Journal: bioRxiv

Article Title: C9ORF72-derived polyGR polypeptides disrupt passive nucleocytoplasmic transport by tuning protein affinity for the nuclear pore barrier

doi: 10.64898/2026.03.16.711670

Figure Lengend Snippet: a, Schematic of the experimental design for assessing polyGR association with nuclear pores using two-colour STORM microscopy in U2OS-CRISPR–NUP96–mEGFP cells. Angled beam imaging was used to selectively excite single molecules of ATTO655-labelled GR 20 or ATTO655 dye alone and Nup96-mEGFP labelled with anti-GFP nanobodies by DNA-PAINT at the basal nuclear envelope. b, Averaged NPC localisations from STORM imaging of fixed cells showing that ATTO655 dye alone does not localize to NPCs (upper panel), whereas ATTO655–GR 20 exhibits localisation in close proximity to NPCs, not co-localising with the structural component Nup96, but instead in surrounding regions including within the central channel. Scale bars = 50 nm.

Article Snippet: U2OS-CRISPR-NUP96-mEGFP cells (Cytion, #300174) were cultured in McCoy’s 5A (Modified) Medium (Thermo Fisher Scientific, 26600023) supplemented with 10% fetal bovine serum (Pan Biotech, P30-3031), 1% non-essential amino acids (Thermo Fisher Scientific, 11140035), 1% penicillin/streptomycin (Thermo Fisher Scientific, 15140122), and 1% sodium pyruvate (Thermo Fisher Scientific, 11360039).

Techniques: Microscopy, CRISPR, Imaging

Journal: Nature Methods

Article Title: Optimal precision and accuracy in 4Pi-STORM using dynamic spline PSF models

doi: 10.1038/s41592-022-01465-8

Figure Lengend Snippet: Mic60 is a component of the MICOS complex, and is involved in the formation and maintenance of crista junctions that connect the crista membrane with the inner boundary membrane. a , Mic60 in a U-2 OS cell, labeled with primary and secondary antibodies. The Mic60 signals appear as structured, punctate clusters. The localizations are color coded according to their z coordinate (identical color scales in a – d ). Scale bar, 200 nm. b , Magnified view of the boxed region in a . Scale bar, 50 nm. c , Mic60 in a COS-7 cell, in which the crista junctions exhibit a linear organization over segments of the inner boundary membrane. Scale bar, 200 nm. d , Magnified view of the boxed region in c . Scale bar, 50 nm. e , f , Unwrapped views of the Mic60 localization density around the surface of the mitochondria, showing the nanoscale distribution of Mic60. In U-2 OS cells, Mic60 appears predominantly punctate, with pairs or clusters of signal density separated by 20–40 nm (Extended Data Fig. and Supplementary Fig. ). In COS-7 cells, Mic60 appears to have a zigzag or double-line arrangement, with a typical width of approximately 25 nm (Extended Data Fig. and Supplementary Fig. ). Dashed lines indicate the extent of the data in f . g , Two-color image of Mic60 (blue) and mitochondrial nucleoids (yellow) in a COS-7 cell, stained with antibodies labeled with Alexa Fluor 647 and Cy5.5, respectively. Scale bar, 1 µm. h , Detailed view of the boxed region in g . Lower density of Mic60 close to the DNA signal, suggesting fewer crista junctions in these regions. i , Cross-section ( x – z ) through the region indicated by the dashed lines in h , showing Mic60 at the inner boundary membrane, and a DNA cluster in the center of the mitochondrion. j , A 3D perspective view of the mitochondrion shown in h and i , where the Mic60 and DNA signals have been rendered as isosurfaces. Scale bars, 250 nm ( h – j ).

Article Snippet: Experiments were performed using either standard COS-7 cells or U-2 OS cells obtained from American Type Culture Collection (ATCC), or gene-edited U-2 OS cells expressing a SNAP-tagged version of the nucleoporin Nup107 (CLS Cell Lines Service, U-2OS-ZFN-SNAP-Nup107 clone 294) or Nup96 (CLS Cell Lines Service, U-2OS-CRISPR-NUP96-SNAP clone 33) .

Techniques: Labeling, Staining

Journal: bioRxiv

Article Title: Mechanical forces drive mitochondrial matrix extrusion and apoptotic pore growth

doi: 10.1101/2025.05.12.653510

Figure Lengend Snippet: A) Schematic of the workflow including cell seeding on carbon-coated coordinate system gridded dishes, labeling of HALO-BAK with JFX650 HALO-Tag ligand, induction of apoptosis and cell fixation. The stoichiometry of apoptotic BAK is then measured by photon-counting confocal microscopy, followed by STED microscopy to examine the nanoscale structural organization of BAK. Samples are resin-embedded, cut and contrasted for transmission electron microscopy to visualize mitochondrial ultrastructure. B) Representative photon-counting confocal (left), STED (middle), and transmission electron microscopy (right) images of U2OS HALO-BAK cells labeled with JFX650 HALO-Tag ligand (red) one hour after apoptosis induction. Mitochondria were visualized using MitoTracker orange (cyan). Enlarged images correspond to cropped regions of the overview image as indicated, showing representative photon-counting confocal and STED images of line, arc, and ring structures. Dashed rectangle indicates region shown in transmission electron microscopy image. Scale bar 10 µm (zoomed images 500 nm) for stoichiometry and STED images and 1 µm (zoomed images 500 nm) for transmission electron microscopy images. Data are representative of n = 8 independent experiments. C) Overlaid STED-CLEM image of a single mitochondrion showing MitoTracker (cyan, confocal) and BAK (red, STED) fluorescence signal and EM image (grayscale, left panel), BAK signal and EM image (middle panel), and single EM image (right panel). Arrowhead indicates position of MOM discontinuity. Images correspond to the magnified (rotated and flipped) region of the image in (B, left panel) as indicated by the rectangle. Scale bar 250 nm. D) STED-CLEM overlay image of BAK (red, STED) and EM signal (grayscale) of the mitochondrion shown in (C). White circle indicates region of interest for quantification of BAK stoichiometry (as indicated) from photon-counting confocal microscopy image (not shown). Dashed line indicates the region of BAK fluorescence and EM intensity measurement (shown in E) along the MOM discontinuity. Scale bar 250 nm. E) Quantification of BAK fluorescence (blue) and EM intensity (gray) at the line profile indicated by the dashed line in (D). F) 3D rendered MOM (blue), MIM (purple), and cristae membranes (green) overlaid with EM image (grayscale, right panel) in top view (middle panel) and tilted view (right panel) of the image shown in (C). Arrowheads indicate the position of the MOM and MIM discontinuities. Scale bar 250 nm.

Article Snippet: The U2OS NUP96-HALO (U-2_OS-CRISPR-NUP96-Halo_clone_no252) CRISPR cell line was obtained from CLS Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Labeling, Confocal Microscopy, Microscopy, Transmission Assay, Electron Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Mechanical forces drive mitochondrial matrix extrusion and apoptotic pore growth

doi: 10.1101/2025.05.12.653510

Figure Lengend Snippet: A) MOM opening at the tip of the mitochondrion, B) MOM opening with extrusion of the MIM, C) MOM opening at the side of the mitochondrion, and D) MOM opening at a potential post-fission site, as indicated by the schematic representation (BAK shown in red). Microscopy images show representative STED microscopy (top), STED-CLEM overlay (middle) and TEM (bottom) examples of mitochondria from U2OS HALO-BAK cells one hour after apoptosis induction labeled with JFX650 Halo-Tag ligand (red, STED) for each category. Mitochondria were labeled with MitoTracker Orange (cyan, confocal). Arrowheads indicate the position of the MOM discontinuity. n indicates the number of cases assigned to the corresponding category out of a total of n = 6 independent experiments. Scale bar 500 nm.

Article Snippet: The U2OS NUP96-HALO (U-2_OS-CRISPR-NUP96-Halo_clone_no252) CRISPR cell line was obtained from CLS Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Microscopy, Labeling

Journal: bioRxiv

Article Title: Mechanical forces drive mitochondrial matrix extrusion and apoptotic pore growth

doi: 10.1101/2025.05.12.653510

Figure Lengend Snippet: A) Representative deconvolved confocal/STED microscopy image of U2OS HALO-BAK cells labeled with JFX650 HALO-Tag ligand (magenta, STED) one hour after apoptosis induction. The MOM was stained by immunolabeling against TOM20 (green, confocal) and the mitochondrial matrix was visualized by MitoTracker Orange (blue, confocal). Numbered rectangles indicate cropped regions in (B). Scale bar 10 µm. B) Enlarged images of cropped regions of the overview image shown in (A) as indicated. Scale bar 1 µm. C) Quantification of apoptotic mitochondria with intact MOM (intact) versus mitochondria with discontinuity of the MOM (MOM discont.) or mitochondria with extrusion of MIM through MOM openings (MIM extrusion). Values correspond to the percentage of mitochondria of individual cells (individual data points) as well as the mean (line) ± SD (whiskers) of n = 14 cells. Significance levels were determined by non-parametric one-way ANOVA (Kruskal-Wallis test) and Dunn’s multiple comparison test. P indicates multiplicity adjusted P values (family-wise significance and confidence level set to 0.05). D) Representative confocal microscopy images of the categories quantified in (C) of U2OS HALO-BAK cells prepared as described in (A). Scale bar 1 µm. E) Representative confocal microscopy overview image of valinomycin/nigericin-induced osmotic mitochondrial matrix swelling in healthy U2OS HALO-BAK cells. The MIM/mitochondrial matrix was visualized by MitoTracker Orange (magenta) and the MOM was stained by immunolabeling against TOM20 (green). Scale bar 20 µm. F) Representative confocal/STED microscopy image of U2OS HALO-BAK cells transiently transfected with TOM20-SNAP labeled with JFX650 SNAP-tag ligand (green, deconvolved confocal) one hour after apoptosis induction. The MIM was labeled with PKFO (magenta, STED). Dotted rectangles indicate cropped regions in (G). Scale bar 5 µm. G) Enlarged images of cropped regions of the image shown in (F) as indicated, showing exemplary mitochondria with intact MOM (intact) or mitochondria with MIM extrusion through MOM openings (MIM extrusion). Scale bar 1 µm. H) Quantification of mitochondria with MIM extrusion through MOM openings in apoptotic U2OS HALO-BAK control (Ctrl) cells or apoptotic U2OS HALO-BAK cells treated with valinomycin/nigericin (Val/Nig) to induce osmotic swelling of the mitochondrial matrix. Values correspond to the percentage of mitochondria of individual cells (individual data points) as well as the mean (line) ± SD (whiskers) of n = 10 (ctrl) and n = 12 (Val/Nig) cells. Significance levels were determined by unpaired nonparametric Student’s t-test (Mann-Whitney test, P value as indicated). Data are representative of n = 5 (panels A-D) and n = 4 (panels F-H) independent experiments. I) Gallery of hourglass-shaped MIM extrusion examples from U2OS HALO-BAK cells one hour after apoptosis induction. The MOM was stained by immunolabeling against TOM20 (green, deconcolved) and the mitochondrial matrix was visualized by MitoTracker Orange (magenta). Scale bar 500 nm. J) Representative axisymmetric image of a MIM extrusion as described in (I). The mitochondria are aligned so that the longitudinal axis is orthogonal to the axis of the opening pore separating the regions of exposed MIM (inner) and MIM contained in MOM (outer, dashed line). The parametrization of the local radius R and the tangent angle ψ as a function of the arc length S is presented. Scale bar 500 nm. K) Local mitochondrial radius R and tangent angle ψ as a function of the arc length S, in blue and burgundy, respectively. L) Minimized energy of the experimental mitochondria presented in (J). In black, the sum of the energies is plotted as a function of the arc length S. The lateral tension, pressure, bending, and line energies are plotted as a function of S, in beige, blue, burgundy, and green, respectively. M) Pore rim line tension γ obtained from the minimization routine of n = 18 mitochondria from n = 9 individual cells. Data are presented as the line tension values for individual mitochondria (individual data points) as well as the median (line) ± interquartile range (whiskers). Data are representative of n = 3 independent experiments.

Article Snippet: The U2OS NUP96-HALO (U-2_OS-CRISPR-NUP96-Halo_clone_no252) CRISPR cell line was obtained from CLS Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Microscopy, Labeling, Staining, Immunolabeling, Comparison, Confocal Microscopy, Transfection, Control, MANN-WHITNEY

Journal: Oncotarget

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

doi: 10.18632/oncotarget.23453

Figure Lengend Snippet: Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Cell morphology was observed with a light microscope (A) . Cell proliferation was analyzed using a trypan blue exclusion assay (B) . After drug administration, cellular proteins were prepared for immunoblot analyses. Levels of ERα and ERβ were immunodetected (C and E, top panels) . Amounts of β-actin were analyzed as the internal standard (C and E, bottom panels) . These protein bands were quantified and statistically analyzed (D and F) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the values significantly ( p < 0.05) differed from the respective control group, p < 0.05.

Article Snippet: Briefly, human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, were purchase from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Light Microscopy, Trypan Blue Exclusion Assay, Western Blot, Control

Journal: Oncotarget

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

doi: 10.18632/oncotarget.23453

Figure Lengend Snippet: Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Distribution of the ERα protein in human osteoblasts was immunodetected using an antibody with Cy3-conjugated streptavidin ( A , top panel). Cellular nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (middle panel). The merged signals indicated that the ERα protein had been translocated into nuclei (bottom panel). These merged fluorescent signals were quantified and statistically analyzed (B) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p < 0.05.

Article Snippet: Briefly, human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, were purchase from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Staining, Control

Journal: Oncotarget

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

doi: 10.18632/oncotarget.23453

Figure Lengend Snippet: Human osteoblast-like U2OS cells were treated with 10 nM of estradiol for 48 h. Total RNA were isolated for analysis of mitochondrial energy metabolism genes using a PCR array, containing 84 genomic genes encoding certain mitochondrial enzymes for ATP synthesis and 12 loading controls (A) . Differential expressions of these genes were measured and shown as a hot map in the order of genes indicated in panel A (B) . Percentages of upregulated, downregulated, and unchanged expressions of these genes were further statistically analyzed (C) . Also, the major genomic complex genes upregulated by estradiol in human osteoblasts were summarized (D) .

Article Snippet: Briefly, human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, were purchase from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Isolation

Journal: Oncotarget

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

doi: 10.18632/oncotarget.23453

Figure Lengend Snippet: Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Distribution of the ERα protein in human osteoblasts was immunodetected using an antibody with Cy3-conjugated streptavidin ( A , top panel). Mitochondria of human osteoblasts were stained with 3,3′-dihexyloxacarbocyanine (DiOC6), a positively charged dye (middle panel). Merged signals indicated that the ERα protein had been translocated into mitochondria (bottom panels). These fluorescent signals were quantified and statistically analyzed (B) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p < 0.05.

Article Snippet: Briefly, human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, were purchase from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Staining, Control

Journal: Oncotarget

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

doi: 10.18632/oncotarget.23453

Figure Lengend Snippet: Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 3, 6, 12, 18, and 24 h. Levels of COX I and II mRNA were analyzed using an RT-PCR ( A and C , top panels). Amounts of β-actin mRNA were assayed as the internal standard (bottom panel). These bands were quantified and statistically analyzed (B and D) . A quantitative real-time PCR analysis was carried out to confirm expression of COX I mRNA in U2OS cells and rat calvarial osteoblasts (E) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p < 0.05.

Article Snippet: Briefly, human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, were purchase from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Oncotarget

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

doi: 10.18632/oncotarget.23453

Figure Lengend Snippet: Human osteoblast-like U2OS cells were exposed to 10 nM of estradiol for 1, 6, 12, and 24 h. Levels of COX I and II proteins were immunodetected (A and C, top panels) . Amounts of β-actin were analyzed as the internal standard (A and C, bottom panels) . These protein bands were quantified and statistically analyzed ( B and D ). Mitochondria of human osteoblasts were stained with 3,3′-dihexyloxacarbocyanine (DiOC6), a positively charged dye. The mitochondrial membrane potential (MMP) of human osteoblasts was determined by quantifying DiOC6-positive signals (E) . The mitochondrial enzyme activity was assayed using a colorimetric method (F) . Cellular ATP levels were quantified using a bioluminescence assay (G) . Each value represents the mean ± SEM for n = 6. The symbol * indicates that the value significantly differed from the respective control group, p < 0.05. FI, fluorescent intensity.

Article Snippet: Briefly, human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, were purchase from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Staining, Membrane, Activity Assay, ATP Bioluminescent Assay, Control

Journal: Oncotarget

Article Title: Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

doi: 10.18632/oncotarget.23453

Figure Lengend Snippet: Human osteoblast-like U2OS cells were treated with ERα siRNA for 24 and 48 h. Scrambled siRNA was administered to control cells as the negative standard. Levels of ERα were immunodetected ( A , top panel). Amounts of β-actin were analyzed as the internal standard (bottom panel). These protein bands were quantified and statistically analyzed (B) . After knocking-down ERα translation for 24 h, human osteoblasts were treated with estradiol for another 6 h. A quantitative PCR analysis was conducted to determine COX I mRNA expression (C) . Each value represents the mean ± SEM, n = 3. The symbols * and # indicate that a value significantly ( p < 0.05) differed from the control and estradiol-treated groups, respectively.

Article Snippet: Briefly, human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, were purchase from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Control, Real-time Polymerase Chain Reaction, Expressing

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ALK2-R206H-mediated pSMAD1/5/8 formation is induced by ACVR2B more efficiently than by ACVR2A. U2OS cells were transfected with vectors encoding HA-ALK2-R206H (or HA-ALK2-WT) alone or with myc-tagged ACVR2A, ACVR2B, or ACVR2B-KD. After 24 h, cells were starved (2 h, 1% serum) and stimulated (or not; control) with ActA (4 nM, 60 min, 37 °C). Cells were lysed, subjected to SDS--PAGE, and immunoblotted for pSMAD1/5/8, tSMAD1/5/8, and β-actin. As shown in , the cell-surface levels of the tagged receptors were similar and were not affected by the co-expressed receptors. ( A , B ) Representative blots of ActA signaling to pSMAD1/5/8. ( C , D ) Quantification of ActA-mediated pSMAD1/5/8 formation. The bands were visualized by ECL and quantified by densitometry. Data are mean ± SEM of the pSMAD1/5/8 over tSMAD1/5/8 ratio of 5 ( C ) or 4 ( D ) independent experiments. The value obtained for ActA-stimulated cells co-transfected with HA-ALK2-R206H and myc-ACVR2B was taken as 1. Asterisks show significant differences between the pairs indicated by brackets, using one-way ANOVA and Bonferroni post hoc test (*, p < 0.02; **, p < 4 × 10 −3 ; ***, p < 8 × 10 −4 ; n.s. = not significant).

Article Snippet: The U2OS human cell line was authenticated by STR analysis at Microsynth AG (Balgach, Switzerland).

Techniques: Transfection, Control, SDS Page

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ACVR2B is superior to ACVR2A in eliciting ALK2-R206H-mediated transcriptional activation of the SMAD1/5/8 pathway. U2OS cells were co-transfected with BRE-Luc and pRL-TK, together with HA-ALK2-R206H or HA-ALK2-WT (alone or together with myc-ACVR2A, myc-ACVR2B, or myc-ACVR2B-KD). These constructs were replaced by empty vector for control samples. After 17 h, cells were starved without serum (5 h) and stimulated (or not; control) with ActA (2 nM, 19 h). Relative Luminescence Units (RLU) are expressed as mean fold induction ± SEM (n = 4 independent experiments). The results were normalized for transfection efficiency using Renilla luminescence by the DLR luminescence assay. The value in untreated, unstimulated cells was taken as 1. The cell-surface levels of the tagged receptors were not altered by the co-expressed receptors . Asterisks show significant differences between the pairs indicated by the brackets, using one-way ANOVA and Bonferroni post hoc test (**, p < 1 × 10 −3 ; ***, p < 5 × 10 −4 ; ****, p < 10 −4 ; n.s. = not significant).

Article Snippet: The U2OS human cell line was authenticated by STR analysis at Microsynth AG (Balgach, Switzerland).

Techniques: Activation Assay, Transfection, Construct, Plasmid Preparation, Control, Luminescence Assay