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u2os cells  (ATCC)


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    ATCC u2os cells
    land-ExM visualizes the protein and lipid context of cells. (A) Workflow of land-ExM. (B) Schematic of NHS-biotin-MA linker. (C) Schematic of mCLING. (D) land-ExM image of <t>U2OS</t> cells incubated with NHS-biotin-MA linker. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4. (E) ExM image of U2OS cells incubated with NHS-MA linker and stained with Alexa Fluor 488 NHS ester dye. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.2. (F) ExM image of U2OS cells incubated with GMA linker and stained with SYPRO Orange. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.2. (G) Bar chart comparing signal-to-noise ratios of protein context images obtained with different ExM methods shown in D–F. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 10 cells. (H–J) Different views of land-ExM images of a breast cancer cell, UCI082014, stained with mCLING for lipid content. The orange dashed lines in H show where the orthogonal views (I and J) align. Scale bar: 5 µm (H), 2 µm (I and J) in pre-expansion unit. Linear expansion factor: 3.8. (K) Magnified images of H. (L) Magnified images of I. The orange dashed line in K shows where the orthogonal view (L) aligns. Scale bar: 0.5 µm in pre-expansion unit. Linear expansion factor: 3.8. All images were taken with an Airyscan microscope. Images D–F were adjusted to the same contrast. Image in D is also shown in .
    U2os Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 8849 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 8849 article reviews
    u2os cells - by Bioz Stars, 2026-02
    99/100 stars

    Images

    1) Product Images from "Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles"

    Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.202502035

    land-ExM visualizes the protein and lipid context of cells. (A) Workflow of land-ExM. (B) Schematic of NHS-biotin-MA linker. (C) Schematic of mCLING. (D) land-ExM image of U2OS cells incubated with NHS-biotin-MA linker. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4. (E) ExM image of U2OS cells incubated with NHS-MA linker and stained with Alexa Fluor 488 NHS ester dye. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.2. (F) ExM image of U2OS cells incubated with GMA linker and stained with SYPRO Orange. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.2. (G) Bar chart comparing signal-to-noise ratios of protein context images obtained with different ExM methods shown in D–F. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 10 cells. (H–J) Different views of land-ExM images of a breast cancer cell, UCI082014, stained with mCLING for lipid content. The orange dashed lines in H show where the orthogonal views (I and J) align. Scale bar: 5 µm (H), 2 µm (I and J) in pre-expansion unit. Linear expansion factor: 3.8. (K) Magnified images of H. (L) Magnified images of I. The orange dashed line in K shows where the orthogonal view (L) aligns. Scale bar: 0.5 µm in pre-expansion unit. Linear expansion factor: 3.8. All images were taken with an Airyscan microscope. Images D–F were adjusted to the same contrast. Image in D is also shown in .
    Figure Legend Snippet: land-ExM visualizes the protein and lipid context of cells. (A) Workflow of land-ExM. (B) Schematic of NHS-biotin-MA linker. (C) Schematic of mCLING. (D) land-ExM image of U2OS cells incubated with NHS-biotin-MA linker. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4. (E) ExM image of U2OS cells incubated with NHS-MA linker and stained with Alexa Fluor 488 NHS ester dye. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.2. (F) ExM image of U2OS cells incubated with GMA linker and stained with SYPRO Orange. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.2. (G) Bar chart comparing signal-to-noise ratios of protein context images obtained with different ExM methods shown in D–F. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 10 cells. (H–J) Different views of land-ExM images of a breast cancer cell, UCI082014, stained with mCLING for lipid content. The orange dashed lines in H show where the orthogonal views (I and J) align. Scale bar: 5 µm (H), 2 µm (I and J) in pre-expansion unit. Linear expansion factor: 3.8. (K) Magnified images of H. (L) Magnified images of I. The orange dashed line in K shows where the orthogonal view (L) aligns. Scale bar: 0.5 µm in pre-expansion unit. Linear expansion factor: 3.8. All images were taken with an Airyscan microscope. Images D–F were adjusted to the same contrast. Image in D is also shown in .

    Techniques Used: Incubation, Staining, Microscopy

    mCLING optimization for lipid staining of cells. (A–D) Airyscan images of U2OS cells stained with different batches of mCLING at different dilution factors. Scale bars: 20 µm. Red arrowheads indicate lipid structures in the cytoplasm. All images were taken with an Airyscan microscope.
    Figure Legend Snippet: mCLING optimization for lipid staining of cells. (A–D) Airyscan images of U2OS cells stained with different batches of mCLING at different dilution factors. Scale bars: 20 µm. Red arrowheads indicate lipid structures in the cytoplasm. All images were taken with an Airyscan microscope.

    Techniques Used: Staining, Microscopy

    Alternative land-ExM workflow to avoid cross talk between NHS-biotin-MA and mCLING. (A) Alternative workflow of land-ExM. (B) i and ii: land-ExM images of U2OS cells stained first with NHS-biotin-MA and then mCLING. iii to v: Magnified images of boxes in i and ii. vi: Normalized intensity profile along the orange line in v. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0 (i and ii). 0.5 µm in pre-expansion unit. Linear expansion factor: 4.0 (iii to v). (C) i and ii: land-ExM images of U2OS cells stained first with mCLING and then NHS-biotin-MA. iii to v: Magnified images of orange boxes in i and ii. vi: Normalized intensity profile along the orange line in v. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4 (i and ii). 0.5 µm in pre-expansion unit. Linear expansion factor: 4.0 (iii to v). All images were taken with an Airyscan microscope.
    Figure Legend Snippet: Alternative land-ExM workflow to avoid cross talk between NHS-biotin-MA and mCLING. (A) Alternative workflow of land-ExM. (B) i and ii: land-ExM images of U2OS cells stained first with NHS-biotin-MA and then mCLING. iii to v: Magnified images of boxes in i and ii. vi: Normalized intensity profile along the orange line in v. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0 (i and ii). 0.5 µm in pre-expansion unit. Linear expansion factor: 4.0 (iii to v). (C) i and ii: land-ExM images of U2OS cells stained first with mCLING and then NHS-biotin-MA. iii to v: Magnified images of orange boxes in i and ii. vi: Normalized intensity profile along the orange line in v. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4 (i and ii). 0.5 µm in pre-expansion unit. Linear expansion factor: 4.0 (iii to v). All images were taken with an Airyscan microscope.

    Techniques Used: Staining, Microscopy

    land-ExM using proteinase K digestion. (A) Workflow of land-ExM using proteinase K digestion to homogenize cells instead of heat denaturation. (B) land-ExM protein image of U2OS cells with proteinase K digestion (proK). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (C) land-ExM protein image of U2OS cells with heat denaturation (heat). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (D) land-ExM lipid image of U2OS cell with proteinase K digestion (proK). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (E) land-ExM lipid image of U2OS cells with heat denaturation (heat). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. All images were taken with an Airyscan microscope. Image in C is also shown in .
    Figure Legend Snippet: land-ExM using proteinase K digestion. (A) Workflow of land-ExM using proteinase K digestion to homogenize cells instead of heat denaturation. (B) land-ExM protein image of U2OS cells with proteinase K digestion (proK). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (C) land-ExM protein image of U2OS cells with heat denaturation (heat). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (D) land-ExM lipid image of U2OS cell with proteinase K digestion (proK). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (E) land-ExM lipid image of U2OS cells with heat denaturation (heat). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. All images were taken with an Airyscan microscope. Image in C is also shown in .

    Techniques Used: Microscopy

    land-ExM labeling and anchoring strategies improve the signal of TREx and pan-ExM. (A) Workflow of land-pan-ExM, which only replaces the labeling strategy of pan-ExM with the labeling strategy of land-ExM. (B) land-TREx protein channel of U2OS cells, where proteins were labeled and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7. (C) TREx protein channel of U2OS cells, where proteins were anchored with acryloyl-X SE and stained with Alexa Fluor 488 NHS ester. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7. (D) Bar chart comparing the signal-to-noise ratio of the protein channel in land-TREx and TREx. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (E) land-TREx lipid channel of U2OS cells, where lipids were labeled by mCLING and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7.0. (F) TREx lipid channel of U2OS cells, where lipids were anchored with acryloyl-X SE and stained with mCLING. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7.0. (G) Bar chart comparing the signal-to-noise ratio of the lipid channel of land-TREx and TREx. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (H) land-pan-ExM protein channel of U2OS cells, where proteins were labeled and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (I) Pan-ExM protein channel of U2OS cells labeled with Alexa Fluor 488 NHS ester. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (J) Bar chart comparing the signal-to-noise ratio of the protein channel in land-pan-ExM and pan-ExM. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (K) land-pan-ExM lipid channel of U2OS cells, where lipids were stained following the workflow (A). Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (L) Pan-ExM lipid channel of U2OS cells labeled with mCLING. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (M) Bar chart comparing the signal-to-noise ratio of the lipid (mCLING) channel in land-pan-ExM and pan-ExM. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. All images were taken with an Airyscan microscope.
    Figure Legend Snippet: land-ExM labeling and anchoring strategies improve the signal of TREx and pan-ExM. (A) Workflow of land-pan-ExM, which only replaces the labeling strategy of pan-ExM with the labeling strategy of land-ExM. (B) land-TREx protein channel of U2OS cells, where proteins were labeled and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7. (C) TREx protein channel of U2OS cells, where proteins were anchored with acryloyl-X SE and stained with Alexa Fluor 488 NHS ester. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7. (D) Bar chart comparing the signal-to-noise ratio of the protein channel in land-TREx and TREx. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (E) land-TREx lipid channel of U2OS cells, where lipids were labeled by mCLING and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7.0. (F) TREx lipid channel of U2OS cells, where lipids were anchored with acryloyl-X SE and stained with mCLING. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7.0. (G) Bar chart comparing the signal-to-noise ratio of the lipid channel of land-TREx and TREx. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (H) land-pan-ExM protein channel of U2OS cells, where proteins were labeled and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (I) Pan-ExM protein channel of U2OS cells labeled with Alexa Fluor 488 NHS ester. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (J) Bar chart comparing the signal-to-noise ratio of the protein channel in land-pan-ExM and pan-ExM. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (K) land-pan-ExM lipid channel of U2OS cells, where lipids were stained following the workflow (A). Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (L) Pan-ExM lipid channel of U2OS cells labeled with mCLING. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (M) Bar chart comparing the signal-to-noise ratio of the lipid (mCLING) channel in land-pan-ExM and pan-ExM. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. All images were taken with an Airyscan microscope.

    Techniques Used: Labeling, Staining, Microscopy

    land-ExM visualizes phase-separated and membrane organelles. (A–G) land-ExM protein images of membraneless phase separation structures. The proteins were labeled with NHS-biotin-MS and after gelation stained with streptavidin-Alexa Fluor 488. (A) land-ExM protein image of nucleoli in a U2OS cell. Red arrowheads indicate the fibrillar center (FC) or dense fibrillar component (DFC) of the nucleolus. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (B) land-ExM protein image of nuclear bodies of breast cancer cell, UCI082014. Red arrowheads indicate the nuclear bodies. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.2. (C) land-ExM protein image of SGs of a U2OS cell treated with NaAsO 2 for 20 min. The red arrowhead indicates a SG. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (D) land-ExM protein image of chromatin of a breast cancer cell. Scale bar: 500 nm in pre-expansion unit. Linear expansion factor: 4.2. (E) land-ExM protein image of NPCs of a breast cancer cell. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.2. (F and G) land-ExM protein images of mitochondria and cytoskeleton of a U2OS cell. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (H–P) land-ExM lipid images of membrane structures. The lipids were labeled with mCLING-Atto647N. (H) land-ExM lipid image of breast cancer cell. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0. (I–M) magnified images of H showing different membrane structures: lipid vesicles (I), mitochondria (J), filopodia (K), nuclear invagination (L), and Golgi apparatus (M). Scale bar: 1 µm (I–M) in pre-expansion unit. (N) 3D land-ExM lipid image of a breast cancer cell after maximum intensity projection, showing the cell membrane. Color-coded by the z-dimension slices from bottom to top. Color bar: purple to white: 0–6 µm in pre-expansion unit. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0. (O and P) magnified images of N showing detailed structures of the cell membrane. Scale bar: 1 µm in pre-expansion unit. All images were taken with an Airyscan microscope.
    Figure Legend Snippet: land-ExM visualizes phase-separated and membrane organelles. (A–G) land-ExM protein images of membraneless phase separation structures. The proteins were labeled with NHS-biotin-MS and after gelation stained with streptavidin-Alexa Fluor 488. (A) land-ExM protein image of nucleoli in a U2OS cell. Red arrowheads indicate the fibrillar center (FC) or dense fibrillar component (DFC) of the nucleolus. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (B) land-ExM protein image of nuclear bodies of breast cancer cell, UCI082014. Red arrowheads indicate the nuclear bodies. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.2. (C) land-ExM protein image of SGs of a U2OS cell treated with NaAsO 2 for 20 min. The red arrowhead indicates a SG. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (D) land-ExM protein image of chromatin of a breast cancer cell. Scale bar: 500 nm in pre-expansion unit. Linear expansion factor: 4.2. (E) land-ExM protein image of NPCs of a breast cancer cell. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.2. (F and G) land-ExM protein images of mitochondria and cytoskeleton of a U2OS cell. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (H–P) land-ExM lipid images of membrane structures. The lipids were labeled with mCLING-Atto647N. (H) land-ExM lipid image of breast cancer cell. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0. (I–M) magnified images of H showing different membrane structures: lipid vesicles (I), mitochondria (J), filopodia (K), nuclear invagination (L), and Golgi apparatus (M). Scale bar: 1 µm (I–M) in pre-expansion unit. (N) 3D land-ExM lipid image of a breast cancer cell after maximum intensity projection, showing the cell membrane. Color-coded by the z-dimension slices from bottom to top. Color bar: purple to white: 0–6 µm in pre-expansion unit. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0. (O and P) magnified images of N showing detailed structures of the cell membrane. Scale bar: 1 µm in pre-expansion unit. All images were taken with an Airyscan microscope.

    Techniques Used: Membrane, Labeling, Staining, Microscopy

    land-ExM coupled with immunostaining LR-ExM for lipid vesicle identification. (A–C) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Lamp2 antibodies (yellow). The anti-Lamp2 antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–G) Magnified images of A–C showing details of lysosomes. Scale bar: 500 nm in pre-expansion unit. (H) Intensity profile along the gray line across the lysosome in image (D). (I–K) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-clathrin antibodies (yellow). The anti-clathrin antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (L–O) Magnified images of I–K showing details of clathrin-coated pits. Scale bar: 500 nm in pre-expansion unit. (P) Intensity profile along the gray line across the clathrin-coated pit in image (L). All images were taken with an Airyscan microscope.
    Figure Legend Snippet: land-ExM coupled with immunostaining LR-ExM for lipid vesicle identification. (A–C) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Lamp2 antibodies (yellow). The anti-Lamp2 antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–G) Magnified images of A–C showing details of lysosomes. Scale bar: 500 nm in pre-expansion unit. (H) Intensity profile along the gray line across the lysosome in image (D). (I–K) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-clathrin antibodies (yellow). The anti-clathrin antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (L–O) Magnified images of I–K showing details of clathrin-coated pits. Scale bar: 500 nm in pre-expansion unit. (P) Intensity profile along the gray line across the clathrin-coated pit in image (L). All images were taken with an Airyscan microscope.

    Techniques Used: Immunostaining, Labeling, Microscopy

    land-ExM coupled with immunostaining LR-ExM for membrane-bound organelle visualization. (A–C) land-ExM total lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Tom20 antibodies (yellow). The anti-Tom20 antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–G) Magnified images of A–C showing details of mitochondria. Scale bar: 1 µm in pre-expansion unit. (H) Intensity profile along the cyan line across the mitochondria in image (D). (I–K) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Sec61b antibodies (yellow). The anti-Sec61b antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (L–O) Magnified in images of I–K showing details of ER. Scale bar: 1 µm in pre-expansion unit. (P) Intensity profile along the cyan line across the ER in image (L). All images were taken with an Airyscan microscope.
    Figure Legend Snippet: land-ExM coupled with immunostaining LR-ExM for membrane-bound organelle visualization. (A–C) land-ExM total lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Tom20 antibodies (yellow). The anti-Tom20 antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–G) Magnified images of A–C showing details of mitochondria. Scale bar: 1 µm in pre-expansion unit. (H) Intensity profile along the cyan line across the mitochondria in image (D). (I–K) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Sec61b antibodies (yellow). The anti-Sec61b antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (L–O) Magnified in images of I–K showing details of ER. Scale bar: 1 µm in pre-expansion unit. (P) Intensity profile along the cyan line across the ER in image (L). All images were taken with an Airyscan microscope.

    Techniques Used: Immunostaining, Membrane, Labeling, Microscopy

    land-ExM reveals SGs at different locations of cells. (A–C) land-ExM images of U2OS cells untreated or treated with NaAsO2 for 20 or 60 min, then immunostained with anti-G3BP1 antibody. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–F) land-ExM images of U2OS cells stained with mCLING (magenta) and NHS ester dye (cyan) and immunostained with anti-G3BP1 (yellow) and anti-Sec61b (white) antibodies. Cells were untreated or treated with NaAsO2 for 20 min or 60 min. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (G) Magnified images of E showing SGs formed adjacent to ER (orange arrowheads). Scale bar: 1 µm in pre-expansion unit. (H) Analysis of the number of nuclear tunnels per cell with or without 60 min NaAsO2 treatment. Each bar represents the mean ± standard error of more than 18 cells. The ns indicates P > 0.05 by Welch’s t test. (I) Analysis of the diameter of nuclear tunnels in cells with or without 60 min NaAsO2 treatment. Each bar represents the mean ± standard error of more than 20 cells. ns indicates P > 0.05 by Welch’s t test. All images were taken with an Airyscan microscope. The cell shown in F is also shown in .
    Figure Legend Snippet: land-ExM reveals SGs at different locations of cells. (A–C) land-ExM images of U2OS cells untreated or treated with NaAsO2 for 20 or 60 min, then immunostained with anti-G3BP1 antibody. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–F) land-ExM images of U2OS cells stained with mCLING (magenta) and NHS ester dye (cyan) and immunostained with anti-G3BP1 (yellow) and anti-Sec61b (white) antibodies. Cells were untreated or treated with NaAsO2 for 20 min or 60 min. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (G) Magnified images of E showing SGs formed adjacent to ER (orange arrowheads). Scale bar: 1 µm in pre-expansion unit. (H) Analysis of the number of nuclear tunnels per cell with or without 60 min NaAsO2 treatment. Each bar represents the mean ± standard error of more than 18 cells. The ns indicates P > 0.05 by Welch’s t test. (I) Analysis of the diameter of nuclear tunnels in cells with or without 60 min NaAsO2 treatment. Each bar represents the mean ± standard error of more than 20 cells. ns indicates P > 0.05 by Welch’s t test. All images were taken with an Airyscan microscope. The cell shown in F is also shown in .

    Techniques Used: Staining, Microscopy

    The nuclear tunnel forms a triple-organellar contact site that includes the SG, the nucleolus, and itself. (A) land-ExM protein (gray) image of U2OS cells immunostained with anti-G3BP1 (red) antibody. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (B–D) Different views of SG in the white dashed box of A. Scale bar: 1 µm in pre-expansion unit. (E) 3D rendering of SG in the white dashed box of A. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (F) land-ExM protein (gray) and lipid (blue) image of U2OS cells immunostained with anti-G3BP1 (red) antibody. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (G–I) Different views of SG in the white dashed box 1 of F. Scale bar: 1 µm in pre-expansion unit. (J) 3D rendering of SG in the white dashed box 1 of F. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in the pre-expansion unit. (K) 3D rendering of SGs in the white dashed box 1–4 of F. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in the pre-expansion unit. (L) land-ExM protein (gray) image of U2OS cells immunostained with anti-G3BP1 (red) and anti-Sec61b (yellow) antibodies. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (M–O) Different views of SG in the white dashed box 1 of L. Scale bar: 1 µm in pre-expansion unit. (P) 3D rendering of SG in the white dashed box 1 of L. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (Q) 3D rendering of SGs in the white dashed box 1–4 of L. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (R) Pie chart of nuclear tunnels with or without SGs. Total tunnels analyzed: 114. (S) Pie chart of SG-filled nuclear tunnels that contact nucleoli versus those that do not. Total tunnel analyzed: 83. All images were taken with an Airyscan microscope. The cell shown in A, F, and L is also shown in .
    Figure Legend Snippet: The nuclear tunnel forms a triple-organellar contact site that includes the SG, the nucleolus, and itself. (A) land-ExM protein (gray) image of U2OS cells immunostained with anti-G3BP1 (red) antibody. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (B–D) Different views of SG in the white dashed box of A. Scale bar: 1 µm in pre-expansion unit. (E) 3D rendering of SG in the white dashed box of A. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (F) land-ExM protein (gray) and lipid (blue) image of U2OS cells immunostained with anti-G3BP1 (red) antibody. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (G–I) Different views of SG in the white dashed box 1 of F. Scale bar: 1 µm in pre-expansion unit. (J) 3D rendering of SG in the white dashed box 1 of F. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in the pre-expansion unit. (K) 3D rendering of SGs in the white dashed box 1–4 of F. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in the pre-expansion unit. (L) land-ExM protein (gray) image of U2OS cells immunostained with anti-G3BP1 (red) and anti-Sec61b (yellow) antibodies. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (M–O) Different views of SG in the white dashed box 1 of L. Scale bar: 1 µm in pre-expansion unit. (P) 3D rendering of SG in the white dashed box 1 of L. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (Q) 3D rendering of SGs in the white dashed box 1–4 of L. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (R) Pie chart of nuclear tunnels with or without SGs. Total tunnels analyzed: 114. (S) Pie chart of SG-filled nuclear tunnels that contact nucleoli versus those that do not. Total tunnel analyzed: 83. All images were taken with an Airyscan microscope. The cell shown in A, F, and L is also shown in .

    Techniques Used: Microscopy



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    land-ExM visualizes the protein and lipid context of cells. (A) Workflow of land-ExM. (B) Schematic of NHS-biotin-MA linker. (C) Schematic of mCLING. (D) land-ExM image of U2OS cells incubated with NHS-biotin-MA linker. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4. (E) ExM image of U2OS cells incubated with NHS-MA linker and stained with Alexa Fluor 488 NHS ester dye. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.2. (F) ExM image of U2OS cells incubated with GMA linker and stained with SYPRO Orange. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.2. (G) Bar chart comparing signal-to-noise ratios of protein context images obtained with different ExM methods shown in D–F. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 10 cells. (H–J) Different views of land-ExM images of a breast cancer cell, UCI082014, stained with mCLING for lipid content. The orange dashed lines in H show where the orthogonal views (I and J) align. Scale bar: 5 µm (H), 2 µm (I and J) in pre-expansion unit. Linear expansion factor: 3.8. (K) Magnified images of H. (L) Magnified images of I. The orange dashed line in K shows where the orthogonal view (L) aligns. Scale bar: 0.5 µm in pre-expansion unit. Linear expansion factor: 3.8. All images were taken with an Airyscan microscope. Images D–F were adjusted to the same contrast. Image in D is also shown in .

    Journal: The Journal of Cell Biology

    Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

    doi: 10.1083/jcb.202502035

    Figure Lengend Snippet: land-ExM visualizes the protein and lipid context of cells. (A) Workflow of land-ExM. (B) Schematic of NHS-biotin-MA linker. (C) Schematic of mCLING. (D) land-ExM image of U2OS cells incubated with NHS-biotin-MA linker. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4. (E) ExM image of U2OS cells incubated with NHS-MA linker and stained with Alexa Fluor 488 NHS ester dye. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.2. (F) ExM image of U2OS cells incubated with GMA linker and stained with SYPRO Orange. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.2. (G) Bar chart comparing signal-to-noise ratios of protein context images obtained with different ExM methods shown in D–F. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 10 cells. (H–J) Different views of land-ExM images of a breast cancer cell, UCI082014, stained with mCLING for lipid content. The orange dashed lines in H show where the orthogonal views (I and J) align. Scale bar: 5 µm (H), 2 µm (I and J) in pre-expansion unit. Linear expansion factor: 3.8. (K) Magnified images of H. (L) Magnified images of I. The orange dashed line in K shows where the orthogonal view (L) aligns. Scale bar: 0.5 µm in pre-expansion unit. Linear expansion factor: 3.8. All images were taken with an Airyscan microscope. Images D–F were adjusted to the same contrast. Image in D is also shown in .

    Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

    Techniques: Incubation, Staining, Microscopy

    mCLING optimization for lipid staining of cells. (A–D) Airyscan images of U2OS cells stained with different batches of mCLING at different dilution factors. Scale bars: 20 µm. Red arrowheads indicate lipid structures in the cytoplasm. All images were taken with an Airyscan microscope.

    Journal: The Journal of Cell Biology

    Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

    doi: 10.1083/jcb.202502035

    Figure Lengend Snippet: mCLING optimization for lipid staining of cells. (A–D) Airyscan images of U2OS cells stained with different batches of mCLING at different dilution factors. Scale bars: 20 µm. Red arrowheads indicate lipid structures in the cytoplasm. All images were taken with an Airyscan microscope.

    Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

    Techniques: Staining, Microscopy

    Alternative land-ExM workflow to avoid cross talk between NHS-biotin-MA and mCLING. (A) Alternative workflow of land-ExM. (B) i and ii: land-ExM images of U2OS cells stained first with NHS-biotin-MA and then mCLING. iii to v: Magnified images of boxes in i and ii. vi: Normalized intensity profile along the orange line in v. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0 (i and ii). 0.5 µm in pre-expansion unit. Linear expansion factor: 4.0 (iii to v). (C) i and ii: land-ExM images of U2OS cells stained first with mCLING and then NHS-biotin-MA. iii to v: Magnified images of orange boxes in i and ii. vi: Normalized intensity profile along the orange line in v. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4 (i and ii). 0.5 µm in pre-expansion unit. Linear expansion factor: 4.0 (iii to v). All images were taken with an Airyscan microscope.

    Journal: The Journal of Cell Biology

    Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

    doi: 10.1083/jcb.202502035

    Figure Lengend Snippet: Alternative land-ExM workflow to avoid cross talk between NHS-biotin-MA and mCLING. (A) Alternative workflow of land-ExM. (B) i and ii: land-ExM images of U2OS cells stained first with NHS-biotin-MA and then mCLING. iii to v: Magnified images of boxes in i and ii. vi: Normalized intensity profile along the orange line in v. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0 (i and ii). 0.5 µm in pre-expansion unit. Linear expansion factor: 4.0 (iii to v). (C) i and ii: land-ExM images of U2OS cells stained first with mCLING and then NHS-biotin-MA. iii to v: Magnified images of orange boxes in i and ii. vi: Normalized intensity profile along the orange line in v. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4 (i and ii). 0.5 µm in pre-expansion unit. Linear expansion factor: 4.0 (iii to v). All images were taken with an Airyscan microscope.

    Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

    Techniques: Staining, Microscopy

    land-ExM using proteinase K digestion. (A) Workflow of land-ExM using proteinase K digestion to homogenize cells instead of heat denaturation. (B) land-ExM protein image of U2OS cells with proteinase K digestion (proK). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (C) land-ExM protein image of U2OS cells with heat denaturation (heat). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (D) land-ExM lipid image of U2OS cell with proteinase K digestion (proK). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (E) land-ExM lipid image of U2OS cells with heat denaturation (heat). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. All images were taken with an Airyscan microscope. Image in C is also shown in .

    Journal: The Journal of Cell Biology

    Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

    doi: 10.1083/jcb.202502035

    Figure Lengend Snippet: land-ExM using proteinase K digestion. (A) Workflow of land-ExM using proteinase K digestion to homogenize cells instead of heat denaturation. (B) land-ExM protein image of U2OS cells with proteinase K digestion (proK). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (C) land-ExM protein image of U2OS cells with heat denaturation (heat). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (D) land-ExM lipid image of U2OS cell with proteinase K digestion (proK). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. (E) land-ExM lipid image of U2OS cells with heat denaturation (heat). Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.0. All images were taken with an Airyscan microscope. Image in C is also shown in .

    Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

    Techniques: Microscopy

    land-ExM labeling and anchoring strategies improve the signal of TREx and pan-ExM. (A) Workflow of land-pan-ExM, which only replaces the labeling strategy of pan-ExM with the labeling strategy of land-ExM. (B) land-TREx protein channel of U2OS cells, where proteins were labeled and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7. (C) TREx protein channel of U2OS cells, where proteins were anchored with acryloyl-X SE and stained with Alexa Fluor 488 NHS ester. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7. (D) Bar chart comparing the signal-to-noise ratio of the protein channel in land-TREx and TREx. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (E) land-TREx lipid channel of U2OS cells, where lipids were labeled by mCLING and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7.0. (F) TREx lipid channel of U2OS cells, where lipids were anchored with acryloyl-X SE and stained with mCLING. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7.0. (G) Bar chart comparing the signal-to-noise ratio of the lipid channel of land-TREx and TREx. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (H) land-pan-ExM protein channel of U2OS cells, where proteins were labeled and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (I) Pan-ExM protein channel of U2OS cells labeled with Alexa Fluor 488 NHS ester. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (J) Bar chart comparing the signal-to-noise ratio of the protein channel in land-pan-ExM and pan-ExM. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (K) land-pan-ExM lipid channel of U2OS cells, where lipids were stained following the workflow (A). Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (L) Pan-ExM lipid channel of U2OS cells labeled with mCLING. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (M) Bar chart comparing the signal-to-noise ratio of the lipid (mCLING) channel in land-pan-ExM and pan-ExM. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. All images were taken with an Airyscan microscope.

    Journal: The Journal of Cell Biology

    Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

    doi: 10.1083/jcb.202502035

    Figure Lengend Snippet: land-ExM labeling and anchoring strategies improve the signal of TREx and pan-ExM. (A) Workflow of land-pan-ExM, which only replaces the labeling strategy of pan-ExM with the labeling strategy of land-ExM. (B) land-TREx protein channel of U2OS cells, where proteins were labeled and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7. (C) TREx protein channel of U2OS cells, where proteins were anchored with acryloyl-X SE and stained with Alexa Fluor 488 NHS ester. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7. (D) Bar chart comparing the signal-to-noise ratio of the protein channel in land-TREx and TREx. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (E) land-TREx lipid channel of U2OS cells, where lipids were labeled by mCLING and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7.0. (F) TREx lipid channel of U2OS cells, where lipids were anchored with acryloyl-X SE and stained with mCLING. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7.0. (G) Bar chart comparing the signal-to-noise ratio of the lipid channel of land-TREx and TREx. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (H) land-pan-ExM protein channel of U2OS cells, where proteins were labeled and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (I) Pan-ExM protein channel of U2OS cells labeled with Alexa Fluor 488 NHS ester. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (J) Bar chart comparing the signal-to-noise ratio of the protein channel in land-pan-ExM and pan-ExM. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (K) land-pan-ExM lipid channel of U2OS cells, where lipids were stained following the workflow (A). Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (L) Pan-ExM lipid channel of U2OS cells labeled with mCLING. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (M) Bar chart comparing the signal-to-noise ratio of the lipid (mCLING) channel in land-pan-ExM and pan-ExM. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. All images were taken with an Airyscan microscope.

    Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

    Techniques: Labeling, Staining, Microscopy

    land-ExM visualizes phase-separated and membrane organelles. (A–G) land-ExM protein images of membraneless phase separation structures. The proteins were labeled with NHS-biotin-MS and after gelation stained with streptavidin-Alexa Fluor 488. (A) land-ExM protein image of nucleoli in a U2OS cell. Red arrowheads indicate the fibrillar center (FC) or dense fibrillar component (DFC) of the nucleolus. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (B) land-ExM protein image of nuclear bodies of breast cancer cell, UCI082014. Red arrowheads indicate the nuclear bodies. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.2. (C) land-ExM protein image of SGs of a U2OS cell treated with NaAsO 2 for 20 min. The red arrowhead indicates a SG. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (D) land-ExM protein image of chromatin of a breast cancer cell. Scale bar: 500 nm in pre-expansion unit. Linear expansion factor: 4.2. (E) land-ExM protein image of NPCs of a breast cancer cell. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.2. (F and G) land-ExM protein images of mitochondria and cytoskeleton of a U2OS cell. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (H–P) land-ExM lipid images of membrane structures. The lipids were labeled with mCLING-Atto647N. (H) land-ExM lipid image of breast cancer cell. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0. (I–M) magnified images of H showing different membrane structures: lipid vesicles (I), mitochondria (J), filopodia (K), nuclear invagination (L), and Golgi apparatus (M). Scale bar: 1 µm (I–M) in pre-expansion unit. (N) 3D land-ExM lipid image of a breast cancer cell after maximum intensity projection, showing the cell membrane. Color-coded by the z-dimension slices from bottom to top. Color bar: purple to white: 0–6 µm in pre-expansion unit. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0. (O and P) magnified images of N showing detailed structures of the cell membrane. Scale bar: 1 µm in pre-expansion unit. All images were taken with an Airyscan microscope.

    Journal: The Journal of Cell Biology

    Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

    doi: 10.1083/jcb.202502035

    Figure Lengend Snippet: land-ExM visualizes phase-separated and membrane organelles. (A–G) land-ExM protein images of membraneless phase separation structures. The proteins were labeled with NHS-biotin-MS and after gelation stained with streptavidin-Alexa Fluor 488. (A) land-ExM protein image of nucleoli in a U2OS cell. Red arrowheads indicate the fibrillar center (FC) or dense fibrillar component (DFC) of the nucleolus. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (B) land-ExM protein image of nuclear bodies of breast cancer cell, UCI082014. Red arrowheads indicate the nuclear bodies. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.2. (C) land-ExM protein image of SGs of a U2OS cell treated with NaAsO 2 for 20 min. The red arrowhead indicates a SG. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (D) land-ExM protein image of chromatin of a breast cancer cell. Scale bar: 500 nm in pre-expansion unit. Linear expansion factor: 4.2. (E) land-ExM protein image of NPCs of a breast cancer cell. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.2. (F and G) land-ExM protein images of mitochondria and cytoskeleton of a U2OS cell. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (H–P) land-ExM lipid images of membrane structures. The lipids were labeled with mCLING-Atto647N. (H) land-ExM lipid image of breast cancer cell. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0. (I–M) magnified images of H showing different membrane structures: lipid vesicles (I), mitochondria (J), filopodia (K), nuclear invagination (L), and Golgi apparatus (M). Scale bar: 1 µm (I–M) in pre-expansion unit. (N) 3D land-ExM lipid image of a breast cancer cell after maximum intensity projection, showing the cell membrane. Color-coded by the z-dimension slices from bottom to top. Color bar: purple to white: 0–6 µm in pre-expansion unit. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0. (O and P) magnified images of N showing detailed structures of the cell membrane. Scale bar: 1 µm in pre-expansion unit. All images were taken with an Airyscan microscope.

    Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

    Techniques: Membrane, Labeling, Staining, Microscopy

    land-ExM coupled with immunostaining LR-ExM for lipid vesicle identification. (A–C) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Lamp2 antibodies (yellow). The anti-Lamp2 antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–G) Magnified images of A–C showing details of lysosomes. Scale bar: 500 nm in pre-expansion unit. (H) Intensity profile along the gray line across the lysosome in image (D). (I–K) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-clathrin antibodies (yellow). The anti-clathrin antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (L–O) Magnified images of I–K showing details of clathrin-coated pits. Scale bar: 500 nm in pre-expansion unit. (P) Intensity profile along the gray line across the clathrin-coated pit in image (L). All images were taken with an Airyscan microscope.

    Journal: The Journal of Cell Biology

    Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

    doi: 10.1083/jcb.202502035

    Figure Lengend Snippet: land-ExM coupled with immunostaining LR-ExM for lipid vesicle identification. (A–C) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Lamp2 antibodies (yellow). The anti-Lamp2 antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–G) Magnified images of A–C showing details of lysosomes. Scale bar: 500 nm in pre-expansion unit. (H) Intensity profile along the gray line across the lysosome in image (D). (I–K) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-clathrin antibodies (yellow). The anti-clathrin antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (L–O) Magnified images of I–K showing details of clathrin-coated pits. Scale bar: 500 nm in pre-expansion unit. (P) Intensity profile along the gray line across the clathrin-coated pit in image (L). All images were taken with an Airyscan microscope.

    Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

    Techniques: Immunostaining, Labeling, Microscopy

    land-ExM coupled with immunostaining LR-ExM for membrane-bound organelle visualization. (A–C) land-ExM total lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Tom20 antibodies (yellow). The anti-Tom20 antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–G) Magnified images of A–C showing details of mitochondria. Scale bar: 1 µm in pre-expansion unit. (H) Intensity profile along the cyan line across the mitochondria in image (D). (I–K) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Sec61b antibodies (yellow). The anti-Sec61b antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (L–O) Magnified in images of I–K showing details of ER. Scale bar: 1 µm in pre-expansion unit. (P) Intensity profile along the cyan line across the ER in image (L). All images were taken with an Airyscan microscope.

    Journal: The Journal of Cell Biology

    Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

    doi: 10.1083/jcb.202502035

    Figure Lengend Snippet: land-ExM coupled with immunostaining LR-ExM for membrane-bound organelle visualization. (A–C) land-ExM total lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Tom20 antibodies (yellow). The anti-Tom20 antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–G) Magnified images of A–C showing details of mitochondria. Scale bar: 1 µm in pre-expansion unit. (H) Intensity profile along the cyan line across the mitochondria in image (D). (I–K) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Sec61b antibodies (yellow). The anti-Sec61b antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (L–O) Magnified in images of I–K showing details of ER. Scale bar: 1 µm in pre-expansion unit. (P) Intensity profile along the cyan line across the ER in image (L). All images were taken with an Airyscan microscope.

    Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

    Techniques: Immunostaining, Membrane, Labeling, Microscopy

    land-ExM reveals SGs at different locations of cells. (A–C) land-ExM images of U2OS cells untreated or treated with NaAsO2 for 20 or 60 min, then immunostained with anti-G3BP1 antibody. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–F) land-ExM images of U2OS cells stained with mCLING (magenta) and NHS ester dye (cyan) and immunostained with anti-G3BP1 (yellow) and anti-Sec61b (white) antibodies. Cells were untreated or treated with NaAsO2 for 20 min or 60 min. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (G) Magnified images of E showing SGs formed adjacent to ER (orange arrowheads). Scale bar: 1 µm in pre-expansion unit. (H) Analysis of the number of nuclear tunnels per cell with or without 60 min NaAsO2 treatment. Each bar represents the mean ± standard error of more than 18 cells. The ns indicates P > 0.05 by Welch’s t test. (I) Analysis of the diameter of nuclear tunnels in cells with or without 60 min NaAsO2 treatment. Each bar represents the mean ± standard error of more than 20 cells. ns indicates P > 0.05 by Welch’s t test. All images were taken with an Airyscan microscope. The cell shown in F is also shown in .

    Journal: The Journal of Cell Biology

    Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

    doi: 10.1083/jcb.202502035

    Figure Lengend Snippet: land-ExM reveals SGs at different locations of cells. (A–C) land-ExM images of U2OS cells untreated or treated with NaAsO2 for 20 or 60 min, then immunostained with anti-G3BP1 antibody. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–F) land-ExM images of U2OS cells stained with mCLING (magenta) and NHS ester dye (cyan) and immunostained with anti-G3BP1 (yellow) and anti-Sec61b (white) antibodies. Cells were untreated or treated with NaAsO2 for 20 min or 60 min. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (G) Magnified images of E showing SGs formed adjacent to ER (orange arrowheads). Scale bar: 1 µm in pre-expansion unit. (H) Analysis of the number of nuclear tunnels per cell with or without 60 min NaAsO2 treatment. Each bar represents the mean ± standard error of more than 18 cells. The ns indicates P > 0.05 by Welch’s t test. (I) Analysis of the diameter of nuclear tunnels in cells with or without 60 min NaAsO2 treatment. Each bar represents the mean ± standard error of more than 20 cells. ns indicates P > 0.05 by Welch’s t test. All images were taken with an Airyscan microscope. The cell shown in F is also shown in .

    Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

    Techniques: Staining, Microscopy

    The nuclear tunnel forms a triple-organellar contact site that includes the SG, the nucleolus, and itself. (A) land-ExM protein (gray) image of U2OS cells immunostained with anti-G3BP1 (red) antibody. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (B–D) Different views of SG in the white dashed box of A. Scale bar: 1 µm in pre-expansion unit. (E) 3D rendering of SG in the white dashed box of A. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (F) land-ExM protein (gray) and lipid (blue) image of U2OS cells immunostained with anti-G3BP1 (red) antibody. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (G–I) Different views of SG in the white dashed box 1 of F. Scale bar: 1 µm in pre-expansion unit. (J) 3D rendering of SG in the white dashed box 1 of F. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in the pre-expansion unit. (K) 3D rendering of SGs in the white dashed box 1–4 of F. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in the pre-expansion unit. (L) land-ExM protein (gray) image of U2OS cells immunostained with anti-G3BP1 (red) and anti-Sec61b (yellow) antibodies. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (M–O) Different views of SG in the white dashed box 1 of L. Scale bar: 1 µm in pre-expansion unit. (P) 3D rendering of SG in the white dashed box 1 of L. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (Q) 3D rendering of SGs in the white dashed box 1–4 of L. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (R) Pie chart of nuclear tunnels with or without SGs. Total tunnels analyzed: 114. (S) Pie chart of SG-filled nuclear tunnels that contact nucleoli versus those that do not. Total tunnel analyzed: 83. All images were taken with an Airyscan microscope. The cell shown in A, F, and L is also shown in .

    Journal: The Journal of Cell Biology

    Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

    doi: 10.1083/jcb.202502035

    Figure Lengend Snippet: The nuclear tunnel forms a triple-organellar contact site that includes the SG, the nucleolus, and itself. (A) land-ExM protein (gray) image of U2OS cells immunostained with anti-G3BP1 (red) antibody. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (B–D) Different views of SG in the white dashed box of A. Scale bar: 1 µm in pre-expansion unit. (E) 3D rendering of SG in the white dashed box of A. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (F) land-ExM protein (gray) and lipid (blue) image of U2OS cells immunostained with anti-G3BP1 (red) antibody. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (G–I) Different views of SG in the white dashed box 1 of F. Scale bar: 1 µm in pre-expansion unit. (J) 3D rendering of SG in the white dashed box 1 of F. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in the pre-expansion unit. (K) 3D rendering of SGs in the white dashed box 1–4 of F. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in the pre-expansion unit. (L) land-ExM protein (gray) image of U2OS cells immunostained with anti-G3BP1 (red) and anti-Sec61b (yellow) antibodies. Cells were treated with NaAsO 2 for 1 h. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (M–O) Different views of SG in the white dashed box 1 of L. Scale bar: 1 µm in pre-expansion unit. (P) 3D rendering of SG in the white dashed box 1 of L. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (Q) 3D rendering of SGs in the white dashed box 1–4 of L. In the reference grid, the spacing of major and minor tick marks is 0.5 and 0.1 µm in pre-expansion unit. (R) Pie chart of nuclear tunnels with or without SGs. Total tunnels analyzed: 114. (S) Pie chart of SG-filled nuclear tunnels that contact nucleoli versus those that do not. Total tunnel analyzed: 83. All images were taken with an Airyscan microscope. The cell shown in A, F, and L is also shown in .

    Article Snippet: U2OS cells (cat#HTB-96; ATCC) were cultured in Mccoy’s 5A medium (Cat#16600082; Gibco) supplemented with 10% Fetal Bovine Serum (cat#10082147; Gibco) and 1% penicillin-streptomycin-amphotericin B (cat#A5955; Sigma-Aldrich).

    Techniques: Microscopy

    The IDR of TRIP12 is responsible for the formation of chromatin condensates (A) Prediction of TRIP12 3D-structure by AlphaFold model. Predicted local distance difference test (pLDDT) indicates the confidence level of the predicted structure. Dark blue, light blue, orange, and yellow represent very high, confident, low, and very low model confidence, respectively. (B) Graphical representation of TRIP12 different domains fused to the GFP reporter protein. Dark blue rectangles locate endogenous or artificial NLSs. IDR, intrinsically disordered region; ARM, armadillo domain; WWE, tryptophan-tryptophan-glutamate-rich domain; HECT, homologous to E6-AP carboxyl terminus; and GFP, green fluorescent protein. (C) Representative images of TRIP12-domains fused to GFP expressing HeLa S3 cells by immunofluorescence. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. Insertion of an artificial NLS in ARM-WWE-GFP, ΔIDR/ARM-WWE/HECT-GFP, HECT-GFP, and ΔIDR-GFP constructs allowed for nuclear localization with a faint presence in the cytoplasm. Scale bars represent 2 μm. (D) Determination of DNA granularity relative to GFP expression level in TRIP12-domains fused to GFP expressing HeLa S3 cells. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on over 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. A Spearman r coefficient test and a two tailed-p value are indicated. (E) Representative images of chromatin condensates induced by TRIP12-IDR overexpression in HCT-116, U2OS, and hTert-RPE1 cell lines by immunofluorescence. Nuclei were counterstained with DAPI. Scale bars represent 2 μm. (F) Comparison of isoelectric point (pI) and capacity to form chromatin condensates (slope) of the different TRIP12-GFP constructs. The pI of the different TRIP12 fragments was determined using ProtParam on Expasy website. The capacity to form chromatin condensates is indicated by the slope values obtained in D, 2F, and C. A Spearman r coefficient test and a two tailed-p value are indicated. (G) Representative images of DNA organization in IDR-GFP deletion constructs in high expressing HeLa S3 cells (left). The cytoplasmic expression of 325-445-GFP constructs is explained by the loss of NLS sequences. Determination of DNA granularity relative to GFP expression level for the different IDR-GFP deletion constructs (right). The DAPI granularity and GFP expression were determined as described in “ ” on more than 40 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. Scale bars represent 2 μm. A Spearman r coefficient test and a two tailed-p value are indicated. (H) Comparison of isoelectric point (pI), capacity to form chromatin condensates (slope), and the length (in aa) of the different IDR-GFP deletion constructs. The pI and the length of the different TRIP12 fragments were determined using ProtParam on Expasy website. The capacity to form chromatin condensates was assessed from the slope values obtained in G. A Spearman r coefficient test and a two tailed-p value are indicated. (I) Representative image of cellular fractionation of HeLa S3 cells expressing 1-107-GFP, 108-207-GFP, 208-324-GFP, and 325-445-GFP constructs. GFP expression in soluble and chromatin-bound fractions was determined by western blot analysis. The level of HSP90 and PanH3 protein expression were used as loading and enrichment controls. The graph represents the percentage of GFP expression in the different fraction. Results are expressed as mean ± SEM of four different experiments. (J) Electric net charge of [1–107], [108–207], [208–324], and [325–445] fragments of TRIP12 IDR at pH 7.4 determined by Prot pi|Protein Tool (left graph). Percentage of basic, acidic, and uncharged residues in [1–107], [108–207], [208–324], and [325–445] fragments of TRIP12 IDR determined by Prot pi|Protein Tool (right graph). (K) Graphical representation of MED1-IDR fused to GFP protein. The dark blue rectangle indicates artificial NLSs. Representative images of MED1-IDR-GFP expressing HeLa S3 cells obtained by immunofluorescence. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on more than 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is represented in black. Scale bars represent 2 μm. A Spearman r coefficient test and a two tailed-p value are indicated.

    Journal: iScience

    Article Title: The TRIP12’s intrinsically disordered region induces chromatin condensates and interferes with nuclear processes

    doi: 10.1016/j.isci.2025.114592

    Figure Lengend Snippet: The IDR of TRIP12 is responsible for the formation of chromatin condensates (A) Prediction of TRIP12 3D-structure by AlphaFold model. Predicted local distance difference test (pLDDT) indicates the confidence level of the predicted structure. Dark blue, light blue, orange, and yellow represent very high, confident, low, and very low model confidence, respectively. (B) Graphical representation of TRIP12 different domains fused to the GFP reporter protein. Dark blue rectangles locate endogenous or artificial NLSs. IDR, intrinsically disordered region; ARM, armadillo domain; WWE, tryptophan-tryptophan-glutamate-rich domain; HECT, homologous to E6-AP carboxyl terminus; and GFP, green fluorescent protein. (C) Representative images of TRIP12-domains fused to GFP expressing HeLa S3 cells by immunofluorescence. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. Insertion of an artificial NLS in ARM-WWE-GFP, ΔIDR/ARM-WWE/HECT-GFP, HECT-GFP, and ΔIDR-GFP constructs allowed for nuclear localization with a faint presence in the cytoplasm. Scale bars represent 2 μm. (D) Determination of DNA granularity relative to GFP expression level in TRIP12-domains fused to GFP expressing HeLa S3 cells. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on over 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. A Spearman r coefficient test and a two tailed-p value are indicated. (E) Representative images of chromatin condensates induced by TRIP12-IDR overexpression in HCT-116, U2OS, and hTert-RPE1 cell lines by immunofluorescence. Nuclei were counterstained with DAPI. Scale bars represent 2 μm. (F) Comparison of isoelectric point (pI) and capacity to form chromatin condensates (slope) of the different TRIP12-GFP constructs. The pI of the different TRIP12 fragments was determined using ProtParam on Expasy website. The capacity to form chromatin condensates is indicated by the slope values obtained in D, 2F, and C. A Spearman r coefficient test and a two tailed-p value are indicated. (G) Representative images of DNA organization in IDR-GFP deletion constructs in high expressing HeLa S3 cells (left). The cytoplasmic expression of 325-445-GFP constructs is explained by the loss of NLS sequences. Determination of DNA granularity relative to GFP expression level for the different IDR-GFP deletion constructs (right). The DAPI granularity and GFP expression were determined as described in “ ” on more than 40 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. Scale bars represent 2 μm. A Spearman r coefficient test and a two tailed-p value are indicated. (H) Comparison of isoelectric point (pI), capacity to form chromatin condensates (slope), and the length (in aa) of the different IDR-GFP deletion constructs. The pI and the length of the different TRIP12 fragments were determined using ProtParam on Expasy website. The capacity to form chromatin condensates was assessed from the slope values obtained in G. A Spearman r coefficient test and a two tailed-p value are indicated. (I) Representative image of cellular fractionation of HeLa S3 cells expressing 1-107-GFP, 108-207-GFP, 208-324-GFP, and 325-445-GFP constructs. GFP expression in soluble and chromatin-bound fractions was determined by western blot analysis. The level of HSP90 and PanH3 protein expression were used as loading and enrichment controls. The graph represents the percentage of GFP expression in the different fraction. Results are expressed as mean ± SEM of four different experiments. (J) Electric net charge of [1–107], [108–207], [208–324], and [325–445] fragments of TRIP12 IDR at pH 7.4 determined by Prot pi|Protein Tool (left graph). Percentage of basic, acidic, and uncharged residues in [1–107], [108–207], [208–324], and [325–445] fragments of TRIP12 IDR determined by Prot pi|Protein Tool (right graph). (K) Graphical representation of MED1-IDR fused to GFP protein. The dark blue rectangle indicates artificial NLSs. Representative images of MED1-IDR-GFP expressing HeLa S3 cells obtained by immunofluorescence. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on more than 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is represented in black. Scale bars represent 2 μm. A Spearman r coefficient test and a two tailed-p value are indicated.

    Article Snippet: U2OS , ATCC , HTB-96.

    Techniques: Expressing, Immunofluorescence, Construct, Transfection, Two Tailed Test, Over Expression, Comparison, Cell Fractionation, Western Blot