human cervix epithelial hela cells  (ATCC)

Journal: Journal of Virology

Article Title: A novel antiviral role of ankyrin repeat and LEM domain-containing 2 (ANKLE2) in restricting vaccinia virus through barrier to autointegration factor (BAF)

doi: 10.1128/jvi.01549-25

Figure Lengend Snippet: Impact of ANKLE2 depletion on BAF phosphorylation and WT or ΔB1 vaccinia virus infection in HeLa cells. ( A ) HeLa cells were transfected with siCTRL or siANKLE2 for 3 days, followed by immunoblotting analysis of ANKLE2. An immunoblot of GAPDH was included as a loading control. ( B ) Cytoplasmic BAF from siANKLE2-transfected HeLa cells was obtained by subcellular fractionation and analyzed by immunoblotting. Closed arrow indicates phosphorylated BAF. The open arrow indicates unphosphorylated BAF. An immunoblot of GAPDH was included as a loading control. ( C ) Relative phospho-BAF protein levels were quantified using ImageLab software (Bio-Rad) from the four independent replicates. The values for siCTRL-transfected cells were normalized to 1. ( D ) DNA accumulation and ( E ) viral yield were measured for WT or ΔB1 vaccinia virus at 24 hpi in siRNA-transfected HeLa cells infected at an MOI of 3. Data are from more than three independent experimental replicates. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Relative fold differences are given under each bracket.

Article Snippet: Human cervix epithelial adenocarcinoma HeLa cells were purchased and obtained directly from the ATCC.

Techniques: Phospho-proteomics, Virus, Infection, Transfection, Western Blot, Control, Fractionation, Software

Journal: Journal of Virology

Article Title: A novel antiviral role of ankyrin repeat and LEM domain-containing 2 (ANKLE2) in restricting vaccinia virus through barrier to autointegration factor (BAF)

doi: 10.1128/jvi.01549-25

Figure Lengend Snippet: Characterization of expression and localization of endogenous and mutant ANKLE2 in siRNA-treated transduced HeLa cells. ( A ) Schematic representation of human full-length ANKLE2 and its N-terminal truncation mutants used in this study, showing annotated domains and amino acid positions indicated by black arrows. ( B ) HeLa cells were transduced with lentivirus expressing various siRNA-resistant ANKLE2 truncations, followed by transfection with siCTRL or siANKLE2. Cells were then treated with 1 µg/mL doxycycline for 3 days to induce protein expression and analyzed by immunoblotting. ( C ) Immunofluorescence analysis of HeLa cells transduced with empty vector (CTRL), full-length ANKLE2-3XFLAG, and N-terminal truncation mutants Δ2–53, Δ2–117, and Δ2–252. ANKLE2-transduced HeLa cells were stained with anti-FLAG antibody (red) and DAPI (blue). The representative images shown were taken using an inverted confocal microscope at ×60 magnification. Scale bars represent 25 µm.

Article Snippet: Human cervix epithelial adenocarcinoma HeLa cells were purchased and obtained directly from the ATCC.

Techniques: Expressing, Mutagenesis, Transduction, Transfection, Western Blot, Immunofluorescence, Plasmid Preparation, Staining, Microscopy

Journal: Journal of Virology

Article Title: A novel antiviral role of ankyrin repeat and LEM domain-containing 2 (ANKLE2) in restricting vaccinia virus through barrier to autointegration factor (BAF)

doi: 10.1128/jvi.01549-25

Figure Lengend Snippet: DNA accumulation and viral yield analysis of WT and ΔB1 vaccinia virus in ANKLE2-transduced HeLa cells following endogenous ANKLE2 depletion. DNA accumulation (left panels) and viral yield (right panels) were measured for ( A ) WT and ( B ) ΔB1 vaccinia virus at a low MOI of 0.1 at 48 hpi, as well as for ( C ) ΔB1 vaccinia virus at a higher MOI of 3 with 24 hpi. Data are from more than three independent experimental replicates. Numbers below the significance bars represent the average fold-change value. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Human cervix epithelial adenocarcinoma HeLa cells were purchased and obtained directly from the ATCC.

Techniques: Virus

Journal: Journal of Virology

Article Title: A novel antiviral role of ankyrin repeat and LEM domain-containing 2 (ANKLE2) in restricting vaccinia virus through barrier to autointegration factor (BAF)

doi: 10.1128/jvi.01549-25

Figure Lengend Snippet: Abundance of viral proteins I3, A11, L4, and F17 during expression of ANKLE2 mutants. ANKLE2-transduced HeLa cells were transfected with siRNAs to deplete endogenous ANKLE2, followed by doxycycline inducement and infection with WT or ΔB1 vaccinia virus at an MOI of 3 for 24 hpi. Whole cell lysates were analyzed by immunoblot using antibodies specific to the proteins on the right. A representative immunoblot is shown with lane numbers shown below each column.

Article Snippet: Human cervix epithelial adenocarcinoma HeLa cells were purchased and obtained directly from the ATCC.

Techniques: Expressing, Transfection, Infection, Virus, Western Blot

Journal: Journal of Virology

Article Title: A novel antiviral role of ankyrin repeat and LEM domain-containing 2 (ANKLE2) in restricting vaccinia virus through barrier to autointegration factor (BAF)

doi: 10.1128/jvi.01549-25

Figure Lengend Snippet: Immunofluorescence analysis of doxycycline-treated ANKLE2-transduced HeLa cells infected with ( A ) WT or ( B ) ΔB1 vaccinia virus at an MOI of 5 for 6 hours at 37°C prior to fixation. Viral DNA replication factories were detected by I3 staining (green) using anti-I3 antibody, whereas transduced ANKLE2 was detected using anti-FLAG primary antibody (red). The representative images shown were taken using an inverted confocal microscope at ×60 magnification. Scale bars represent 25 µm.

Article Snippet: Human cervix epithelial adenocarcinoma HeLa cells were purchased and obtained directly from the ATCC.

Techniques: Immunofluorescence, Infection, Virus, Staining, Microscopy

Journal: Journal of Virology

Article Title: A novel antiviral role of ankyrin repeat and LEM domain-containing 2 (ANKLE2) in restricting vaccinia virus through barrier to autointegration factor (BAF)

doi: 10.1128/jvi.01549-25

Figure Lengend Snippet: Protein immunoblotting of cytoplasmic BAF in doxycycline-treated ANKLE2-transduced HeLa cells following subcellular fractionation. Cytoplasmic and membrane fractions were analyzed by immunoblot using antibodies specific to the proteins on the right. When using an antibody recognizing total BAF, the closed arrow indicates the migration position of phosphorylated BAF, and the open arrow indicates the unphosphorylated BAF. A representative immunoblot is shown with lane numbers shown below each column.

Article Snippet: Human cervix epithelial adenocarcinoma HeLa cells were purchased and obtained directly from the ATCC.

Techniques: Western Blot, Fractionation, Membrane, Migration

Journal: Journal of Virology

Article Title: A novel antiviral role of ankyrin repeat and LEM domain-containing 2 (ANKLE2) in restricting vaccinia virus through barrier to autointegration factor (BAF)

doi: 10.1128/jvi.01549-25

Figure Lengend Snippet: Immunoblotting of FLAG-tag immunoprecipitated lysates from doxycycline-treated ANKLE2-transduced HeLa cells. Confluent monolayers of cells in 10 cm 2 dishes were harvested, pelleted, and lysed with a lysis buffer. Complexes of transduced ANKLE2 containing 3XFLAG tag were immunoprecipitated overnight at 4°C and eluted by incubation at 37°C for 24 h. Samples were then analyzed by immunoblotting using antibodies against ANKLE2, FLAG, PP2A, and total BAF proteins. INPUT lysates were included to confirm the similar levels of ANKLE2, PP2A, and BAF proteins in each lysate. A representative immunoblot is shown with lane numbers shown below each column.

Article Snippet: Human cervix epithelial adenocarcinoma HeLa cells were purchased and obtained directly from the ATCC.

Techniques: Western Blot, FLAG-tag, Immunoprecipitation, Lysis, Incubation