Journal: Journal of Cell Science

Article Title: OptoLoop – an optogenetic tool to probe the functional role of genome organization

doi: 10.1242/jcs.264574

Figure Lengend Snippet: Optogenetic manipulation of proximity between repetitive genomic loci. (A) Scheme of OptoLoop consisting of a fusion between dCas9 and the optogenetic protein CRY2. OptoLoop is targeted to specific genomic loci by introducing specific sgRNAs. CRY2–CRY2 interactions activated by blue light bridge targeted loci to form a chromatin loop. (B) Left panel, region of chromosome 19 showing sgIDR3 and sgTCF3 target sites, representative Hi-C contact map (data from ) and BACs used in DNA-FISH to label the IDR3 (magenta) and TCF3 loci (green). Right panel, mCherry channel images of U2OS dCas9–3XmCherry–CRY2 cells transfected with sgIDR3 and sgTCF3, kept in dark or illuminated with blue light for 3 h (1 s pulses every 10 s), and fixed. Scale bars: 5 µm. (C) Left panel, representative image of DNA-FISH for IDR3 and TCF3 with specific BAC FISH probes in U2OS cells. Right panel represents a single cell highlighted in left panel (yellow box); the expansion shows a single allele in this cell. Dashed line denotes the distance between the two FISH signals. Scale bars: 20 µm (left panel), 5 µm (right panel), 1 µm (expansion). (D) IDR3–TCF3 distances, calculated for U2OS dCas9–mCherry–CRY2 polyclonal cells transfected with indicated combinations of sgIDR3 and sgTCF3, kept under dark or illuminated for 3 h (1 s pulses every 10 s). Violin plot corresponds to a representative experiment, with black lines representing median distances. Bar plot represents means of two independent experiments. Each dot represents the median of typically 5000–10,000 alleles analyzed per experiment. (E) Fraction of alleles with IDR3-TCF3 distance <0.27 µm measured from DNA-FISH images for U2OS dCas9–mCherry–CRY2 polyclonal cells and three clones of U2OS dCas9–3XmCherry–CRY2 cells, transfected with indicated combinations of sgIDR3 and sgTCF3, and kept in dark or illuminated for 3 h (1 s pulses every 10 s). Each dot represents the fraction of typically 5000–10,000 alleles analyzed per experiment. Bars represent means of two or three independent experiments. (F) Measurement of cell-to-cell heterogeneity in loop formation. Bars with green shades: observed fraction of cells with none, one or both alleles with IDR3–TCF3 distance <0.27 µm obtained from a representative experiment shown in E with 2500–5000 cells analyzed per sample. Bars with magenta shades: expected fraction of cells with none, one or both alleles with IDR3–TCF3 distance <0.27 µm assuming that alleles from a same cell are independent between each other (Eqn 2). * P <0.05; *** P <0.001; ns, not significant [two-way ANOVAs followed by post-hoc Tukey tests (D,E); paired two-tailed t -test (E); chi-squared test (F)].

Article Snippet: NIH3T3 (mouse fibroblasts, ATCC #CRL-1658), U2OS (from human osteosarcoma, ATCC #HTB-96), HeLa (from human cervical adenocarcinoma, ATCC #CRM-CCL-2) and Lenti-X HEK-293T (from human embryonic kidney, cat. #632180 from Takara Bio, Japan) cell lines were cultured in Dulbecco's modified Eagle's medium (Gibco, Waltham, MA, USA) supplemented with 10% (15% for NIH3T3) fetal bovine serum (Gibco, Waltham, MA, USA) plus 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco, Waltham, MA, USA) at 37°C in a humidified atmosphere with 5% CO 2 .

Techniques: Hi-C, Transfection, Single Cell, Clone Assay, Two Tailed Test

Journal: Journal of Cell Science

Article Title: OptoLoop – an optogenetic tool to probe the functional role of genome organization

doi: 10.1242/jcs.264574

Figure Lengend Snippet: Benchmarking OptoLoop against a previous optogenetic manipulation tool. (A) Scheme of LADL consisting of soluble CRY2wt and a fusion between dCas9 and the CRY2 partner CIBN. dCas9–CIBN tethers specific genomic loci and light-activation induces both CRY2–CRY2 and CRY2–CIBN interactions bridging the targeted loci to form a loop. (B) Images of U2OS cells expressing dCas9–3XGFP–CIBN and mCherry fused to CRY2wt or CRY2olig, transfected with sgIDR3 and sgTCF3, and fixed after being kept under dark or illuminated with blue light pulses for 3 h (1 s pulses every 10 s). White arrows indicate FISH signals corresponding to IDR3–TCF3 loci. Scale bar: 5 µm. (C) Fraction of alleles with IDR3–TCF3 distance <0.27 µm measured from DNA-FISH images of U2OS cell lines stably expressing dCas9-3XGFP-CRY2 and mCherry-CRY2olig (LADL, clone #7) or dCas9-3XmCherry-CRY2 (OptoLoop, clone #3), transfected with sgIDR3 and sgTCF3, and kept under dark or illuminated with blue light for 3 h (1 s pulses every 10 s). Each dot represents the fraction of 5000–7000 alleles analyzed per experiment. Bars represent the means of three independent experiments. * P <0.05; ** P <0.01; *** P <0.001 (two-way ANOVA followed by post-hoc Tukey test).

Article Snippet: NIH3T3 (mouse fibroblasts, ATCC #CRL-1658), U2OS (from human osteosarcoma, ATCC #HTB-96), HeLa (from human cervical adenocarcinoma, ATCC #CRM-CCL-2) and Lenti-X HEK-293T (from human embryonic kidney, cat. #632180 from Takara Bio, Japan) cell lines were cultured in Dulbecco's modified Eagle's medium (Gibco, Waltham, MA, USA) supplemented with 10% (15% for NIH3T3) fetal bovine serum (Gibco, Waltham, MA, USA) plus 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco, Waltham, MA, USA) at 37°C in a humidified atmosphere with 5% CO 2 .

Techniques: Activation Assay, Expressing, Transfection, Stable Transfection

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

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

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

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

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

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

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

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

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

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

Journal: bioRxiv

Article Title: Proximity labelling of the BAK macropore uncovers a new role for SLC35A4-MP in mitochondrial dynamics

doi: 10.64898/2026.03.22.713508

Figure Lengend Snippet: A . SDS-PAGE separation and immunoblotting of mitochondria isolated from WT, SLC35A4–MP KO and SLC35A4-MP KO + SLC35A4-MP FLAG U2OS cells. SLC35A4-MP FLAG expression was induced with 5 ng/ml doxycycline for 48 h. SDHA serves as a loading control. Representative of n = 3 biological replicates. B. Representative confocal images of SLC35A4-MP KO + SLC35A4-MP FLAG U2OS cells. SLC35A4-MP FLAG expression was induced with 5 ng/ml doxycycline for 48 h. Immunofluorescence was used to label mitochondria (Anti-TOM20, magenta), and determine SLC35A4-MP localisation (anti-FLAG, yellow). Scale bar indicates 5 µm. C. Electron microscopy of WT and SLC35A4-MP KO U2OS cells. Scale bars indicate 500 nm. D. The steady-state whole cell proteome of SLC35A4-MP KO vs WT. MitoCarta 3.0 proteins are highlighted in black, and those with OMM or IMM localisations are highlighted in blue and pink respectively. E. Whole cell proteome of SLC35A4-MP KO vs WT U2OS in apoptotic vs non-apoptotic conditions. The log 2 (fold change) of significantly altered ( p < 0.05) WT and KO proteins following 1 h apoptotic treatment was plotted against each other; WT (x-axis) and KO (y-axis). Mitochondrial proteins are highlighted in black, and those with OMM or IMS/IMM localisation are highlighted in blue or pink respectively.

Article Snippet: Human osteosarcoma cells (U2OS) were purchased from the ATCC, and a clonal line was established.

Techniques: SDS Page, Western Blot, Isolation, Expressing, Control, Immunofluorescence, Electron Microscopy

Journal: bioRxiv

Article Title: Proximity labelling of the BAK macropore uncovers a new role for SLC35A4-MP in mitochondrial dynamics

doi: 10.64898/2026.03.22.713508

Figure Lengend Snippet: A . The steady-state SLC35A4-MP interactome. Proteins with a log 2 (SLC35A4-MP FLAG 0 h / WT 0 h) > 1, p value < 0.05, were considered significantly enriched. Proteins with MitoCarta 3.0 annotations have been coloured as indicated B. Comparison of log 2 (SLC35A4-MP FLAG / WT) enrichment of the SLC35A4-MP FLAG interactome at 0 h and 1 h apoptosis. Mitochondrial proteins are coloured in black. Proteins preferentially enriched at 0 h or 1 h post induction of apoptosis are highlighted by the blue and red regions respectively. Stable interactors are highlighted by the yellow region (refer to Fig. S4B) C-D. BN-PAGE analysis of 1% digitonin soluble complexes from isolated mitochondria, immunoblotting using antibodies against C) MIC10 (MICOS complex) and D) OPA1 respectively. NDUFA9 (CI) and SDHA (CII) serve as a loading controls. Representative of n = 3 independent experiments. For densitometric quantification of soluble MIC10 or OPA1 containing complexes, complex abundance was normalised to NDUFA9 or SDHA respectively and displayed relative to WT levels. Data represents n = 3 independent experiments. Error bars indicate mean ± SEM. * = p value < 0.05, ** = p value < 0.01 by one-way ANOVA with Tukey’s multiple comparison test. E. SDS-PAGE analysis of OPA1 isoforms in mitochondria isolated from control, SLC35A4-MP KO and SLC35A4-MP rescue U2OS cells (SLC35A4-MP FLAG) . SLC35a4-MP FLAG expression was induced with doxycycline for 48 h (concentrations as indicated). LONP1 serves as a loading control. Representative of n = 3 independent experiments. F-G. Densitometric quantification of L-OPA1 ‘a’ isoform, and S-OPA1 isoforms ‘c’ and ‘e’ in F) SLC35A4-MP KO (without doxycycline treatment), or G) SLC35A4-MP KO cells overexpressing SLC35A4-MP FLAG with increasing concentrations of doxycycline. OPA1 isoform levels were normalised to LONP1 and displayed relative to WT (without doxycycline treatment). n = 3 independent experiments. Error bars indicate mean ± SEM. F: *** = p value < 0.005 by Student’s t-test. G: ** = p value < 0.01 *** = p value < 0.005 by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Human osteosarcoma cells (U2OS) were purchased from the ATCC, and a clonal line was established.

Techniques: Comparison, Isolation, Western Blot, SDS Page, Control, Expressing

Journal: bioRxiv

Article Title: Proximity labelling of the BAK macropore uncovers a new role for SLC35A4-MP in mitochondrial dynamics

doi: 10.64898/2026.03.22.713508

Figure Lengend Snippet: A. Live-cell imaging of WT, SLC35A4-MP KO and SLC35A4-MP rescue U2OS cells (SLC35A4-MP FLAG) stably expressing TOM20 Halo following treatment with ABT-737 [10 µM], S63845 [2 µM] and 1 h pre-treatment with QVD-OPh [20 µM]. Representative images from n=3 independent experiments. Scale bars indicate 5 µm. B-C. Quantification of apoptosis induced mtDNA release and mitochondrial fragmentation respectively. Quantification was performed on 10-15 cells from n = 3 independent experiments. Data points indicate mean ± SEM. Dashed line indicates the start of image acquisition D. Live cell imaging of SLC35A4-MP KO U2OS cells following CCCP [20µM] treatment. Representative images from n = 3 independent experiments. Scale bars indicate 10 µm. E. Quantification of mitochondrial fragmentation. Quantification was performed on 10-15 cells from n=3 independent experiments. Dashed line indicates start of image acquisition.

Article Snippet: Human osteosarcoma cells (U2OS) were purchased from the ATCC, and a clonal line was established.

Techniques: Live Cell Imaging, Stable Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: MDS/AML-associated DDX41 helicase facilitates homologous recombination repair by potentially resolving R-loops

doi: 10.1093/nar/gkag219

Figure Lengend Snippet: DDX41 loss diminishes HR repair. ( A ) Western blot assays of DDX41 siRNA and siRNA control in U2OS GFP reporter cell lines with indicated antibodies. β-Actin serves as a loading control. Quantification of relative protein level for DDX41 and I-SecI is shown on the right. ( B ) Percentages of GFP positive cells as assessed by flow cytometry 36 h after U2OS GFP reporter cell lines treated with siRNA control, siRNA control + I-SceI plasmid, or DDX41 siRNA + I-SceI plasmid. ( C ) Cell survival assays of WT and DDX41–KO HT1080 cells treated with Olaparib. ( D and E ) Immunofluorescence staining of WT and DDX41–KO HT1080 cells with γH2AX, RPA32 (D) or RAD51 (E), and DAPI without bleomycin treatment (UT) or 4 h post BLM treatment (30 μg/ml for 1 h). Quantification of RPA32 and RAD51 foci is shown in the middle. Data represent the mean ± SEM of three independent experiments. * P < 0.05, *** P < 0.001, and **** P < 0.0001.

Article Snippet: U2OS (HTB-96, ATCC) and U2OS GFP reporter cells [ ] (a gift from Jeremy Stark, City of Hope) were grown in Mccoy 5A media (16600082, Thermo Fisher) supplemented with 10% fetal bovine serum.

Techniques: Western Blot, Control, Flow Cytometry, Plasmid Preparation, Immunofluorescence, Staining

Journal: iScience

Article Title: Predicting DNA damage response using synthetic cell painting profiles and experimental analysis

doi: 10.1016/j.isci.2026.115000

Figure Lengend Snippet: Application of the model to external datasets and experimental testing (A) Principal component analysis plot for the idr-0080 and cpg-0012 datasets. (B) Classification of cpg-0012 compounds into high-DDR (9,923) and low-DDR (20,694) groups. The high-DDR group includes known DNA damage inducers. Test compounds without prior DDR annotation were selected from the high-DDR (tetrindole, KF38789, and LY2183240) and low-DDR (amoxapine, acetazolamide, and captopril) groups. Flow cytometric (C) and Western blot (D) analyses of γH2AX expression in U2OS cells following 18 h treatment with 10 μM test compounds. (E) U2OS cells were treated with high-DDR candidates at increasing doses or with 10 μM low-DDR candidates for 72 h, and cell viability was measured using the CellTiter-Glo assay. Data are shown as means ± SD from independent experiments. One-way ANOVA with LSD post hoc test was used for statistical analysis. ∗ , p < 0.05; ∗∗∗ , p < 0.001 versus vehicle.

Article Snippet: U2OS human osteosarcoma cells (ATCC, HTB-96), originally derived from a moderately differentiated sarcoma of the tibia of a 15-year-old White female osteosarcoma patient, were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and authenticated by ATCC-provided STR profiling.

Techniques: Western Blot, Expressing, Glo Assay

Journal: iScience

Article Title: Predicting DNA damage response using synthetic cell painting profiles and experimental analysis

doi: 10.1016/j.isci.2026.115000

Figure Lengend Snippet:

Article Snippet: U2OS human osteosarcoma cells (ATCC, HTB-96), originally derived from a moderately differentiated sarcoma of the tibia of a 15-year-old White female osteosarcoma patient, were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and authenticated by ATCC-provided STR profiling.

Techniques: Recombinant, Flow Cytometry, Software