kat2a primary cell based screening assays cell culture ht1080 human fibrosarcoma cell line ht1080  (ATCC)

Journal: Nucleic acids research

Article Title: Timed chromatin invasion during mitosis governs prototype foamy virus integration site selection and infectivity.

doi: 10.1093/nar/gkaf449

Figure Lengend Snippet: Figure 3. Conserved Gag CBS residues are essential for timely mitotic chromatin capture. Gag localization during PFV infection. G2 / M-phase synchronized HT1080 cells were transduced with PFV particles encoding WT, R540Q, or Y537Q Gag and fixed at different time points after drug release, corresponding to different mitotic phases indicated in orange. Gag proteins were detected using polyclonal anti-PFV Gag antiserum (green); the nuclear en v elope w as stained with anti-Lamin A / C antibodies (red) and cellular DNA with DAPI (gra y). White arro ws sho w MTOC accumulation of PFV Gag. Scale bars: 20 μm. Percentages of chromatin-bound Gag are indicated for each mitotic phase. See “Materials and methods” section for detailed information on quantification. Results are representative of those observed across at least five independent experiments.

Article Snippet: For each time oint measured, data of this second control (binding of Blitz uffer on biosensors loaded with biotinylated Gag WT pepide) were subtracted to every condition and were plotted on urves, using Prism 9 software. ell culture uman fibrosarcoma cell line HT1080 (ATCC CCL-121) and roteoglycan-deficient packaging cell line 293T-25A [ 26 ] were aintained in Dulbecco’s modified Eagle medium (DMEM; isher), supplemented with 50 μg / ml of gentamicin (Fisher cientific) and 10% fetal calf serum (FCS) (Eurobio Scientific). iral vector production FV particles were produced using a four-component system, ased on a protocol described in [ 26 ].

Techniques: Infection, Transduction, Staining

Journal: Nucleic acids research

Article Title: Timed chromatin invasion during mitosis governs prototype foamy virus integration site selection and infectivity.

doi: 10.1093/nar/gkaf449

Figure Lengend Snippet: Figure 4. Conserved PFV Gag CBS residues are required for optimal infectivity. ( A ) Six days post-infection, GFP-positive cells were counted by flow cytometry as relative measures of infectivity. Error bars are SDs determined from at least three independent infections; the WT values in each e xperiment w ere set to 10 0%. ( B ) Quantit ativ e PCR of integrated vDNA, 6 da y s after HT1080 infection, with PFV v ector particles carrying WT, R540Q, or Y537Q Gag with WT IN or WT Gag containing virus with catalytically inert D185N / E221Q IN (IN-NQ). Results are expressed as percentage relative to the WT condition, which was set to 100%. Statistical analyses were performed using the ordinary one-way ANO V A, with Tukey’s multiple comparisons tests (*** P < .0 0 05; **** P < .0 0 01).

Article Snippet: For each time oint measured, data of this second control (binding of Blitz uffer on biosensors loaded with biotinylated Gag WT pepide) were subtracted to every condition and were plotted on urves, using Prism 9 software. ell culture uman fibrosarcoma cell line HT1080 (ATCC CCL-121) and roteoglycan-deficient packaging cell line 293T-25A [ 26 ] were aintained in Dulbecco’s modified Eagle medium (DMEM; isher), supplemented with 50 μg / ml of gentamicin (Fisher cientific) and 10% fetal calf serum (FCS) (Eurobio Scientific). iral vector production FV particles were produced using a four-component system, ased on a protocol described in [ 26 ].

Techniques: Infection, Flow Cytometry, Virus