u2os  (CLS Cell Lines Service GmbH)

Journal: Viruses

Article Title: Super-Resolution Imaging of Nuclear Pore Responses to Mechanical Stress and Energy Depletion

doi: 10.3390/v18020167

Figure Lengend Snippet: ATP depletion of U2OS NUP96-SNAP cells. Cells were incubated in complete DMEM with 10% FBS ((+)Gluc), in HEPES-based imaging buffer without glucose ((−)Gluc), or in ATP depletion buffer: 10 mM of sodium azide with 6 mM of 2-deoxy-D-glucose (NaN 3 ) or 10 µM of Antimycin A with 20 mM of 2-deoxy-D-glucose (Ant-A) at different times. The cellular ATP concentration (in µM) was measured in triplicate samples using a kit and plotted as mean ± SD. Statistical analysis was done with two-way ANOVA. (See for an independent replicate).

Article Snippet: U2OS NUP96-SNAP clone #33, in which SNAP was knocked-in at the C-terminus of NUP96 using CRISPR/Cas9 technology, was obtained from Cell Lines Services (CLS GmbH #300444, Eppelheim, Germany; see Thevathasan et al., 2019 [ ]).

Techniques: Incubation, Imaging, Concentration Assay

Journal: Viruses

Article Title: Super-Resolution Imaging of Nuclear Pore Responses to Mechanical Stress and Energy Depletion

doi: 10.3390/v18020167

Figure Lengend Snippet: STORM imaging of NPCs on ATP depleted samples. ( A ) EM density (grey) images of the NPC with NUP96 highlighted (magenta) viewed from the side, top, and tilted. Structure was taken from the Protein Data Bank (PDB): 7TBJ. ( B ) Top : Confocal images of U2OS NUP96-SNAP cells incubated in complete medium ((+)Gluc) and ATP-depletion media, NaN 3 or Ant-A, for 30 min at 37 °C. Cells were then fixed with PFA, permeabilized, and NUP96-SNAP was stained with the SNAP-AF647 dye. Shown are representative images of confocal slices at the bottom of the nuclear envelope. Middle : STORM images (AF647 localizations) of NPCs at the bottom of the nuclear envelope. Scale bar: 0.5 µm. Inset : zoomed in of one NPC (scale bar 50 nm). Bottom : Images of averaged NPCs from STORM images. NPCs were selected manually, and averaging was performed using a MATLAB script. ( C ) Distribution of the number of SNAP-AF647 dye localizations per NPC obtained by STORM (panel ( B )). Table shows mean localizations per NPC ± SD and the number of NPCs analyzed. ( D ) Mean number of localizations for each biological replicate. (See for independent replicates). Statistical analysis was done with Brown-Forsythe and Welch ANOVA. ( E ) Mean nuclear volumes (µm 3 ) for (+)Gluc, NaN 3 , and Ant-A treated samples. Shown are mean ± SD from three biological replicates. Brown-Forsythe and Welch ANOVA were used for statistical analysis.

Article Snippet: U2OS NUP96-SNAP clone #33, in which SNAP was knocked-in at the C-terminus of NUP96 using CRISPR/Cas9 technology, was obtained from Cell Lines Services (CLS GmbH #300444, Eppelheim, Germany; see Thevathasan et al., 2019 [ ]).

Techniques: Imaging, Incubation, Staining

Journal: Viruses

Article Title: Super-Resolution Imaging of Nuclear Pore Responses to Mechanical Stress and Energy Depletion

doi: 10.3390/v18020167

Figure Lengend Snippet: Osmotic swelling of nuclear membrane. ( A , B ) U2OS NUP96-SNAP cells were transfected with the membrane tension sensor, cPLA2-GFP (green). Twenty-four hours after transfection, cells were permeabilized for 5 min with 25 µg/mL digitonin in base medium in the presence of 5% Polyvinylpyrrolidone (PVP360) to prevent cell swelling. Scale bar 5 μm. ( A , B ). Permeabilization medium was removed, and cells were further incubated with 5% PVP360 for 5 min ( A ) or in 0% PVP360 for 15 min ( B ). Pre-permeabilized samples were fixed and stained with SNAP-AF647 dye (magenta). Left : Representative images of a middle section of the nuclear envelope (NE). Right : Images of individual cPLA2 and SNAP channels. Below each image are line histograms of the normalized intensities of cPLA2 (green) and SNAP (NE, magenta), corresponding to lines drawn across the nuclei on the confocal images. ( C ) Distributions of nuclear volumes (in µm 3 ) of stretched (0% PVP360) and non-stretched (5% PVP360) nuclei. Shown are means ± SD, n = Number of nuclei. Statistical analysis was done using Welch’s t -test.

Article Snippet: U2OS NUP96-SNAP clone #33, in which SNAP was knocked-in at the C-terminus of NUP96 using CRISPR/Cas9 technology, was obtained from Cell Lines Services (CLS GmbH #300444, Eppelheim, Germany; see Thevathasan et al., 2019 [ ]).

Techniques: Membrane, Transfection, Incubation, Staining

Journal: Viruses

Article Title: Super-Resolution Imaging of Nuclear Pore Responses to Mechanical Stress and Energy Depletion

doi: 10.3390/v18020167

Figure Lengend Snippet: Stretching the nuclear envelope does not change the NPC radius. ( A ) STORM images of U2OS NUP96-SNAP cells either fixed before permeabilization (Pre-fixed) or pre-permeabilized with 25 µg/mL of digitonin and incubated under swelling (0% PVP360) or not swelling (5% PVP360) conditions, as in . Cells fixed prior to permeabilization were used as a control. NUP96-SNAP was stained with the SNAP-AF647 dye. Shown are representative images at the bottom of the nuclear envelope. Scale bar: 0.5 µm. ( B – D ) Same as in . Top : Scatter plots showing the radial distributions of single-molecule localization densities of superimposed NPCs selected from ( A ), aligned at their center of mass. Shown right is the pseudo color scale for the localization density. Bottom : SML radial distance histograms were fitted with a Gaussian function (red). The µ and σ were calculated from the fitted Gaussian distribution ( Top and Bottom , green lines). ( E ) The table shows µ ± σ calculated on ( B – D ), number of NPCs analyzed for each condition and p -values. ( F ) Mean SML radial distances from the center for three biological replicates. Mean ± SD are plotted. (See for analysis of independent replicates).

Article Snippet: U2OS NUP96-SNAP clone #33, in which SNAP was knocked-in at the C-terminus of NUP96 using CRISPR/Cas9 technology, was obtained from Cell Lines Services (CLS GmbH #300444, Eppelheim, Germany; see Thevathasan et al., 2019 [ ]).

Techniques: Incubation, Control, Staining