bj5ta cells  (ATCC)

Journal: bioRxiv

Article Title: Mechanisms by which barrier-to-autointegration factor regulates dynamics of nucleocytoplasmic leakage and membrane repair following nuclear envelope rupture

doi: 10.1101/2023.12.21.572811

Figure Lengend Snippet: Development of a nuclear transport-independent NE rupture reporter. A) Illustration of the transport-dependent BAF-IRES-GFP-NLS rupture reporter system for co-expression with BAF variants. P LTR , long terminal repeat promoter. B) IF images of BJ5ta cells expressing either empty vector- or WT-BAF-IRES-GFP-NLS probed with BAF antibody after treatment with siControl or siBAF. C) Representative western blots of cell lysates from cells in A probed with a BAF antibody. MW; molecular weight. D) Illustration of workflow for testing GFP-NLS diffusion and repair of NE ruptures in cells expressing BAF variants. E) Graphical representation of measuring early rupture diffusion and repair (blue line) or inhibited repair (red line) in cells from D. F) Illustration of the transport-independent BAF-IRES-Hsp90-GFP rupture reporter system for co-expression of BAF variants, along with IF images (F), and western blots (H). I) Illustration of workflow for testing Hsp90-GFP rupture diffusion and repair, as well as graphical representation of Hsp90-GFP rupture curves in cells expressing BAF variants. Scale bar; 10 µm.

Article Snippet: The culture media was collected and filtered through a 0.45μm filter and added to BJ5ta cells (target cells), along with polybrene (2.5 μg/mL; Santa Cruz Biotechnology), and incubated at 37°C for 24 hr.

Techniques: Expressing, Plasmid Preparation, Western Blot, Molecular Weight, Diffusion-based Assay

Journal: bioRxiv

Article Title: Mechanisms by which barrier-to-autointegration factor regulates dynamics of nucleocytoplasmic leakage and membrane repair following nuclear envelope rupture

doi: 10.1101/2023.12.21.572811

Figure Lengend Snippet: Endogenous BAF is sufficient to respond to NE ruptures with gradual depletion leading to a progressive loss of that response. A) Representative images of BJ5ta cells expressing either Empty- or WT-BAF-IRES-Hsp90-GFP and cGAS-mCherry, treated with either siControl or siBAF for 96 hr and followed through NE rupture to observe the extent of leakage. The enrichment of cGAS (yellow arrows) along the nuclear periphery (outlined in red for clarity) signal the location and time of NE rupture. Scale bar, 10 µm. B) Quantification of the nuclear- to-cytoplasmic increase in Hsp90-GFP following laser-induced NE rupture from cells. The graph represents mean values ± SEM of Empty siControl (n= 18), Empty siBAF (n=18), WT-BAF siControl (n=5) and WT-BAF siBAF (n=11) cells from at least three independent experiments. C) Rates of Hsp90-GFP leakage into the nucleus during the first 30 seconds following NE rupture. D) Repair check, measured as a percent increase in nuclear-to-cytoplasmic Hsp90-GFP diffusion into the nucleus following nuclear photobleaching (performed 10 minutes after NE rupture) to check functional repair. The graph represents mean values ± SEM of Empty siControl (n = 4), WT-BAF siControl (n = 5), WT-BAF siBAF (n = 8) and Empty siBAF (n = 10). E) Representative Western blot of cell lysates from cells treated with 0, 0.1, 1 and 10 pmol of BAF siRNAs for 72 hr, with tubulin used as a loading control. F) Quantification of the nuclear-to cytoplasmic increase in Hsp90-GFP following laser-induced NE rupture from cells treated with 0, 0.1, 1 and 10 pmol of BAF siRNAs (n= 12, 14, 9, and 11 cells, respectively) from at least two independent experiments. G) Initial rates of Hsp90-GFP leakage into the nucleus during the first 30 seconds following NE rupture from E. H) Repair check of cells from F, 10 min post-rupture. Statistical significance: *, P<0.05; **<0.005; ***, P<0.0005 using a one way ANOVA with Dunnett’s post hoc multiple comparison test.

Article Snippet: The culture media was collected and filtered through a 0.45μm filter and added to BJ5ta cells (target cells), along with polybrene (2.5 μg/mL; Santa Cruz Biotechnology), and incubated at 37°C for 24 hr.

Techniques: Expressing, Diffusion-based Assay, Functional Assay, Western Blot, Control, Comparison

Journal: bioRxiv

Article Title: Mechanisms by which barrier-to-autointegration factor regulates dynamics of nucleocytoplasmic leakage and membrane repair following nuclear envelope rupture

doi: 10.1101/2023.12.21.572811

Figure Lengend Snippet: BAF controls rupture diffusion in a size-dependent manner. A) Measurements of the width of the NE rupture gap in BJ5ta cells expressing GFP-Sec61β. Number of cells analyzed: siControl, n=15; siBAF, n=14. Error bars indicate ± SEM from triplicate experiments. B) Representative images of BJ5ta cells expressing either Hsp90-GFP or α-Tubulin-GFP after laser-induced NE rupture. Scale bar; 10 µm. C) Quantification of the nuclear-to-cytoplasmic ratio of cells expressing either Hsp90-GFP or α-tubulin-GFP treated with either siControl (n = 29 and 14, respectively) or siBAF (n = 21 and 16, respectively) from triplicate experiments. Error bars indicate ± SEM. D) Initial rate of increase into the nucleus following NE rupture for cells in B. E) Representative images of BJ5ta cells expressing Hsp90-GFP or α-tubulin-GFP during FLIP. Green circle indicates area of photobleaching in the cytoplasm. Red circle indicates area of measurement. F) FLIP measurements showing relative mobility of Hsp90-GFP (n = 16 cells) or α-tubulin-GFP (n = 16 cells). The slope of each line x was used to factor relative mobility between Hsp90-GFP and α-tubulin-GFP. G) Mobility of Hsp90-GFP and α-tubulin-GFP (measured as the best line-of-fit slope for each individual cell). H) Initial rate of increase into the nucleus following NE rupture for cells in D normalized by mobility. Error bars indicate ± SEM. Statistical significance: *, P<0.05; **<0.005; ***, P<0.0005 using an unpaired student t-test.

Article Snippet: The culture media was collected and filtered through a 0.45μm filter and added to BJ5ta cells (target cells), along with polybrene (2.5 μg/mL; Santa Cruz Biotechnology), and incubated at 37°C for 24 hr.

Techniques: Diffusion-based Assay, Expressing