaky signal from VSV-N channel. Data are shown as mean ± SD. in ( b )-( k ). Scale bar: 10 μm for ( m ) and ( n ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Infection, Control, Transfection, Isolation, Binding Assay, Western Blot, Expressing, Labeling, Staining

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images to show the colocalization between GFP-PARP10 foci and VSV inclusions. b , Representative images of GFP-PARP10 inducible U2OS cells stimulated with different doxycycline doses. Higher expression induced more foci formation. c , Western blot showing the protein expression of GFP-PARP10 with different doxycycline doses. d , Calculation of the cellular threshold concentration of GFP-PARP10 to form cytoplasmic foci. The absolute concentration in cells is converted by a standard curve created by measuring the intensity of recombinant PARP10 protein. Each black dot indicates one cell with condensates, the red dot indicates the cell without condensates, and the line marks the threshold concentration for condensate formation. At least 100 cells were measured and analyzed. e , f , FRAP experiments for GFP-PARP10 foci in cells. The fluorescent intensity in the region of interest (ROI) in ( e ) is quantified in ( f ). g , Representative images for the GFP-PARP10 droplets fusion process. h , Representative images of GFP-PARP10 foci with LysoTracker, lipid dye, RAB7 staining, and mCherry-RAB5 co-expression. i , Representative images for GFP-PARP10 foci in cells stained with G3BP1 and LSM14A. j , Correlative light electron microscopy (CLEM) images of GFP-PARP10 foci. GFP-PARP10 foci were identified by the high electron density and correlation with GFP fluorescence. MitoTracker in red indicates the location of mitochondria. k , Representative images of recombinant PARP10 protein condensation induced by 10% Ficoll under the indicated salt and protein concentrations. l , Western blot showing the ADP-ribosylation of recombinant PARP10 with the indicated concentrations of NAD + in vitro . The ADPr-modified PARP10 was blotted with eAf1521. m , Representative images of Ficoll-induced PARP10 condensation in vitro with the treatment of 1,6-Hexanediol or 2,5-Hexanediol. Data are shown as mean ± SD. in ( d ) and ( f ). Scale bar: 10 μm for ( a ), ( b ), ( e ), ( g )-( i ). 2 μm for ( j ) and 500 nm for the inset. 20 μm for ( k ) and ( m ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Expressing, Western Blot, Concentration Assay, Recombinant, Staining, Electron Microscopy, Fluorescence, In Vitro, Modification

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of endogenous PARP10 staining in WT and PARP10 KO U2OS cells. b , c , Western blot showing the expression of endogenous PARP10 in U2OS cells and verification of PARP10 KO. PARP10 was stained with commercial antibody from Proteintech ( b ) and homemade antibody ( c ). d , Western blot showing the induction of endogenous PARP10 in U2OS with IFNβ, IFNγ, and TNFα stimulation. Scale bar: 10 μm for ( a ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Staining, Western Blot, Expressing

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , The schematic diagram for PARP10 protein and predicted intrinsically disordered region (IDR) and net charge per residue (NCPR) pattern are shown below. b , The schematic diagram for PARP10 mutants used. c , Representative images of cells stably expressing PARP10 mutants in ( b ). d , Quantification of the percentage of cells with foci in ( c ). e , Western blot showing the relative protein expression of each construct used in ( c ). f , Representative images of oligo-dT FISH staining in GFP-PARP10 condensate. g , Representative images of GFP-PARP10 condensates with poly(I:C) treatment. Cells with G3BP1 foci were indicative of successful poly(I:C) transfection. Arrow indicates cells transfected with poly(I:C) and arrowhead indicates cells absent of poly(I:C). h , Representative images of GFP-PARP10 condensates with RNase A (100 μg/mL) treatment for 20 min. PBS was used as control buffer. i , Representative images of PARP10 WT condensation in the presence of indicated concentrations of polyA RNA or total RNA with or without 10% Ficoll. Data are shown as mean ± SD. in ( d ). Scale bar: 10 μm for ( c ), ( f )-( h ). 20 μm for ( i ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Residue, Stable Transfection, Expressing, Western Blot, Construct, Staining, Transfection, Control

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of ADPr staining with eAf1521 and mitochondria staining with Tom20. b , RRM sequence alignment of PARP10 with TDP43 and hnRNPA1. c , Diagram showing the constructs used in the assay, and the representative images are shown below for each construct. Scale bar: 10 μm. d , Quantification of percentage of cells with foci formation in ( c ). e , Western blot showing the interaction between PARP10 domains (RRM2/3, KH1, or KH2) and different fragments in ( c ), determined by co-immunoprecipitation assay.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Staining, Sequencing, Construct, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative 2D classes of the recombinant, full-length PARP10 protein under cryo-EM. Scale bar: 200 Å. b , Cryo-EM map of PARP10 RRM1-3 pentamer obtained from 3D reconstruction and refinement. c , Structural model of PARP10 RRM1-3 pentamer, which is generated by docking AlphaFold-predicted models of PARP10 RRM1-3 domains into the cryo-EM map shown in ( b ). The PARP10 RRM1-3 oligomer adopts an open ring conformation with an angle of 140 degrees. d , Detailed illustration of the RRM1-3 dimer interface at two orientations. One dimeric interface of RRM1-3 is zoomed in, and residues in the interface between two RRM1-3 protomers are shown in sticks. e , Two protomers of PARP10 RRM1-3 contact with each other by electrostatic interactions. One protomer is represented with electrostatic potential surface, and the other is shown in cartoon. f,g , Representative images of cells ( f ) stably expressing PARP10 mutants depicted in ( g ). Scale bar: 10 μm. h , Quantification of the percentage of cells with foci in ( f ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Recombinant, Cryo-EM Sample Prep, Generated, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , A representative cryo-EM micrograph of full-length PARP10 protein. Scale bar: 500 Å. b , 2D class averages were selected for 3D reconstruction. Scale bar: 180 Å. c , Flow chart for cryo-EM data analysis. d , Maps of two classes of PARP10 RRM rings with different twisted angles. e , 3D variability analysis and non-uniform refinement of RRM oligomers. f - h , Map local resolution ( f ), Fourier shell correlation (FSC) curves ( g ), and particle distribution used in 3D reconstruction ( h ) of PARP10 RRM1-3 pentamer. i , j , 2D classes and corresponding models to show the two types of higher-order oligomerization of RRM1-3. Scale bar: 200 Å.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , b , Representative images for GFP-PARP10 condensate in cells stained with ADPr recognition antibodies (E6F6A and 10H) ( a ) and reagent (hsPARP14 macro2-3-rFC) ( b ). The catalytic deficient mutant GFP-PARP10 G888W served as a negative control. c , d , FRAP experiments for GFP-PARP10 and mCherry-eAf1521 co-expression cells. The fluorescent intensity in the region of interest (ROI) in ( c ) is quantified in ( d ). e , f , Representative images ( e ) and line quantification ( f ) of fluorescein-NAD + enrichment in PARP10-WT but not G888W condensates induced by PEG-8000 in vitro . g-i , Representative images of the reactions to show the PEG-8000 induced PARP10 condensation ( g ). Western blot showing ADP-ribosylation of 10 μM PARP10 with or without 10% PEG-8000 addition ( h ). The quantification of the ADP-ribosylation activity was shown in ( i ). n = 2. j , k , Western blot showing the ADP-ribosylation activity of PARP10 WT and condensation-deficient mutants 3RE and 3EA in cells ( j ). ADP-ribosylation levels were blotted with eAf1521. The quantification of the ADP-ribosylation activity was shown in ( k ). n = 3. Data are shown as mean ± SD. in ( d ), ( i ), ( k ). Scale bar: 10 μm for ( a )-( c ); 20 μm for ( e ) and ( g ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Staining, Mutagenesis, Negative Control, Expressing, In Vitro, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of cells stably expressing GFP-PARP10 WT and catalytic deficient mutants (G888W and ΔCD). ADPr signals stained by eAf1521. b , Western blot showing the relative protein expression of each construct used in ( a ). c , Box plot for the partition coefficient (PC) of PARP10 WT and mutants in ( a ). Each symbol represents one condensate. At least 25 condensates were measured and analyzed. d , Phase separation threshold concentration was measured for PARP10 WT and mutants in ( a ). The absolute concentration in cells is converted by a standard curve created by measuring the intensity of recombinant PARP10 protein. Each symbol represents one cell. At least 100 cells were measured and analyzed. Each black dot indicates one cell with condensates, the red dot indicates the cell without condensates, and the line marks the threshold concentration for condensate formation. e , f , Representative images ( e ) and quantification ( f ) for FRAP experiments on PARP10 WT ( n = 25), G888W ( n = 11), and H887E ( n = 17). The arrow indicated the bleached foci. Data are shown as mean ± SD. g , h , Representative images of GFP-PARP10 WT ( g ) and G888W ( h ) mutant treated with PARP10 inhibitor OUL-35. i , Box plot quantification of the diameter of PARP10 condensate in ( g ) and ( h ). j , Representative images of GFP-PARP10 co-expression with mCherry-MACROD2/TARG1/ARH3/PARG103. k , Box plot quantification of the diameter of PARP10 condensate in ( j ). Data are shown as mean ± SD. l , Representative images of PARP10 WT/H887E condensation induced by 10% Ficoll with or without NAD + addition. m , Western blot showing the ADP-ribosylation and protein levels of PARP10 WT and H887E in ( l ). n , Western blot showing CHX chase assay for IFNβ-induced endogenous PARP10 protein level in U2OS cells coupled with MG132 or Bafilomycin A1 treatments. o , Western blot showing ADP-ribosylation of GFP-PARP10 WT in cells coupled with MG132 treatment for the indicated times. The accumulation of ubiquitin signal was detected with P4D1 ubiquitin antibody. The catalytic deficient mutant GFP-PARP10 G888W serves as a negative control. p , Western blot showing the accumulation of GFP-PARP10 protein in cells treated with different concentrations of PARP10 activity inhibitor OUL35. q , Quantification of the GFP-PARP10 protein levels in ( p ). Data are shown as mean ± SD. in ( q ). Scale bar: 10 μm for ( a ), ( e ), ( g ), ( h ), ( j ). 20 μm for ( l ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Stable Transfection, Expressing, Staining, Western Blot, Construct, Concentration Assay, Recombinant, Mutagenesis, Ubiquitin Proteomics, Negative Control, Activity Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , List of ADPr hydrolases used in the study. b , Western blot showing the effect of mCherry-tagged ADPr hydrolase and its catalytic deficient (CD) mutants on the GFP-PARP10 auto-ADP-ribosylation in HEK293T cells. The auto-ADP-ribosylation was probed by eAf1521. c , Representative images of GFP-PARP10 condensates stained for FK2. d , TUBE pulldown assay showing the ubiquitination of PARP10 in response to MG132 and IFNβ treatment. GST bead only and PARP10 KO cells were used as controls. e , Western blot showing the ubiquitination of PARP10 in cells. f , Representative images of GFP-PARP10 WT/G888W stable cells treated with CHX for different times. g , Box plot quantification of the diameter of PARP10 condensate in ( f ). Scale bar: 10 μm for ( c ) and ( f ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Western Blot, Staining, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a - d , Representative images of cells co-transfected with GFP/GFP-PARP10 WT/G888W and mCherry-DTX2 ( a ), DTX3L ( b ), DTX3 ( c ), DTX4 ( d ). e , Representative images of cells transfected with GFP-PARP10 WT/G888W and stained with DTX1 antibody. f , Representative images of cells co-transfected with GFP-PARP10 WT and mCherry-DTX2 WT or 5 Mut which disrupts the ADPr binding. g , Western blot showing the interaction between GFP-PARP10 WT and mCherry/mCherry-DTX2 WT/5 Mut, determined by co-immunoprecipitation assay. h , Representative images and summary of GFP-PARP10 condensates colocalized with FK2-positive TRIMs, but not with the FK2-negative catalytic mutants. Scale bar: 10 μm for ( a )-( f ) and ( h ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Transfection, Staining, Binding Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Normalized VSV mRNA levels in HEK293T cells transfected with GFP/GFP-PARP10 WT/G888W/3RE and infected with VSV (8 hpi, MOI = 1.5). n = 6. b , Normalized VSV mRNA levels in Parp10 +/+ , Parp10 -/- , Parp10 G837W/G837W and Parp10 3E/3E BMDM with or without VSV infection (12 hpi, MOI = 1.5). n = 5. c , NAD + levels in HEK293T cells transfected with GFP/GFP-PARP10 WT/G888W, with or without VSV infection (8 hpi, MOI = 1.5), measured by MS. n = 3. d , NAD + levels in WT U2OS, HEK293T and BMDM cells infected with VSV (MOI = 1.5) for the indicated times, measured by MS. n = 3. e , Heatmap of gene expression in BMDM infected with VSV (12 hpi, MOI = 1.5) or not. n = 3. Genes related to NAD + metabolic process were shown. f , Fold changes of NAD + consuming genes in BMDM with VSV (12 hpi, MOI = 1.5) infection relative to non-infected control. n = 3. g , NAD + levels in Parp10 +/+ (WT), Parp10 -/- , and Parp10 3E/3E BMDM with or without VSV infection (12 hpi, MOI = 1.5), measured by MS. n = 3. h , Schematic diagram of the NAD + salvage pathway and the effect of NAMPT inhibitor FK866. i , Normalized VSV mRNA levels in HEK293T cells transfected with GFP/GFP-PARP10 WT, followed by the supplement of the indicated concentrations of NR and VSV infection (8 hpi, MOI = 1.5). n = 3. j , NAD + levels of cells in ( i ), determined by bioluminescent assays. n = 3. k , Normalized VSV mRNA levels in HEK293T cells treated with FK866 or DMSO (control), followed by VSV infection (8 hpi, MOI = 1.5). n = 3. l , NAD + levels of cells in ( k ), determined by bioluminescent assays. n = 3. m , Normalized VSV mRNA levels in HEK293T cells transfected with GFP vector/GFP-SARM1-TIR WT/E642A, followed by VSV infection (8 hpi, MOI = 1.5). n = 3. n , NAD + levels of cells in ( m ), determined by bioluminescent assays. n = 3. o , NAD + consumption by recombinant PARP10 with or without the addition of PEG-8000 for condensation induction in vitro over time, determined by bioluminescent assays. p , Representative images of GFP-PARP10 condensate with or without VSV infection (24 hpi, MOI = 1.5). Scale bar: 10 μm. q , Box plot quantification of the diameter of PARP10 condensate in ( p ). r , Schematic diagram showing that PARP10 inhibits VSV infection by regulating NAD + homeostasis. Data are shown as mean ± SD. in ( a )-( d ), ( g ), ( i )-( n ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Transfection, Infection, Gene Expression, Control, Plasmid Preparation, Recombinant, In Vitro

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of cells transfected with mouse PARP10 WT and mutants. ADPr signals were detected using eAf1521. Scale bar: 10 μm. b , Schematic diagram showing the generation of Parp10 G837W/G837W and Parp10 3E/3E mice with verification by Sanger sequencing. c , Normalized Parp10 mRNA levels in Parp10 +/+ , Parp10 -/- , Parp10 G837W/G837W and Parp10 3E/3E BMDM infected with VSV (12 hpi, MOI = 1.5) or not. n = 3. Data are shown as mean ± SD. d , Western blot showing the PARP10 protein expression in Parp10 +/+ , Parp10 -/- , Parp10 G837W/G837W and Parp10 3E/3E BMDM infected with VSV (12 hpi, MOI = 1.5) or not. e , KEGG analysis of metabolism pathways affected by PARP10 expression in HEK293T cells. f , Heatmap of metabolite levels in HEK293T cells transfected with GFP/GFP-PARP10 WT/G888W/3RE, with or without VSV infection (8 hpi, MOI = 1.5), measured by MS. n = 3. Metabolites of particular interest are colored by category. Metabolites with significant changes ( p < 0.05) are shown. g , Western blot showing the ADP-ribosylation of GFP-PARP10 in cells infected with VSV (MOI = 1.5) for the indicated times. Cells transfected with GFP vector and catalytic-deficient mutant (G888W) served as negative controls. PARP10 ADP-ribosylation was probed with eAf1521. h , GSEA showing the enriched gene set in HEK293T cells expressing GFP-PARP10 relative to GFP vector. i , Heat map of gene expression in ( h ) gene set. The expression profile for GFP, GFP-PARP10 WT, G888W, and 3RE mutants was shown. j , k , Heat map of gene expression related to NAD + catalytic process ( j ) and NAD + consumption ( k ) from the whole blood samples in healthy ( n = 17) and COVID-19 ( n = 20) populations. l , Comparison of PARP10 expression between healthy ( n = 17) and COVID-19 ( n = 20) populations. Data are shown as mean ± SD.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Transfection, Sequencing, Infection, Western Blot, Expressing, Plasmid Preparation, Mutagenesis, Gene Expression, Comparison

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , A flow chart for BMDM isolation and RNA-seq analysis, created through BioRender. b , Volcano plot showing the upregulated and downregulated genes in BMDM infected with VSV (12 hpi, MOI = 1.5) compared to non-infected control. Blue dot indicates downregulated genes, red dot indicates upregulated genes, green dot indicates upregulated interferon-stimulated genes (ISGs). c , KEGG analysis of upregulated genes in ( b ). d , Amino acid sequence comparison between human and mouse PARP10. e , Schematic diagram showing the KO of the mouse Parp10 gene. f , g , Western blot showing the PARP10 protein expression in multiple tissues from Parp10 +/+ and Parp10 -/- mice. Normalization to GAPDH is shown in ( g ). h , Relative expression of PARP10 from human single-cell RNA-seq data extracted from https://www.proteinatlas.org/ . i , Western blot validating the MAVS KO in HEK293T cells. j , Normalized mRNA levels of Ifnb1 / Ccl5 / Isg56 in WT/BX795-treated/MAVS KO HEK293T cells transfected with GFP vector or GFP-PARP10, followed by VSV infection (8 hpi, MOI = 1.5). n = 3. k , Western blot to determine the levels of p-IRF3, total IRF3, and PARP10 in Parp10 +/+ and Parp10 -/- BMDM at different time points post-VSV (MOI = 1.5) infection. l , Schematic diagram showing the EGFP KI strategy for the generation of the Parp10 mEGFP/mEGFP mice.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Isolation, RNA Sequencing, Infection, Control, Sequencing, Comparison, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Fold changes of differentially expressed ARTD genes in BMDM with VSV (12 hpi, MOI = 1.5) infection compared to non-infected control. n = 3. b , Normalized VSV mRNA levels in HEK293T cells transfected with different ARTD genes and infected with VSV (8 hpi, MOI = 1.5). n = 3. c , Normalized PARP10 mRNA levels in Parp10 +/+ and Parp10 -/- BMDM at different time points post-VSV (MOI = 1.5) infection. n = 3. d , Normalized VSV mRNA levels in Parp10 +/+ and Parp10 -/- BMDM infected with VSV (12 hpi, MOI = 1.5). n = 3. e , f , Normalized VSV mRNA levels in spleen ( e ) and liver ( f ) samples isolated from VSV (1x10 8 PFU/mouse, 24 hpi) infected Parp10 +/+ ( n = 4) and Parp10 -/- ( n = 5) mice. g , Normalized SFV mRNA levels in Parp10 +/+ and Parp10 -/- BMDM infected with SFV (12 hpi, MOI = 0.5). n = 3. h , Normalized Ifna1 and Ifnb1 mRNA levels in Parp10 +/+ and Parp10 -/- BMDM infected with VSV (12 hpi, MOI = 1.5). n = 3. i , Normalized VSV mRNA levels in BHK-21 cells transfected with GFP or GFP-PARP10 and infected with VSV (8 hpi, MOI = 1.5). n = 3. j , Normalized VSV mRNA levels in WT/BX795-treated/MAVS KO HEK293T cells transfected with GFP or GFP-PARP10, followed by VSV infection (8 hpi, MOI = 1.5). n = 3. k , Normalized VSV mRNA levels in HEK293T cells transfected with GFP or GFP-PARP10 during viral binding, entry, and endocytosis steps. The endocytosis steps were blocked with 10 μM Chloroquine. l , Western blot showing the expression of mEGFP-PARP10 in Parp10 +/+ and Parp10 mEGFP/mEGFP BMDM with or without VSV infection (MOI = 1.5) for the indicated times. m , Representative images of colocalization between endogenous PARP10 and VSV inclusion labeled by N protein in Parp10 mEGFP/mEGFP BMDM infected with VSV (6 hpi, MOI = 1.5) or in non-infection control. n , Control IF experiment as ( m ) except without primary antibody staining for PARP10, thus excluding the leaky signal from VSV-N channel. Data are shown as mean ± SD. in ( b )-( k ). Scale bar: 10 μm for ( m ) and ( n ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Infection, Control, Transfection, Isolation, Binding Assay, Western Blot, Expressing, Labeling, Staining

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images to show the colocalization between GFP-PARP10 foci and VSV inclusions. b , Representative images of GFP-PARP10 inducible U2OS cells stimulated with different doxycycline doses. Higher expression induced more foci formation. c , Western blot showing the protein expression of GFP-PARP10 with different doxycycline doses. d , Calculation of the cellular threshold concentration of GFP-PARP10 to form cytoplasmic foci. The absolute concentration in cells is converted by a standard curve created by measuring the intensity of recombinant PARP10 protein. Each black dot indicates one cell with condensates, the red dot indicates the cell without condensates, and the line marks the threshold concentration for condensate formation. At least 100 cells were measured and analyzed. e , f , FRAP experiments for GFP-PARP10 foci in cells. The fluorescent intensity in the region of interest (ROI) in ( e ) is quantified in ( f ). g , Representative images for the GFP-PARP10 droplets fusion process. h , Representative images of GFP-PARP10 foci with LysoTracker, lipid dye, RAB7 staining, and mCherry-RAB5 co-expression. i , Representative images for GFP-PARP10 foci in cells stained with G3BP1 and LSM14A. j , Correlative light electron microscopy (CLEM) images of GFP-PARP10 foci. GFP-PARP10 foci were identified by the high electron density and correlation with GFP fluorescence. MitoTracker in red indicates the location of mitochondria. k , Representative images of recombinant PARP10 protein condensation induced by 10% Ficoll under the indicated salt and protein concentrations. l , Western blot showing the ADP-ribosylation of recombinant PARP10 with the indicated concentrations of NAD + in vitro . The ADPr-modified PARP10 was blotted with eAf1521. m , Representative images of Ficoll-induced PARP10 condensation in vitro with the treatment of 1,6-Hexanediol or 2,5-Hexanediol. Data are shown as mean ± SD. in ( d ) and ( f ). Scale bar: 10 μm for ( a ), ( b ), ( e ), ( g )-( i ). 2 μm for ( j ) and 500 nm for the inset. 20 μm for ( k ) and ( m ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Expressing, Western Blot, Concentration Assay, Recombinant, Staining, Electron Microscopy, Fluorescence, In Vitro, Modification

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of endogenous PARP10 staining in WT and PARP10 KO U2OS cells. b , c , Western blot showing the expression of endogenous PARP10 in U2OS cells and verification of PARP10 KO. PARP10 was stained with commercial antibody from Proteintech ( b ) and homemade antibody ( c ). d , Western blot showing the induction of endogenous PARP10 in U2OS with IFNβ, IFNγ, and TNFα stimulation. Scale bar: 10 μm for ( a ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Staining, Western Blot, Expressing

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , The schematic diagram for PARP10 protein and predicted intrinsically disordered region (IDR) and net charge per residue (NCPR) pattern are shown below. b , The schematic diagram for PARP10 mutants used. c , Representative images of cells stably expressing PARP10 mutants in ( b ). d , Quantification of the percentage of cells with foci in ( c ). e , Western blot showing the relative protein expression of each construct used in ( c ). f , Representative images of oligo-dT FISH staining in GFP-PARP10 condensate. g , Representative images of GFP-PARP10 condensates with poly(I:C) treatment. Cells with G3BP1 foci were indicative of successful poly(I:C) transfection. Arrow indicates cells transfected with poly(I:C) and arrowhead indicates cells absent of poly(I:C). h , Representative images of GFP-PARP10 condensates with RNase A (100 μg/mL) treatment for 20 min. PBS was used as control buffer. i , Representative images of PARP10 WT condensation in the presence of indicated concentrations of polyA RNA or total RNA with or without 10% Ficoll. Data are shown as mean ± SD. in ( d ). Scale bar: 10 μm for ( c ), ( f )-( h ). 20 μm for ( i ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Residue, Stable Transfection, Expressing, Western Blot, Construct, Staining, Transfection, Control

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of ADPr staining with eAf1521 and mitochondria staining with Tom20. b , RRM sequence alignment of PARP10 with TDP43 and hnRNPA1. c , Diagram showing the constructs used in the assay, and the representative images are shown below for each construct. Scale bar: 10 μm. d , Quantification of percentage of cells with foci formation in ( c ). e , Western blot showing the interaction between PARP10 domains (RRM2/3, KH1, or KH2) and different fragments in ( c ), determined by co-immunoprecipitation assay.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Staining, Sequencing, Construct, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative 2D classes of the recombinant, full-length PARP10 protein under cryo-EM. Scale bar: 200 Å. b , Cryo-EM map of PARP10 RRM1-3 pentamer obtained from 3D reconstruction and refinement. c , Structural model of PARP10 RRM1-3 pentamer, which is generated by docking AlphaFold-predicted models of PARP10 RRM1-3 domains into the cryo-EM map shown in ( b ). The PARP10 RRM1-3 oligomer adopts an open ring conformation with an angle of 140 degrees. d , Detailed illustration of the RRM1-3 dimer interface at two orientations. One dimeric interface of RRM1-3 is zoomed in, and residues in the interface between two RRM1-3 protomers are shown in sticks. e , Two protomers of PARP10 RRM1-3 contact with each other by electrostatic interactions. One protomer is represented with electrostatic potential surface, and the other is shown in cartoon. f,g , Representative images of cells ( f ) stably expressing PARP10 mutants depicted in ( g ). Scale bar: 10 μm. h , Quantification of the percentage of cells with foci in ( f ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Recombinant, Cryo-EM Sample Prep, Generated, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , A representative cryo-EM micrograph of full-length PARP10 protein. Scale bar: 500 Å. b , 2D class averages were selected for 3D reconstruction. Scale bar: 180 Å. c , Flow chart for cryo-EM data analysis. d , Maps of two classes of PARP10 RRM rings with different twisted angles. e , 3D variability analysis and non-uniform refinement of RRM oligomers. f - h , Map local resolution ( f ), Fourier shell correlation (FSC) curves ( g ), and particle distribution used in 3D reconstruction ( h ) of PARP10 RRM1-3 pentamer. i , j , 2D classes and corresponding models to show the two types of higher-order oligomerization of RRM1-3. Scale bar: 200 Å.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , b , Representative images for GFP-PARP10 condensate in cells stained with ADPr recognition antibodies (E6F6A and 10H) ( a ) and reagent (hsPARP14 macro2-3-rFC) ( b ). The catalytic deficient mutant GFP-PARP10 G888W served as a negative control. c , d , FRAP experiments for GFP-PARP10 and mCherry-eAf1521 co-expression cells. The fluorescent intensity in the region of interest (ROI) in ( c ) is quantified in ( d ). e , f , Representative images ( e ) and line quantification ( f ) of fluorescein-NAD + enrichment in PARP10-WT but not G888W condensates induced by PEG-8000 in vitro . g-i , Representative images of the reactions to show the PEG-8000 induced PARP10 condensation ( g ). Western blot showing ADP-ribosylation of 10 μM PARP10 with or without 10% PEG-8000 addition ( h ). The quantification of the ADP-ribosylation activity was shown in ( i ). n = 2. j , k , Western blot showing the ADP-ribosylation activity of PARP10 WT and condensation-deficient mutants 3RE and 3EA in cells ( j ). ADP-ribosylation levels were blotted with eAf1521. The quantification of the ADP-ribosylation activity was shown in ( k ). n = 3. Data are shown as mean ± SD. in ( d ), ( i ), ( k ). Scale bar: 10 μm for ( a )-( c ); 20 μm for ( e ) and ( g ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Staining, Mutagenesis, Negative Control, Expressing, In Vitro, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of cells stably expressing GFP-PARP10 WT and catalytic deficient mutants (G888W and ΔCD). ADPr signals stained by eAf1521. b , Western blot showing the relative protein expression of each construct used in ( a ). c , Box plot for the partition coefficient (PC) of PARP10 WT and mutants in ( a ). Each symbol represents one condensate. At least 25 condensates were measured and analyzed. d , Phase separation threshold concentration was measured for PARP10 WT and mutants in ( a ). The absolute concentration in cells is converted by a standard curve created by measuring the intensity of recombinant PARP10 protein. Each symbol represents one cell. At least 100 cells were measured and analyzed. Each black dot indicates one cell with condensates, the red dot indicates the cell without condensates, and the line marks the threshold concentration for condensate formation. e , f , Representative images ( e ) and quantification ( f ) for FRAP experiments on PARP10 WT ( n = 25), G888W ( n = 11), and H887E ( n = 17). The arrow indicated the bleached foci. Data are shown as mean ± SD. g , h , Representative images of GFP-PARP10 WT ( g ) and G888W ( h ) mutant treated with PARP10 inhibitor OUL-35. i , Box plot quantification of the diameter of PARP10 condensate in ( g ) and ( h ). j , Representative images of GFP-PARP10 co-expression with mCherry-MACROD2/TARG1/ARH3/PARG103. k , Box plot quantification of the diameter of PARP10 condensate in ( j ). Data are shown as mean ± SD. l , Representative images of PARP10 WT/H887E condensation induced by 10% Ficoll with or without NAD + addition. m , Western blot showing the ADP-ribosylation and protein levels of PARP10 WT and H887E in ( l ). n , Western blot showing CHX chase assay for IFNβ-induced endogenous PARP10 protein level in U2OS cells coupled with MG132 or Bafilomycin A1 treatments. o , Western blot showing ADP-ribosylation of GFP-PARP10 WT in cells coupled with MG132 treatment for the indicated times. The accumulation of ubiquitin signal was detected with P4D1 ubiquitin antibody. The catalytic deficient mutant GFP-PARP10 G888W serves as a negative control. p , Western blot showing the accumulation of GFP-PARP10 protein in cells treated with different concentrations of PARP10 activity inhibitor OUL35. q , Quantification of the GFP-PARP10 protein levels in ( p ). Data are shown as mean ± SD. in ( q ). Scale bar: 10 μm for ( a ), ( e ), ( g ), ( h ), ( j ). 20 μm for ( l ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Stable Transfection, Expressing, Staining, Western Blot, Construct, Concentration Assay, Recombinant, Mutagenesis, Ubiquitin Proteomics, Negative Control, Activity Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , List of ADPr hydrolases used in the study. b , Western blot showing the effect of mCherry-tagged ADPr hydrolase and its catalytic deficient (CD) mutants on the GFP-PARP10 auto-ADP-ribosylation in HEK293T cells. The auto-ADP-ribosylation was probed by eAf1521. c , Representative images of GFP-PARP10 condensates stained for FK2. d , TUBE pulldown assay showing the ubiquitination of PARP10 in response to MG132 and IFNβ treatment. GST bead only and PARP10 KO cells were used as controls. e , Western blot showing the ubiquitination of PARP10 in cells. f , Representative images of GFP-PARP10 WT/G888W stable cells treated with CHX for different times. g , Box plot quantification of the diameter of PARP10 condensate in ( f ). Scale bar: 10 μm for ( c ) and ( f ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Western Blot, Staining, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a - d , Representative images of cells co-transfected with GFP/GFP-PARP10 WT/G888W and mCherry-DTX2 ( a ), DTX3L ( b ), DTX3 ( c ), DTX4 ( d ). e , Representative images of cells transfected with GFP-PARP10 WT/G888W and stained with DTX1 antibody. f , Representative images of cells co-transfected with GFP-PARP10 WT and mCherry-DTX2 WT or 5 Mut which disrupts the ADPr binding. g , Western blot showing the interaction between GFP-PARP10 WT and mCherry/mCherry-DTX2 WT/5 Mut, determined by co-immunoprecipitation assay. h , Representative images and summary of GFP-PARP10 condensates colocalized with FK2-positive TRIMs, but not with the FK2-negative catalytic mutants. Scale bar: 10 μm for ( a )-( f ) and ( h ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Transfection, Staining, Binding Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Normalized VSV mRNA levels in HEK293T cells transfected with GFP/GFP-PARP10 WT/G888W/3RE and infected with VSV (8 hpi, MOI = 1.5). n = 6. b , Normalized VSV mRNA levels in Parp10 +/+ , Parp10 -/- , Parp10 G837W/G837W and Parp10 3E/3E BMDM with or without VSV infection (12 hpi, MOI = 1.5). n = 5. c , NAD + levels in HEK293T cells transfected with GFP/GFP-PARP10 WT/G888W, with or without VSV infection (8 hpi, MOI = 1.5), measured by MS. n = 3. d , NAD + levels in WT U2OS, HEK293T and BMDM cells infected with VSV (MOI = 1.5) for the indicated times, measured by MS. n = 3. e , Heatmap of gene expression in BMDM infected with VSV (12 hpi, MOI = 1.5) or not. n = 3. Genes related to NAD + metabolic process were shown. f , Fold changes of NAD + consuming genes in BMDM with VSV (12 hpi, MOI = 1.5) infection relative to non-infected control. n = 3. g , NAD + levels in Parp10 +/+ (WT), Parp10 -/- , and Parp10 3E/3E BMDM with or without VSV infection (12 hpi, MOI = 1.5), measured by MS. n = 3. h , Schematic diagram of the NAD + salvage pathway and the effect of NAMPT inhibitor FK866. i , Normalized VSV mRNA levels in HEK293T cells transfected with GFP/GFP-PARP10 WT, followed by the supplement of the indicated concentrations of NR and VSV infection (8 hpi, MOI = 1.5). n = 3. j , NAD + levels of cells in ( i ), determined by bioluminescent assays. n = 3. k , Normalized VSV mRNA levels in HEK293T cells treated with FK866 or DMSO (control), followed by VSV infection (8 hpi, MOI = 1.5). n = 3. l , NAD + levels of cells in ( k ), determined by bioluminescent assays. n = 3. m , Normalized VSV mRNA levels in HEK293T cells transfected with GFP vector/GFP-SARM1-TIR WT/E642A, followed by VSV infection (8 hpi, MOI = 1.5). n = 3. n , NAD + levels of cells in ( m ), determined by bioluminescent assays. n = 3. o , NAD + consumption by recombinant PARP10 with or without the addition of PEG-8000 for condensation induction in vitro over time, determined by bioluminescent assays. p , Representative images of GFP-PARP10 condensate with or without VSV infection (24 hpi, MOI = 1.5). Scale bar: 10 μm. q , Box plot quantification of the diameter of PARP10 condensate in ( p ). r , Schematic diagram showing that PARP10 inhibits VSV infection by regulating NAD + homeostasis. Data are shown as mean ± SD. in ( a )-( d ), ( g ), ( i )-( n ).

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Transfection, Infection, Gene Expression, Control, Plasmid Preparation, Recombinant, In Vitro

Journal: bioRxiv

Article Title: PARP10 condensation inhibits viral infection via targeting NAD + homeostasis

doi: 10.64898/2025.12.22.696120

Figure Lengend Snippet: a , Representative images of cells transfected with mouse PARP10 WT and mutants. ADPr signals were detected using eAf1521. Scale bar: 10 μm. b , Schematic diagram showing the generation of Parp10 G837W/G837W and Parp10 3E/3E mice with verification by Sanger sequencing. c , Normalized Parp10 mRNA levels in Parp10 +/+ , Parp10 -/- , Parp10 G837W/G837W and Parp10 3E/3E BMDM infected with VSV (12 hpi, MOI = 1.5) or not. n = 3. Data are shown as mean ± SD. d , Western blot showing the PARP10 protein expression in Parp10 +/+ , Parp10 -/- , Parp10 G837W/G837W and Parp10 3E/3E BMDM infected with VSV (12 hpi, MOI = 1.5) or not. e , KEGG analysis of metabolism pathways affected by PARP10 expression in HEK293T cells. f , Heatmap of metabolite levels in HEK293T cells transfected with GFP/GFP-PARP10 WT/G888W/3RE, with or without VSV infection (8 hpi, MOI = 1.5), measured by MS. n = 3. Metabolites of particular interest are colored by category. Metabolites with significant changes ( p < 0.05) are shown. g , Western blot showing the ADP-ribosylation of GFP-PARP10 in cells infected with VSV (MOI = 1.5) for the indicated times. Cells transfected with GFP vector and catalytic-deficient mutant (G888W) served as negative controls. PARP10 ADP-ribosylation was probed with eAf1521. h , GSEA showing the enriched gene set in HEK293T cells expressing GFP-PARP10 relative to GFP vector. i , Heat map of gene expression in ( h ) gene set. The expression profile for GFP, GFP-PARP10 WT, G888W, and 3RE mutants was shown. j , k , Heat map of gene expression related to NAD + catalytic process ( j ) and NAD + consumption ( k ) from the whole blood samples in healthy ( n = 17) and COVID-19 ( n = 20) populations. l , Comparison of PARP10 expression between healthy ( n = 17) and COVID-19 ( n = 20) populations. Data are shown as mean ± SD.

Article Snippet: Parp10 -/- , Parp10 3E/3E , Parp10 G837W/G837W and mEGFP- Parp10 knock-in ( Parp10 mEGFP/mEGFP ) mice were generated via CRISPR/Cas9 mediated gene editing by Cyagen Bioscience Inc. Mice were maintained under specific-pathogen-free conditions with controlled temperature (18–23 °C), humidity (40–60%) and a 12-hour light/12-hour dark cycle.

Techniques: Transfection, Sequencing, Infection, Western Blot, Expressing, Plasmid Preparation, Mutagenesis, Gene Expression, Comparison

Journal: The EMBO Journal

Article Title: ALYREF condensation stabilizes m 5 C-modified PARP10 mRNA and promotes PI3K-AKT signaling in ovarian cancer

doi: 10.1038/s44318-025-00657-0

Figure Lengend Snippet: ( A ) Western blotting assay detecting PARP10 expression in ovarian cancer cells upon ALYREF knockdown. ( B ) The protein expression was quantified by grayscale in ( A ). ( C ) Nucleocytoplasmic separation assays were confirmed by western blotting. ( D ) Effect of ALYREF deficiency on the nucleocytoplasmic ratio of PARP10 mRNA in ovarian cancer cells. ( E ) Effect of ALYREF deficiency on the stability of PARP10 mRNA in ovarian cancer cells. ( F ) Interaction between MTR4 and PARP10 mRNA in ovarian cancer cells with ALYREF knockdown or not by RIP and RT-qPCR assays. ( G ) Expression of PARP10 mRNA was assessed in ovarian cancer cells upon ALYREF knockdown alone or followed by MTR4 knockdown. ( H ) The stability of PARP10 mRNA was assessed in A2780 ovarian cancer cells upon ALYREF knockdown alone or followed by MTR4 knockdown. ( I ) RT-qPCR assays verifying the enrichment of PARP10 in NSUN2 RIP assays. ( J ) RT-qPCR assays detecting the enrichment of PARP10 in meRIP assays. ( K ) RT-qPCR assays detecting the enrichment of PARP10 in ALYREF RIP assays in ovarian cancer cells upon NSUN2 knockdown. ( L ) Schematic showed the m 5 C modification site of PARP10 according to the RNA-BisSeq. ( M ) Primers used to detect the enrichment of RARP10 mRNA in eCLIP assays shown in ( L ). ( N ) The eCLIP experiments detecting the interaction between ALYREF and PARP10 mRNA. ( O ) Effect of wild-type ALYREF or mutant overexpression on the expression of PARP10 mRNA in ovarian cancer cells. ( P ) Western blotting verifying overexpression of ALY-wt or ALY-mut in A2780 and OVCAR3 cell lines. ( Q ) The expression of the protein was quantified by grayscale in ( P ). ( R ) RT-qPCR assays detecting the enrichment of PARP10 in meRIP assays. ( S ) RT-qPCR assays detecting the enrichment of PARP10 in RIP assays. ( T ) RT-qPCR assays detecting expression changes of PARP10 in ovarian cancer cells with overexpression of PARP10-wt or PARP10-mut upon ALYREF knockdown. ( U ) RT-qPCR assays detecting expression changes of PARP10 in ovarian cancer cells with overexpression of PARP10-wt or PARP10-mut upon NSUN2 knockdown. ( V ) RT-qPCR assays detecting expression changes of PARP10 in ovarian cancer cells with overexpression of PARP10-wt or PARP10-mut upon MTR4 knockdown. n = 3 independent experiments. Data are shown as means ± S.D. P value was calculated by one-way ANOVA test with multiple comparisons ( B , D , F , G , O , Q) or unpaired two-sided Student’s t test ( I – K , M , N , R , S , T – V ). .

Article Snippet: PARP10 , Proteintech , 26072-1-AP.

Techniques: Western Blot, Expressing, Knockdown, Quantitative RT-PCR, Modification, Mutagenesis, Over Expression

Journal: The EMBO Journal

Article Title: ALYREF condensation stabilizes m 5 C-modified PARP10 mRNA and promotes PI3K-AKT signaling in ovarian cancer

doi: 10.1038/s44318-025-00657-0

Figure Lengend Snippet: ( A ) Detecting the protein levels in ovarian cancer cells upon ALYREF knockdown by western blotting. ( B ) The expression of the protein was quantified by grayscale in ( A ). ( C ) RIP assays detecting the interaction between MTR4 and PARP10 mRNA. ( D ) RT-qPCR detecting the expression level of PARP10 mRNA in nucleocytoplasmic separation assays. ( E ) Effect of ALYREF knockdown on the protein level of MTR4 in A2780 and OVCAR3 cells. ( F ) The expression of the protein was quantified by grayscale in ( E ). ( G ) ALYREF and MTR4 were knocked down in OVCAR3 cells, and PARP10 mRNA stability was detected. ( H ) RT-qPCR assays verifying the enrichment of PARP10 in NSUN2 RIP assays in SKOV3 cells. ( I ) RT-qPCR detecting the enrichment of PARP10 mRNA in ALYREF RIP assays in SKOV3 cells. ( J ) Detecting protein expression levels in ovarian cancer cells upon NSUN2 knockdown by western blotting assays. ( K ) The expression of the protein was quantified by grayscale in ( J ). ( L ) The diagram of eCLIP experiments of ALYREF in ovarian cancer cells. ( M ) Effect of ALYREF or NSUN2 knockdown on the stability of wild-type or m 5 C-mutated PARP10 mRNA in ovarian cancer cells. n = 3 independent experiments ( B – D , F – I , K , M ). Data are shown as means ± S.D. P value was calculated by one-way ANOVA test with multiple comparisons ( B , F , K ) or unpaired two-sided Student’s t test ( C , D , H , I ). .

Article Snippet: PARP10 , Proteintech , 26072-1-AP.

Techniques: Knockdown, Western Blot, Expressing, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: ALYREF condensation stabilizes m 5 C-modified PARP10 mRNA and promotes PI3K-AKT signaling in ovarian cancer

doi: 10.1038/s44318-025-00657-0

Figure Lengend Snippet: ( A ) Condensation of ALYREF protein was predicted according to catGRANULE. ( B ) Phase separation of ALYREF was predicted by the π Interaction Score Prediction Tool. ( C ) The phase separation of ALYREF was predicted by https://fuzdrop.bio.unipd.it/predictor . ( D ) Effect of temperature on the phase transition properties of ALYREF protein. ( E ) Fluorescence recovery assays after photobleaching of ALYREF condensates in ovarian cancer cells. The red arrow represents the photobleached region in the droplet. Scale bar, 10 μm. ( F ) Quantitative analysis of condensates in ( E ). The black arrow represents the time point of photobleaching. The red arrow represents the photobleached region in the droplet. The red curve represents the average of the normalized fluorescence intensities in different droplet photobleaching regions ( n = 3 independent experiments). ( G ) Effect of m 5 C-modified RNA on phase separation of ALYREF proteins ( n = 50). Scale bar, 20 μm. ( H ) Co-localization of ALYREF protein and PARP10 RNA by RNA FISH assays in ovarian cancer cells. Scale bar, 10 μm. Data are shown as means ± S.D. P value was calculated by one-way ANOVA test with multiple comparisons ( G ). .

Article Snippet: PARP10 , Proteintech , 26072-1-AP.

Techniques: Sublimation, Fluorescence, Modification

Journal: The EMBO Journal

Article Title: ALYREF condensation stabilizes m 5 C-modified PARP10 mRNA and promotes PI3K-AKT signaling in ovarian cancer

doi: 10.1038/s44318-025-00657-0

Figure Lengend Snippet: ( A ) Schematic diagram of the vector construction of ALYREF-wt, truncations, and mutants. ( B ) In vitro phase separation of ALYREF-wt, truncations, and mutants tagged with GFP was detected. Scale bar, 5 μm. ( C ) Representative images depicting exogenously expressed GFP-ALYREF and its respective truncated forms in OVCAR3 and A2780 cells are shown. A scale bar of 5 µm is provided for reference. ( D ) Statistical analysis of the number of LLPS puncta for each group ( n = 50). ( E ) Schematic diagram of the opto-droplet system. ( F ) Representative images showing the clustering of ALYREF and its truncated form induced by blue light in HEK293T cells. Stimulation was achieved using a 488 nm laser. Scale bar: 5 µm. ( G ) RT-qPCR assays detecting PARP10 expression level in ovarian cancer cells with overexpression of ALYREF-wt, truncations, and mutants, respectively. ( H ) RT-qPCR assays detecting karyoplasmic distribution of PARP10 mRNA in ovarian cancer cells with overexpression of ALYREF-wt, truncations, and mutants, respectively. ( I ) Effect of ALYREF-wt, truncations, and mutants on the stability of PARP10 mRNA in ovarian cancer cells. n = 3 independent experiments ( G – I ). Data are shown as means ± S.D. P value was calculated by one-way ANOVA test with multiple comparisons ( G , H ). .

Article Snippet: PARP10 , Proteintech , 26072-1-AP.

Techniques: Plasmid Preparation, In Vitro, Quantitative RT-PCR, Expressing, Over Expression

Journal: The EMBO Journal

Article Title: ALYREF condensation stabilizes m 5 C-modified PARP10 mRNA and promotes PI3K-AKT signaling in ovarian cancer

doi: 10.1038/s44318-025-00657-0

Figure Lengend Snippet: ( A ) The proliferation of ovarian cancer cells with overexpression of ALYREF-wt, truncations, and mutants, respectively. ( B ) The colony formation assays of ovarian cancer cells with overexpression of ALYREF-wt, truncations, and mutants, respectively. ( C ) The migration and matrigel invasion assays of ovarian cancer cells with overexpression of ALYREF-wt, truncations, and mutants, respectively. Scale bar, 200 μm. ( D ) Representative images of xenograft tumors formed by ovarian cancer cells with overexpression of ALYREF-wt, truncations, and mutants, respectively in nude mice. ( E ) Assessment of the weight of xenograft tumors in nude mice ( n = 5). ( F ) Assessment of the size of xenograft tumors in nude mice ( n = 5). ( G ) The proliferation of ALYREF-depleted ovarian cancer cells with overexpression of PARP10. ( H ) The statistical result of colony formation of ALYREF-depleted ovarian cancer cells with overexpression of PARP10. ( I ) The statistical results of transwell migration and matrigel invasion of ALYREF-depleted ovarian cancer cells with overexpression of PARP10. ( J ) Spearman’s rank correlation between ALYREF mRNA and PARP10 mRNA was carried out in ovarian cancer. ( K ) Expression levels of ALYREF and PARP10 in ovarian cancer tissues and normal fallopian tube epithelium by western blotting. ( L ) Correlation analysis of ALYREF and PARP10 in ovarian cancer tissues. n = 3 independent experiments ( A – C , G – I ). Data are shown as means ± S.D. P value was calculated by one-way ANOVA test with multiple comparisons ( A – C , E , F , G – I ) or unpaired two-sided Student’s t test ( K ). .

Article Snippet: PARP10 , Proteintech , 26072-1-AP.

Techniques: Over Expression, Migration, Expressing, Western Blot

Journal: The EMBO Journal

Article Title: ALYREF condensation stabilizes m 5 C-modified PARP10 mRNA and promotes PI3K-AKT signaling in ovarian cancer

doi: 10.1038/s44318-025-00657-0

Figure Lengend Snippet: ( A ) Detecting the protein level in ALYREF-depleted ovarian cancer cells with PARP10 overexpression by western blotting. ( B ) The expression of the protein was quantified by grayscale in ( A ) ( n = 3 independent experiments). Data are shown as means ± S.D. P value was calculated by one-way ANOVA test with multiple comparisons. ( C ) Colony formation assays of ALYREF-depleted ovarian cancer cells with overexpression of PARP10. ( D ) The transwell migration and matrigel invasion assays of ALYREF-depleted ovarian cancer cells with overexpression of PARP10. Scale bar, 100 μm. ( E ) Correlation analysis between ALYREF and PARP10 expression in various cancers. .

Article Snippet: PARP10 , Proteintech , 26072-1-AP.

Techniques: Over Expression, Western Blot, Expressing, Migration

Journal: The EMBO Journal

Article Title: ALYREF condensation stabilizes m 5 C-modified PARP10 mRNA and promotes PI3K-AKT signaling in ovarian cancer

doi: 10.1038/s44318-025-00657-0

Figure Lengend Snippet: ( A ) Alternation frequencies of ALYREF gene in various cancers including according to analysis in the TCGA pan-cancer database. ( B ) Alternation frequencies of ALYREF gene in ovarian cancers. ( C ) Analysis of the association between ALYREF amplification and its expression in ovarian cancer. The central line within the box represents the median value. The upper and lower edges of the box represent the 75th and 25th percentiles, respectively. The whiskers extend from the box to the maximum and minimum values within 1.5 times the interquartile range, with any data points beyond this range considered as outliers. ( D , E ) Expression of ALYREF in ovarian cancers at different FIGO stages and tumor pathological grades. ( F , G ) Associations of ALYREF expression with ovarian cancer patients’ OS, DFS, and PFS. ( H ) Schematic illustrating the mechanisms of ALYREF-mediated stability of m 5 C-modified PARP10 mRNA by LLPS in ovarian cancer. Data are shown as means ± S.D. P value was calculated by unpaired two-sided Student’s t test ( D , E ) two-sided Log-rank (Mantel-Cox) test ( F , G ). .

Article Snippet: PARP10 , Proteintech , 26072-1-AP.

Techniques: Amplification, Expressing, Modification

Journal: The EMBO Journal

Article Title: ALYREF condensation stabilizes m 5 C-modified PARP10 mRNA and promotes PI3K-AKT signaling in ovarian cancer

doi: 10.1038/s44318-025-00657-0

Figure Lengend Snippet: ( A ) Correlation analysis of ALYREF mRNA expression with its gene amplification. ( B ) Correlation analysis of ALYREF mRNA expression with DNA methylation. ( C ) ALYREF expression in serous ovarian cancer was significantly correlated with tumor FIGO stage. ( D ) Survival analysis of ovarian cancer patients with different PARP10 expressions according to the Kaplan-Meier Plotter. ( E , F ) Survival analysis of subtype ovarian cancer patients with different ALYREF expressions according to the Kaplan-Meier Plotter. ( G ) Representative IHC images of ALYREF expression in ovarian cancer tissues. Scale bar, 100 μm. ( H ) Survival analysis of ovarian cancer patients with different ALYREF expressions or patients at advanced stage (III and IV) with different ALYREF expressions. ( I – K ) Pan-cancer analysis of ALYREF RNA and protein expression in various cancers according to the TCGA ( I ) and CPTAC databases ( J , K ). ( L ) Survival analysis of cancer patients with different ALYREF expressions according to the Kaplan-Meier Plotter. The central line within the box represents the median value ( C , J , K ). The upper and lower edges of the box represent the 75th and 25th percentiles, respectively. The whiskers extend from the box to the maximum and minimum values within 1.5 times the interquartile range, with any data points beyond this range considered as outliers. P value was calculated by two-sided Log-rank (Mantel-Cox) test ( D – F , H , L ) or unpaired two-sided Student’s t test ( J , K ). .

Article Snippet: PARP10 , Proteintech , 26072-1-AP.

Techniques: Expressing, Amplification, DNA Methylation Assay