Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 1. DDB1 interacts with and cleaved by NS3/4A. (A) DDB1 interacts with NS3/4A in overexpression system. The 293 cells were transfected with the indicated plasmids. Coimmunoprecipitation was performed with anti-Flag or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-Flag anti-HA. The lysates were analyzed by immunoblots with anti-DDB1 or anti-HA. (B) Endogenous DDB1 interacts with NS3/4A in JFH-1 infected cells. Huh-7 cells (5 107) were mock-infected or infected with JFH-1 (Multiplicity of Infection, MOI: 0.3) for 3 days. Coimmunoprecipitation was performed with anti-DDB1 or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-DDB1 and anti-NS3. The lysates were analyzed by immunoblots with anti-DDB1 or anti-NS3.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Control, Western Blot, Infection

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 2. NS3/4A cleaves DDB1 at C378. (A) Cleavage of DDB1 by NS3/4A is inhibited by the NS3/4A inhibitor VX-950. The 293 cells were transfected with N-terminal or C-terminal Flag-tagged DDB1 (N-Flag-DDB1 or DDB1-C-Flag respectively) and HA-NS3/4A. The transfected cells were treated with VX-950 (0.2 mM) or left untreated for 1 day before immunoblot analysis with anti-Flag or anti-HA. (B) Alignment of the junction sequences of NS proteins of HCV and the potential NS3/4A cleavage sites in TC-PTP, VISA, TRIF and DDB1. (C) NS3/4A cleaves DDB1 at C378. The 293 cells were transfected with the indicated plasmids and cells lysates were analyzed by immunoblots with anti-Flag or anti-HA. (D) DDB1 N-terminal cleavage product migrated similarly to overexpressed DDB1(1–378) mutant. The 293 cells were transfected with the indicated plasmids, treated with VX-950 or left untreated for 1 day before immunoblot analysis with with anti-Flag or anti-HA.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Transfection, Western Blot, Mutagenesis

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 3. DDB1 plays a critical role in HCV replication. (A) Overexpression of DDB1 potentiates HCV RNA replication. Huh-7 cells (1 106) were transfected with the indicated amounts of Flag-DDB1 plasmid for 24 h and then cells were split and mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-Flag or anti-b-actin. Graphs show mean7SD, n¼3. (B) Knockdown of DDB1 inhibits HCV RNA replication. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (C) Knockdown of DDB1 inhibits HCV protein expression. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 3 days, and the cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (D) Knockdown of DDB1 inhibits production of infectious HCV particles. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show mean7SD, n¼3. (E) Knockdown of DDB1 inhibits RNA replication of HCV subgenomic replicon. Control or DDB1-RNAi knockdown Huh-7 cells and Huh-7 Con1 subgenomic replicon cells were cultured for 3 days. The cells (2 106) were collected and intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. Cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (F) DDB1 has no effects on HCV entry. Control or DDB1-RNAi knockdown Huh-7 cells were infected with HCVpp for 3 days (NC: Negative Control, HCVpp pakaging without HCV E1E2). The lysates of infected cells were assayed by luciferase reporter assays and immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Western Blot, Knockdown, Control, Expressing, Staining, Cell Culture, Negative Control, Luciferase

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 4. DDB1 cleavage is required for HCV replication. (A) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) The indicated stable cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh medium was added for 48 h. The JFH-1-containing medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D) Control or DDB1-RNAi knockdown Huh-7 cells were stably transduced with empty vector, DDB1, DDB1(C378R), off-target nonsense mutants of DDB1 or DDB1(C378R) respectively. Two days later, cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-actin. Graphs show meanþSD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Stable Transfection, Infection, Quantitative RT-PCR, Western Blot, Staining, Control, Knockdown, Transduction, Plasmid Preparation

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 5. DDB1 cleavage products do not affect the HCV infection. (A) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D–F) Control or DDB1-RNAi-#1 (targeted sequence is within the cDNA fragment encoding aa379–1140) transduced Huh-7 cells were further transfected with empty vector, Flag-DDB1(1–378), Flag-DDB1(379–1140*) (*, a RNAi off-target mutant),or a combination of Flag-DDB1(1–378) and Flag-DDB1(379–1140*) by Lipofectamine 2000. One day post transfection, the cells were split and mock infected or infected with JFH-1(MOI: 0.3) for 3 (D, E) or 1 (F) day. Intracellular HCV RNA levels (D), intracellular viral particles (E), or viral titers in the medium (F) were then determined as described in (A–C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Infection, Stable Transfection, Transduction, Quantitative RT-PCR, Western Blot, Staining, Control, Sequencing, Transfection, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Data from AlphaFold 3, predicting (A) NAT10 dimerization and the degree of interaction between (B) NAT10-DDB2, (C) NAT10-p53, and (D) DDB2-p53, were imported to ChimeraX 1.9 software showing individual proteins. NAT10 is highlighted by orange, DDB2 is green, p53 is blue colour. The source data are shown at https://www.ibp.cz/en/research/departments/cellular-biology-and-epigenetics/open-data . This raw data from AlphaFold 3 can be uploaded to ChimeraX 1.9 for more detailed analysis.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Software

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: (A) The following proteins were studied in non-irradiated and UVC-irradiated NAT10 wt and dn cells: NAT10, p53, XPA, XPC, and DDB2. In detail, panel (A) shows western blot analysis of proteins NAT10, p53, XPA, XPC, and DDB2 in non-irradiated and UVC-irradiated cells without and with a depletion of the NAT10 acetyltransferase. Data revealed lower p53, XPA, XPC, and DDB2 protein levels in NAT10 dn cells, with further reductions of XPA, XPC, and DDB2 protein levels upon UVC exposure. UVC irradiation also decreased the pool of p53, XPA, and XPC in NAT10 wt cells. Panels (B-D) show the quantification of western blot data from panel (A) using ImageJ software (NIH freeware, USA). Non-irradiated cells and cells irradiated by UVC light were analyzed. (E) TP53 deficiency led to a decrease in the pool of NAT10 and DDB2 proteins. Panel (F) shows the quantification of western blot data from panel (E) using ImageJ software (NIH freeware, USA). The protein levels were normalized to GAPDH (reference and loading control). The asterisks in panels B-D and F represent statistically significant differences with either a p-value ≤ 0.05 (*) or a p-value ≤ 0.001 (***). AlphaFold 3 models of ( G) NAT10 interaction with p53 (pTM = 0.53), ( H ) NAT10 and DDB2 (pTM = 0.5), and ( I ) DDB2 and p53 protein (pTM = 0.47). Structures are colored with AlphaFold 3 pLDDT confidence score.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Irradiation, Western Blot, Software, Control

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Panels (A, B) show AlphaFold 3 multimer prediction of human NAT10 interaction with (A) NAT10 and p53 and DDB2 proteins (pTM = 0.48) or (B) NAT10 and p53 and DDB1-DDB2 protein complex complex (pTM = 0.53).

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques:

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Interaction properties between p53-DDB2 and NAT10-DDB2 in human NAT10 wt and NAT10 dn cells were studied using PLA. (A) The panel shows an example of PLA methodology, adapted from BioRender software. In (B) NAT10 wt and (C) NAT10 dn cells, after fixation, the PLA was performed using antibodies to detect the p53-DDB2 or NAT10-DDB2 complexes in both non-irradiated and UVC-irradiated NAT10 wt and NAT10 dn cells. Red dots indicate amplified interaction signals, with cell nuclei counterstained with DAPI, used for visualization of DNA content. NAT10-DDB2 in NAT10 dn acted as a control, showing no external impact on PLA outcomes. This section quantifies PLA signals (dots per nucleus) for (D) the p53-DDB2 complex in non-irradiated and UVC-irradiated NAT10 wt cells, (E) p53-DDB2 in non-irradiated and UVC-irradiated NAT10 dn cells. (F) The UVC irradiation significantly increased NAT10-DDB2 PLA signals in NAT10 wt cells, suggesting enhanced interaction. Non-irradiated cells and cells irradiated by UVC light were analyzed. We used ImageJ software (NIH freeware, USA) to analyze the number of PLA signals. The statistical analysis was performed using GraphPad Prism 9 software (USA) and the nonparametric Mann-Whitney U -test. The asterisk in panel F represents statistically significant differences with a p-value ≤ 0.05 (*).

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Software, Irradiation, Amplification, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: (A, B) Animation documents chromatin density inside the cell nucleus (see graphical illustration created by BioRender software). Panel (A) shows cell nucleolus (Nu) and nuclear lamina in dark blue, and panel (B) shows localization of heterochromatin (green) decorating the periphery of cell nucleus and nucleolus, while euchromatin (pale blue) is shown as de-condensed treads inside the cell nucleus. An analysis of the DDB2 nuclear distribution pattern was done using immunohistochemistry in (C) NAT10 wt and NAT10 dn cells. (D) The NAT10 protein (red) was accumulated into tiny, well-visible foci in DAPI-dense (considered as heterochromatin), DAPI-poor (euchromatin, more decondensed) genomic region as well as inside compartment of nucleoli. Panel (E) shows representative images of the DDB2 protein recruitment to UVA-microirradiated chromatin in NAT10 wt and NAT10 dn cells. Microirradiation using a 405 nm laser line was performed, followed by immunofluorescent staining for DDB2 and DNA damage marker γH2A.X. DAPI was used for nuclear counterstaining in blue fluorescence. The scale bar represents 7.5 μm.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Software, Immunohistochemistry, Staining, Marker, Fluorescence

Journal: bioRxiv

Article Title: Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro

doi: 10.1101/2023.07.12.548668

Figure Lengend Snippet: ( a ) The summary of multivalent interactions of XLF and XRCC4 CTRs characterized in this study (pink and blue wavy lines) as well as previously characterized interactions (straight grey lines). ( b ) Condensates may form and enhance the assembly of NHEJ super-complexes, DNA tethering, rates of enzymatic reactions, and the recruitment of other NHEJ effectors through multivalent interactions of the IDRs, extending the proposed synaptic models obtained from single-particle cryo-EM and single molecule analysis ( , ). This occurs when the local concentrations of XLF and X4L4 (in shadow) are above a critical concentration. DNA-PKcs: DNA-dependent protein kinase catalytic subunit, WRN: Werner syndrome protein, Pol μ : DNA polymerase μ , Pol / : DNA polymerase / , TdT: terminal deoxynucleotidyl transferase, APTX: aprataxin, PNKP: bifunctional polynucleotide phosphatase/kinase, TDP1: tyrosyl-DNA phosphodiesterase 1, PAXX: paralog of XRCC4 and XLF, APLF: aprataxin and PNK-like factor, CYREN: cell cycle regulator of non-homologous end joining, NONO: non-POU domain-containing octamer-binding protein, SFPQ: splicing factor, proline- and glutamine-rich, lncRNAs: long non-coding RNA ( , , ).

Article Snippet: Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have recently been obtained by single-particle cryo-electron microscopy analysis.

Techniques: Single Particle, Cryo-EM Sample Prep, Concentration Assay, Non-Homologous End Joining, Binding Assay