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: American Journal of Cancer Research

Article Title: METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m 6 A modifications and splicing factors

doi:

Figure Lengend Snippet: Establishment of stable METTL3-knockdown cell lines using osteosarcoma U2OS cells. A. Using the CRISPR-Cas9 system, METTL3-knockdown cells were established (2-9 and 2-14) along with control cells (NT2 and NT3). METTL3-knockdown efficiency was confirmed by the decreased METTL3 and METTL14 protein expression using western blot. B. Levels of m6A in total RNA isolated from each stable cell line was measured via ELISA.

Article Snippet: U2OS cells were purchased from the Korean Cell Line Bank.

Techniques: Knockdown, CRISPR, Control, Expressing, Western Blot, Isolation, Stable Transfection, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m 6 A modifications and splicing factors

doi:

Figure Lengend Snippet: Enriched RBP motif in alternatively spliced genes. A. rMAPS captured a total of 19 RBPs that were enriched in alternatively spliced genes in METTL3-knockdown cells. Each RBP is represented with a different color based on the type of alternative splicing (alternative 3’ splice site: red, alternative 5’ splice site: blue, cassette exon: black, intron retention: green). B. The Pearson’s correlation coefficients between each RBP and METTL3 expression were analyzed in 12,839 TCGA pan-cancer patients. C. The m6A modification of the 3’UTR of SFPQ mRNA. The m6A sites in SFPQ mRNA was analyzed using SRAMP (https://www.cuilab.cn/sramp; left panel) and a red arrow indicates the predicted m6A site in the 3’UTR. MeRIP-qPCR analysis was performed to validate the predicted m6A site in U2OS cells (right panel). D. The mRNA expression levels of SFPQ and IGF2BP3 after knockdown of IGF2BP3. The mRNA expression levels were estimated by real-time PCR after transfecting 25 nM of IGF2BP3 siRNA for 48 h. E. Enrichment of IGF2BP3 in the 3’UTR of SFPQ mRNA. RIP-qPCR analysis was performed to detect binding of IGF2BP3 to the 3’UTR of SFPQ mRNA. F. The number of SFPQ peaks located within a distance of 1000 bp of alternatively spliced genes was counted.

Article Snippet: U2OS cells were purchased from the Korean Cell Line Bank.

Techniques: Knockdown, Alternative Splicing, Expressing, Modification, Real-time Polymerase Chain Reaction, Binding Assay

Journal: American Journal of Cancer Research

Article Title: METTL3 regulates alternative splicing of cell cycle-related genes via crosstalk between mRNA m 6 A modifications and splicing factors

doi:

Figure Lengend Snippet: Alternatively spliced genes in METTL3-knockdown HULEC-5a and A375 cells. A. Alternative splicing was analyzed using JUM in METTL3-knockdown HULEC-5a and A375 cells using shRNA. The number of alternatively spliced genes is represented using a bar graph (p-value < 0.05 being significant). B. Venn diagram showing the number of common alternatively spliced genes between U2OS, HULEC-5a and A375 cells. C. Top 10 enriched GO terms of alternatively spliced genes in HULEC-5a and A375 cells.

Article Snippet: U2OS cells were purchased from the Korean Cell Line Bank.

Techniques: Knockdown, Alternative Splicing, shRNA