Journal: bioRxiv

Article Title: Single-molecule spatial genomics reveals the multi-scale organization and plasticity of extrachromosomal DNA in glioblastoma

doi: 10.64898/2026.03.05.709911

Figure Lengend Snippet: Coordinated physical expansion, transcriptional activity, and deposition of transcriptional machinery underlie heterogeneity across single ecDNA molecules. (A.) Stratified scatter plot of the percentage of genes transcribed and the radius of gyration (Rg) for ecDNA. Each black dot represents the median per group, and the bar represents the 95% confidence interval. The red line marks the ordinary least squares line. N = 34, 349 ecDNA traces total. From left to right, each group has N = [248, 754, 1986, 3936, 6598, 8341, 7203, 4069, 1214] traces. (B.) Images of a representative cell showing ecDNA traces and immunostaining by mm-MERFISH of phosphorylated-RNA Polymerase II (Pol2S2), spliceosome (SC35), H3K27ac, Lamina A/C, and Nuclear Pore Complex (NUP98). (C.) Representative low Rg (top row) and high Rg (bottom row) single molecule traces in the context of multi-modal measurements. (D.) Correlation between radius of gyration and the normalized Pol2S2 brightness across ecDNA traces with different percentages of transcribed genes. Dots mark medians and error bars mark 95% confidence intervals. N is the same as A). (E.) Split violin plot comparisons of z scored differences across ecDNA from within the same cell vs. from different cells. N = 438,277 simulations. White circles mark medians. A two-sided Mann-Whitney U test was used to compute statistical significance. **** - p < 1e-4. (F.) Scatter plot comparing the mean transcription and global Pol2S2 deposition per cell, colored by the mean compaction per cell. N = 493 cells. (G.) Histogram of ecDNA copy number distribution. N = 493 cells. (H.) Violins comparing the mean transcription, compaction and Pol2S2 deposition between the low copy and high copy cells. A one-sided Mann-Whitney U test was used to compute statistical significance. (I.) Representative images of cells with low copy (left column) and high copy (right column). Top row: Single molecules represented by the rainbow configuration. Nucleus represented by DAPI stain (gray). Bottom row: immunofluorescence image of Pol2S2 (blue). Gray contour marks the cytoplasm boundaries.

Article Snippet: Primary antibodies used: Pol2S2: Rabbit Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody (Abcam, Cat# ab193468, RRID:AB_2905557) SC35: Mouse monoclonal [SC-35] to SC35 - Nuclear Speckle Marker (Abcam, Cat# ab11826, RRID:AB_298608) H3K27ac: Rabbit Histone H3K27ac antibody (pAb) (Active motif, Cat# 39133, RRID:AB_2561016) Lamina A/C: Mouse Lamin A/C (E-1) (Santa Cruz Biotechnology, Cat# sc-376248, RRID:AB_10991536) Nup98: NUP98 (C39A3) Rabbit mAb (Cell Signaling Technology, Cat# 2598, RRID:AB_2267700) Secondary antibodies used: Anti-mouse secondary: Alexa Fluor® 790 AffiniPure Donkey Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Labs, Cat# 715-655-150) Anti-rabbit secondary: Alexa Fluor® 647 AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Labs, Cat# 111-605-144)

Techniques: Activity Assay, Immunostaining, MANN-WHITNEY, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Single-molecule spatial genomics reveals the multi-scale organization and plasticity of extrachromosomal DNA in glioblastoma

doi: 10.64898/2026.03.05.709911

Figure Lengend Snippet: (A.) Mean relative brightness of active and repressive antibody marks across each 5-Kb segment on the ecDNA. Active marks: polII, SC35, H3K27ac. Repressive marks: Lamina, NUP98. (B.) Schematics defining lamina associated ecDNA. If the distance between the centroid of an ecDNA molecule to the nuclear lamina is smaller than 750nm (purple), the ecDNA is considered ‘lamina associated ecDNA’. (C.) Distribution of distance to lamina across all ecDNA molecules. (D.) Violin plots comparing the nascent transcription rate between lamina-associated ecDNA vs. not. White dot marks mean. A one-sided Mann-Whitney U test was used to test for statistical significance. (E.) Schematics defining speckle associated ecDNA. If the distance between the centroid of an ecDNA molecule to the nearest spliceosome is smaller than 750nm (brown), the ecDNA is considered ‘speckle associated ecDNA. (F.) Distribution of distance to speckle across all ecDNA molecules. (G.) Violin plots comparing the nascent transcription between speckle-associated ecDNA vs. not. White dot marks mean. (H.) Histogram of the number of micronuclei per cell. Bar represents the standard error of mean. N = 1193 cells. (I.) Representative images of cells with micronuclei (white arrow). #1: micronucleus with ecDNA and nascent EGFR transcription. #2: micronucleus with only ecDNA and no nascent EGFR. #3: micronucleus without ecDNA and nascent EGFR. (J.) Stacked bar graph showing the status of containing ecDNA and nascent EGFR in cells with micronuclei. N = 238 cells. (K.) Scatter plot showing the average bursting rate per gene across all measured ecDNA molecules when residing inside the main nucleus vs inside the micronucleus. Gray line marks y = x. (L.) Fraction of cells with micronuclei within different ecDNA copy number bins. Black dot represents the median per group, and the bar represents the 95% confidence interval. The red line represents the fitted OLS line.

Article Snippet: Primary antibodies used: Pol2S2: Rabbit Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody (Abcam, Cat# ab193468, RRID:AB_2905557) SC35: Mouse monoclonal [SC-35] to SC35 - Nuclear Speckle Marker (Abcam, Cat# ab11826, RRID:AB_298608) H3K27ac: Rabbit Histone H3K27ac antibody (pAb) (Active motif, Cat# 39133, RRID:AB_2561016) Lamina A/C: Mouse Lamin A/C (E-1) (Santa Cruz Biotechnology, Cat# sc-376248, RRID:AB_10991536) Nup98: NUP98 (C39A3) Rabbit mAb (Cell Signaling Technology, Cat# 2598, RRID:AB_2267700) Secondary antibodies used: Anti-mouse secondary: Alexa Fluor® 790 AffiniPure Donkey Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Labs, Cat# 715-655-150) Anti-rabbit secondary: Alexa Fluor® 647 AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Labs, Cat# 111-605-144)

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Novel single molecule imaging approaches reveal structure-function alterations to the nuclear pore complex in early C9ORF72 -associated TDP-43 proteinopathy

doi: 10.64898/2026.02.17.706337

Figure Lengend Snippet: Neuroblastoma cell line SH-SY5Y untreated (control) or with acute treatment (1hr) of polypeptide GR20 (10µM), as per single molecule tracking experiments, and then immunostained with dye-conjugated antibodies for the NPC components Nup98 and Nup50. a) Example fluorescence image of improved immunostaining detection of individual NPC spot detection of AF647-labelled Nup50 (magenta) and AF488-labelled Nup98 (green) acquired using HILO microscopy, with enlarged view and respective definition of Nup50/98 positions; scale bars are 5 μm and 500nm for enlarged views. b) Representative fluorescence images of untreated (control) and polyGR-treated cells with output spot detection for Nup98 and Nup50 within deep learning nuclear envelope central boundary definition (yellow dashed line); scale bars are 5 μm. c) Schematics of process for paired particle localisation and alignment averaging for Nup98 positioning (green) in reference to Nup50 (magenta). d) Output density heatmap of Nup98 positioning in reference to Nup50 from paired localisation analysis for experimental data of control and polyGR-treated cells with comparative best fit simulation heatmap, with values for mean radii – here, indicating the distance between Nup50 and Nup98 - (±SD) and structural fidelity between experimental and best fit simulation data using the structural similarity index (SSIM) below. e) Best fit simulated distribution of Nup50 (magenta) and Nup98 (green) – now reflecting their effective position in a nuclear pore complex - used to generate heat map in (d) with lines for respective median Nup98 and Nup50 diameter in control and polyGR treated conditions. (f) Quantification for determined median diameter (±95% CI) of Nup98 and Nup50 under control and polyGR-treated conditions using paired localisation and diameter simulation analysis. Statistical testing: unpaired Mann Whitney; ****p<0.0001; N>250; raw numerical data in Source Data File for Fig. 3.

Article Snippet: Cells were washed three times with PBS and blocked with 5% BSA in PBS for 1 hour at room temperature, then incubated overnight at 4°C with conjugated antibodies (1:50) freshly diluted in blocking buffer, including anti-Nup50 (G-4) Alexa Fluor 647–conjugated antibody (sc-398993 AF647, Santa Cruz Biotechnology) and anti-Nup98 (C-5) Alexa Fluor 488–conjugated antibody (sc-74578 AF488, Santa Cruz Biotechnology).

Techniques: Control, Fluorescence, Immunostaining, Microscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: Novel single molecule imaging approaches reveal structure-function alterations to the nuclear pore complex in early C9ORF72 -associated TDP-43 proteinopathy

doi: 10.64898/2026.02.17.706337

Figure Lengend Snippet: Nucleocytoplasmic transport (beige) in physiological and C9ORF72 polyGR (10 µM) disease conditions with width and density of arrow denote transport passage and molecular density, and percentage success rate of tracks. Nuclear pore structure schematic in grey in cross section (upper) and nuclear side view (lower) with Nup98 positions denoted in green and Nup50 in magenta, arrows highlighting change in organisation upon exposure to polyGR, and values for simulated diameter measurement. Associated change in nucleocytoplasmic distribution of disease-associated protein TDP-43 shown in blue. Created in BioRender.

Article Snippet: Cells were washed three times with PBS and blocked with 5% BSA in PBS for 1 hour at room temperature, then incubated overnight at 4°C with conjugated antibodies (1:50) freshly diluted in blocking buffer, including anti-Nup50 (G-4) Alexa Fluor 647–conjugated antibody (sc-398993 AF647, Santa Cruz Biotechnology) and anti-Nup98 (C-5) Alexa Fluor 488–conjugated antibody (sc-74578 AF488, Santa Cruz Biotechnology).

Techniques:

Journal: bioRxiv

Article Title: Quantitative Expansion Microscopy for In Situ Estimation of Endogenous Target Abundance

doi: 10.64898/2026.01.18.700178

Figure Lengend Snippet: (a) The qExM framework targets endogenous proteins or protein complexes using pairs of antibodies against non-overlapping epitopes, leveraging western blot-effective antibodies enabled by expansion microscopy’s improved labeling efficiency. (b) Schematic representation of the labeling outcome categories in a qExM experiment. The total population of targets can be labeled by either antibody A (blue), antibody B (red), both antibodies (dual-labeled), or remain unlabeled. Each label provides a random subset of the total population, allowing assessment of the other label’s efficiency. (c) Mathematical formulation of the estimators used in qExM to calculate labeling efficiency of each antibody and the total abundance of target proteins, derived from the Chapman capture-recapture method. (d , e , f) Fluorescence microscopy images of U2OS cells prepared using three expansion microscopy protocols and collected and displayed with identical parameters: d: chemical fixation with glycidyl methacrylate anchoring (ChemGMA), e: cryo-fixation with formaldehyde/acrylamide anchoring (CryoFAA), and f: cryo-fixation with glycidyl methacrylate anchoring (CryoGMA). Cells were immunolabeled for Nup96-GFP (cyan), mitochondrial MT-CO1 (magenta), and α-tubulin (yellow). Scale bar 5 µm. (g) Signal to Noise measurements comparing the mitochondrial MT-CO1 signal across the three expansion microscopy protocols.

Article Snippet: In this study we used the following primary antibodies: for CI: NDUFS2 (Abcam ab192022), ND2 (Sigma MABS2047), for CIII: UQCRCP1 (Abcam ab14746), UQCRFS1/RISP (Abcam ab11925), for CIV: MT-CO1 (Abcam ab14705), MT-CO2 (Abcam ab79393), GFP (Abcam ab6673), Nup96 (ProteinTech-12329-1-AP), Nup54 (Sigma HPA035929), ELYS (Sigma HPA031658), P5CS (Proteintech 17719-1-AP, 68184-1-IG) All antibodies were used at a dilution of 1:100, except ELYS which was used at a concentration of 1:50.

Techniques: Western Blot, Labeling, Formulation, Derivative Assay, Fluorescence, Microscopy, Immunolabeling

Journal: bioRxiv

Article Title: Quantitative Expansion Microscopy for In Situ Estimation of Endogenous Target Abundance

doi: 10.64898/2026.01.18.700178

Figure Lengend Snippet: (a) qExM STED images of Nup96-mEGFP U2OS cells with mEGFP (red) and Nup96 (blue) labeling. (b) qExM STED images of Nup96-mEGFP U2OS cells with mEGFP (red) and Nup54 (blue) labeling. (c) qExM STED images of Nup96-mEGFP U2OS cells with mEGFP (red) and ELYS (blue) labeling. Smaller images in a-c show individual nuclear pores at higher magnification, demonstrating the characteristic ring-like arrangement of NPC subunits. (d) Structural model of the human nuclear pore complex highlighting the positions of Nup96-mEGFP (green), Nup54 (yellow), and ELYS (magenta) within the 8-fold symmetrical arrangement. (e) Image analysis workflow showing raw data (Image), segmentation mask (Segmentation), instance identification (Centers), and colocalization analysis (Colocalization). (f) Labeling efficiency estimates from qExM mEGFP and each target protein. “Direct Counting” refers to the labeling efficiency defined by the number of observed target per nuclear pore, knowing that there should be 8 total targets. No statistical significance was found between groups (mEGFP p = 0.23, Nup96 p= 0.65, Nup54 p= 0.28, ELYS p = 0.19) (g) Estimated abundance of protein subunits for each experiment, showing means of 7.4, 7.0, and 8.7 for Nup96, Nup54, and ELYS, respectively, closely matching the expected 8-fold symmetry. Scale bars: 500 nm (overview images) and 50 nm (insets). 3 experimental replicates were made for each imaging pair. 45 images of 1365 NPC wlabeled ith Nup96-mEGFP with Nup96. 41 images of 1465 NPC labeled with Nup96-mEGFP with Nup54. 32 images of 1118 NPC labeled with Nup96-mEGFP with ELYS.

Article Snippet: In this study we used the following primary antibodies: for CI: NDUFS2 (Abcam ab192022), ND2 (Sigma MABS2047), for CIII: UQCRCP1 (Abcam ab14746), UQCRFS1/RISP (Abcam ab11925), for CIV: MT-CO1 (Abcam ab14705), MT-CO2 (Abcam ab79393), GFP (Abcam ab6673), Nup96 (ProteinTech-12329-1-AP), Nup54 (Sigma HPA035929), ELYS (Sigma HPA031658), P5CS (Proteintech 17719-1-AP, 68184-1-IG) All antibodies were used at a dilution of 1:100, except ELYS which was used at a concentration of 1:50.

Techniques: Labeling, Imaging