time lapse imaging  (Olympus)

Journal: Microsystems & Nanoengineering

Article Title: High-fidelity bioassembly of organoids and spheroids using inertial droplet microfluidics for precision oncology and tumor microenvironment modeling

doi: 10.1038/s41378-026-01244-x

Figure Lengend Snippet: The automated OsciSphere bioassembly platform and workflow. a 3D model of the OsciSphere platform. The inset shows the core 8-channel droplet generator, which integrates an oscillator, syringe pump, disposable pipette tips, and a cooling block to maintain Matrigel in a liquid state. b Photograph of the automated workstation layout. c Schematic of the OsciSphere workflow. 1) Cells are suspended in Matrigel at 4 °C. 2) The platform’s droplet generator dispenses uniform droplets into a 96-well plate containing a biphasic carrier oil/culture medium overlay. 3) Droplets are solidified at 37 °C for 10 min. 4) Solidified microspheres are transferred into the underlying medium using an antistatic gun or gentle agitation. 5) This automated process yields arrays of µMCTs or µTDOs ready for high-throughput screening applications. d Time-lapse imaging (left) demonstrates the “pull-break-sediment” cycle of droplet formation. The platform reliably produces approximately 100 uniform 30 nL droplets from only 3 µL of Matrigel (right), achieving excellent size uniformity. Scale bar, 500 µm. e Encapsulation uniformity demonstrated by fluorescent beads within Matrigel microspheres (60.9 ± 5.1 beads per droplet, CV = 7.67%, n = 28). f Platform versatility is shown by forming uniform microspheres from agarose and HAMA. g Radar chart comparing OsciSphere’s performance metrics (e.g., uniformity, throughput, automation) against conventional 3D culture methods. OsciSphere enables rapid and uniform 3D culture, including ( h ) µMCTs and i µTDOs. j Dome TDOs exhibit significant spatial heterogeneity, leading to diffusion-limited regions and apoptotic cores by Day 3

Article Snippet: Growth and morphology were monitored via time-lapse phase-contrast imaging (Incucyte S3, Sartorius, Göttingen, Germany).

Techniques: Transferring, Blocking Assay, Gentle, High Throughput Screening Assay, Imaging, Encapsulation, Diffusion-based Assay

Journal: Microsystems & Nanoengineering

Article Title: High-fidelity bioassembly of organoids and spheroids using inertial droplet microfluidics for precision oncology and tumor microenvironment modeling

doi: 10.1038/s41378-026-01244-x

Figure Lengend Snippet: OsciSphere operates in a deterministic regime to produce uniform µMCTs that recapitulate key physiological tumor features. a Scaffold-free ULA culture (Day 4) yields a multimodal population, with a single large central spheroid and numerous highly variable satellite aggregates. b Conventional Matrigel dome culture (Day 3) exhibits significant spatial heterogeneity driven by diffusion gradients, with smaller spheroids in the nutrient-poor center and larger ones at the periphery. c OsciSphere-generated µMCTs (~600 cells/droplet, Day 3) exhibit structural isotropy and consistency. d Quantification of spheroid diameters confirms the superior monodispersity of µMCTs ( n = 100, CV = 4.2%) compared to the high variability of ULA ( n = 244) and dome ( n = 237) cultures (mean ± SD, **** p < 0.0001). e Bright-field time-lapse shows rapid self-assembly of compact HCT116 spheroids by Day 1. f Live/Dead staining at 72 h confirms high cell viability. g Histological analysis (H&E) reveals a dense 3D tissue architecture, with immunohistochemistry for Ki67 confirming robust proliferative activity within the spheroid. h Optimization of formation efficiency reveals a critical density threshold at 600–900 cells per microsphere, achieving 99.6% successful formation by Day 2 ( n = 10, mea n ± SD, red star indicates the optimal 600-cell condition). i Immunofluorescence (IF) of 2D HCT116 cultures shows basal expression of Vimentin and N-cadherin. Scale bar, 10 µm. j µMCTs display pronounced, organized expression of mesenchymal markers (Vimentin, N-cadherin), indicative of EMT. Scale bar, 50 µm. k This invasive phenotype is validated by RT-qPCR, showing significant upregulation of key EMT-associated genes ( N-cadherin , Snail , Slug ) and cancer stem cell-associated genes ( Sox2 , Oct4 ) in µMCTs ( n = 3, mea n ± SD, **** p < 0.0001). l µMCTs exhibit a physiologically relevant reduced proliferation rate compared to 2D cultures, mimicking in vivo tumor kinetics ( n = 5, mean ± SEM, **** p < 0.0001). m Flow cytometry analysis reveals elevated intracellular ROS levels in µMCTs (51.2%) versus 2D cultures (30.2%), consistent with the establishment of metabolic gradients and a hypoxic tumor microenvironment. Statistical significance was analyzed by using one-way analysis of variance (ANOVA)

Article Snippet: Growth and morphology were monitored via time-lapse phase-contrast imaging (Incucyte S3, Sartorius, Göttingen, Germany).

Techniques: Diffusion-based Assay, Generated, Staining, Immunohistochemistry, Activity Assay, Immunofluorescence, Expressing, Quantitative RT-PCR, In Vivo, Flow Cytometry

Journal: Microsystems & Nanoengineering

Article Title: High-fidelity bioassembly of organoids and spheroids using inertial droplet microfluidics for precision oncology and tumor microenvironment modeling

doi: 10.1038/s41378-026-01244-x

Figure Lengend Snippet: OsciSphere-derived µTDOs resolve diffusion limitations to drive superior growth and maturation. a Comparative time-lapse microscopy reveals the impact of culture geometry. Conventional Matrigel domes exhibit severe spatial heterogeneity: while organoids at the nutrient-rich “Edge” grow, those in the diffusion-limited “Core” undergo apoptosis (red arrow) by Day 3. In contrast, OsciSphere µTDOs (bottom row) exhibit uniform, necrosis-free growth independent of spatial position. b Quantification of projected surface area demonstrates significantly accelerated expansion kinetics for µTDOs compared to dome cultures ( n = 58, mean ± SEM, **** p < 0.0001). c Morphogenic analysis reveals enhanced maturation in the OsciSphere format, with a significantly higher frequency of multi-budded organoids observed by Day 2 ( n = 58, mea n ± SEM). d Viability imaging (Calcein-AM/PI) on Day 3 confirms that µTDOs maintain high cell survival without the central necrosis observed in static hydrogel cultures. e Histological validation against native murine intestine. H&E staining demonstrates that µTDOs recapitulate the polarized crypt-villus architecture of the in vivo epithelium. Immunohistochemistry for Ki67 (brown) confirms the preservation of active proliferative zones in the crypt domains of both µTDOs and native tissue. Statistical significance was analyzed by using one-way ANOVA

Article Snippet: Growth and morphology were monitored via time-lapse phase-contrast imaging (Incucyte S3, Sartorius, Göttingen, Germany).

Techniques: Derivative Assay, Diffusion-based Assay, Time-lapse Microscopy, Imaging, Biomarker Discovery, Staining, In Vivo, Immunohistochemistry, Preserving

Journal: Microsystems & Nanoengineering

Article Title: High-fidelity bioassembly of organoids and spheroids using inertial droplet microfluidics for precision oncology and tumor microenvironment modeling

doi: 10.1038/s41378-026-01244-x

Figure Lengend Snippet: OsciSphere enables high-fidelity modeling of the patient-specific tumor-immune microenvironment . a Schematic of the precision immuno-oncology workflow. Patient-derived hCRC tissues are processed into µPDOs via OsciSphere, creating physically permissive scaffolds that support autologous PBMC infiltration. b Bright-field micrographs of source PDO lines established from three independent hCRC patients. c Transcriptomic validation confirms high-fidelity modeling: established PDOs (O) maintain strong gene expression correlations ( R > 0.87) with their matched parental tumor tissue (T). d Genomic profiling demonstrates that µPDOs preserve the patient-specific mutational landscape across a panel of key oncogenic drivers. e Histological comparison reveals that µPDOs (bottom) recapitulate the native tumor architecture (top). H&E staining shows comparable morphology, while Ki67 staining confirms the maintenance of robust proliferative zones in both the parent tissue (brown) and the µPDOs (red). f Failure mode of conventional culture: endpoint imaging reveals that the dense, large-volume Matrigel dome acts as a physical barrier, excluding PBMCs (red) from the tumor core. g OsciSphere overcomes the barrier effect: time-lapse imaging captures the active migration of PBMCs (red) into the µPDO (dashed circle), facilitating sustained tumor-immune interactions (yellow arrows) over 72 h. Scale bar, 100 µm. h Flow cytometry analysis quantifies IFN-γ levels. i Quantif i cation of the mean fluorescence intensity of CD8 + IFN-γ + PBMCs from PBMC-only, PBMCs + µPDOs, PBMC + µPDOs + sintilimab groups ( n = 3, mea n ± SD; * p < 0.05, ** p < 0.01, ns, not significant). Statistical significance was analyzed by using one-way ANOVA

Article Snippet: Growth and morphology were monitored via time-lapse phase-contrast imaging (Incucyte S3, Sartorius, Göttingen, Germany).

Techniques: Derivative Assay, Biomarker Discovery, Gene Expression, Comparison, Staining, Imaging, Migration, Flow Cytometry, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: DYN-1 regulates SPD-2 and PLK-1 localization and mitotic spindle pole organization

doi: 10.1091/mbc.E25-07-0337

Figure Lengend Snippet: PLK-1 persists abnormally at centrosomes during mitotic progression in dyn-1(RNAi) embryos. (A) Time-lapse analysis of different mitotic stages in control RNAi and dyn-1(RNAi) embryos. White arrows: anterior centrosome-localized PLK-1::sfGFP. (B) Percentage of centrosomes retaining PLK-1 at each stage. Fisher's Exact Test; ns = not significant; n = centrosomes analyzed. Experimental replicates: three for all assays. All fluorescence images include a 10 µm scale bar.

Article Snippet: Time-lapse imaging was performed using the cellSens software (Olympus America) by taking 1.5 μm Z-stacks every ∼75 s at a 60X magnification with a 1.42 numerical aperture (NA) objective using a Prime 95B CMOS camera.

Techniques: Control, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: DYN-1 regulates SPD-2 and PLK-1 localization and mitotic spindle pole organization

doi: 10.1091/mbc.E25-07-0337

Figure Lengend Snippet: DYN-1 depletion disrupts Intercellular Bridge (IB) assembly. (A) Time-lapse images of mCherry::tubulin showing normal IB formation in control RNAi embryos (top panel) and abnormal (middle panel) or missing (bottom panel) IBs in dyn-1(RNAi) embryos. White arrows: IB (B) Classification of IB morphology (normal, abnormal, absent). n = embryos analyzed. Experimental replicates: three for all assays. All fluorescence images include a 10 µm scale bar.

Article Snippet: Time-lapse imaging was performed using the cellSens software (Olympus America) by taking 1.5 μm Z-stacks every ∼75 s at a 60X magnification with a 1.42 numerical aperture (NA) objective using a Prime 95B CMOS camera.

Techniques: Control, Fluorescence

Journal: bioRxiv

Article Title: Spindle pole proteins confine chromosomes to ensure their expulsion during female meiosis

doi: 10.64898/2026.03.24.713942

Figure Lengend Snippet: (A) Time-lapse imaging of in utero oocytes during meiosis I (left) and II (right). Each spindle pole protein was endogenously labeled with GFP or mNeonGreen (green). mCherry::tubulin was co-visualized to monitor spindle shortening and rotation during the anaphase transition (magenta). Spindle shortening was set as 0 s. (B) Diagram of ex utero oocytes forming the first polar body facing toward the microscope objective (top). GFP::ZYG-9 (green) encapsulated chromosomes indicated by mCherry::H2B (magenta) during the anaphase transition (middle). Enlarged images of each signal show voids of GFP::ZYG-9 signals occupied by chromosomes at the anaphase transition (bottom). Onset of chromosome segregation was set as 0 s. All scale bars are 5 µm.

Article Snippet: Time-lapse images were taken using an inverted Nikon ECLIPSE Ti2 equipped with Yokogawa Spinning Disk Field Scanning Confocal System (CSU-W1), piezo Z stage, and iXon Ultra EMCDD camera (Andor) controlled by NIS-Elements software.

Techniques: Imaging, In Utero, Labeling, Microscopy

Journal: bioRxiv

Article Title: Spindle pole proteins confine chromosomes to ensure their expulsion during female meiosis

doi: 10.64898/2026.03.24.713942

Figure Lengend Snippet: (A) Super-resolution microscopy of ex utero C. elegans oocytes expressing GFP::ZYG-9 (spindle poles, green) and mCherry::H2B (chromosomes, magenta) in meiosis I (top). Chromosome regions are cropped (bottom). (B) Total fluorescence of spindle-associated GFP::ZYG-9. (C) Distance between the spindle pole and the chromosome bivalent. Data are normalized to maxima and represent mean ± 95% C.I. (n= 10). (D) Super-resolution microscopy of endogenously labeled spindle pole proteins (green) and chromosomes (Sir-DNA, magenta) during chromosome segregation in anaphase (left). Line scans of normalized signal intensity across the long axis of the spindle (right). (E) Time-lapse imaging of spindle pole proteins after nocodazole treatment. (F) Normalized total fluorescence of each spindle pole proteins in (E). Data represent mean ± 95% C.I. of GFP::ASPM-1 (n= 6), CMD-1::GFP (n= 8), LIN-5::mNeonGreen (n= 6), GFP::TAC-1 (n= 5), GFP::ZYG-9 (n= 5). (G) Super-resolution microscopy of ex utero oocytes treated with 20 µM nocodazole for 15 min (left). Enlarged images shown on the right. All scale bars are 5 µm.

Article Snippet: Time-lapse images were taken using an inverted Nikon ECLIPSE Ti2 equipped with Yokogawa Spinning Disk Field Scanning Confocal System (CSU-W1), piezo Z stage, and iXon Ultra EMCDD camera (Andor) controlled by NIS-Elements software.

Techniques: Super-Resolution Microscopy, Expressing, Fluorescence, Labeling, Imaging

Journal: bioRxiv

Article Title: Spindle pole proteins confine chromosomes to ensure their expulsion during female meiosis

doi: 10.64898/2026.03.24.713942

Figure Lengend Snippet: (A) Co-visualization of GFP::ZYG-9 (green) and mCherry::tubulin (magenta) during metaphase I (left). Morphology of the spindle indicated by the tubulin signal (right). (B) Time-lapse imaging of GFP::ZYG-9 (green) and mCherry::histone H2B (magenta) throughout anaphase I. Yellow allow heads indicate regressed chromosomes from the polar body to the oocyte cytoplasm. (C-G) Normalized total fluorescence of spindle-associated GFP::ZYG-9 (C, E), lateral distance of the farthest chromosomes (D, F), and population of oocytes with regressed chromosomes (G) during anaphase I and II. Data are mean ± 95% C.I. (WT, n= 20; 5RG, n= 23). (H) Co-visualization of GFP::ZYG-9 (green) and mCherry::histone H2B (magenta) during interphase (left) and mitosis (right) in 2-cell stage embryos. Yellow arrowheads indicate extra nucleus and ectopic chromosomes. (I) Population of 1-4 cell stage embryos with abnormal chromosomes in (H). Data represent mean ± SD from four independent experiments (WT, n= 63; 5RG, n= 72). p-value was determined by unpaired t-test. (J) Number of hatched eggs from single mother expressing GFP::ZYG-9 WT (n= 8) or GFP::ZYG-9 5RG (n= 8). Data represent mean + SD. p-value was determined by unpaired t-test. (K) Time-lapse imaging of ex utero oocytes treated with 20 µM nocodazole. Yellow arrows indicate de-clustered chromosomes. (L-P) Quantification of total fluorescence of GFP::ZYG-9 condensate (L), distance between the farthest chromosomes (M), number of de-clustered chromosomes (N), normalized mCherry::Histone H2B intensity (O), and chromosome size (P) after nocodazole treatment. Data are mean ± 95% C.I. (WT, n= 8; 5RG, n= 10). All scale bars are 5 µm.

Article Snippet: Time-lapse images were taken using an inverted Nikon ECLIPSE Ti2 equipped with Yokogawa Spinning Disk Field Scanning Confocal System (CSU-W1), piezo Z stage, and iXon Ultra EMCDD camera (Andor) controlled by NIS-Elements software.

Techniques: Imaging, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Viral rewiring of APC/C-CDC20 drives Aurora B hyperubiquitination, mitotic regression and polyploidy

doi: 10.64898/2026.02.13.705602

Figure Lengend Snippet: (A) Cleavage furrow regression in AdV-infected mitotic cells leads to polyploidy. Representative time-lapse images of an AdV-C2-wt-infected HeLa-H2B-mCherry cell undergoing mitosis, showing cytokinesis, cleavage furrow ingression, midbody formation, subsequent furrow regression, and nuclear congression into a single polyploid nucleus. Scale bar, 10 µm. (B) Nuclear envelope remodeling in AdV-induced polyploid cells. Representative confocal images of HeLa cells expressing mRFP-LAP2B show smooth nuclear envelope morphology in non-infected cells, whereas regressed cells generated during AdV infection display pronounced nuclear envelope distortions, including lobulation and membrane invaginations. Scale bar, 10 µm. (C) Viral dose-dependent induction of cleavage furrow regression following AdV5-wt infection. Grey bars indicate the percentage of regressed divisions, and red symbols denote the total number of cell divisions analyzed per condition. Significance was calculated using ordinary one-way ANOVA. **** = p<0.0001. (D) Temporal accumulation of regressed divisions during lytic AdV5-wt infection. Single-cell division times are shown for normal and regressed divisions in non-infected and infected cells. (E) E4orf4 is required for efficient cleavage furrow regression independently of its PP2A-binding function. Deletion of E4orf4 markedly reduces regression frequency, whereas the PP2A-binding-deficient mutant AdV (AdV-C5-E4orf4-R81F84A) retains regression activity. Significance was calculated using one-way ANOVA. ** = p<0.01, ns = p>0.05. (F) E4orf4 expression is sufficient to induce cleavage furrow regression in the absence of productive infection. Time-to-division analysis shows increased regression events upon expression of GFP- or HA-tagged E4orf4 compared with non-infected and vector controls. (G) DNA content analysis of primary human bronchial airway (HBA) basal cells mock-treated or infected with AdV-C5-GFP-E4orf4 at low (MOI 0.3) or moderate (MOI 3) multiplicity. Cells were infected for 48h, followed by fixation, fluorescent microscopy and quantification. Representative histograms show DNA content distributions with G1 peaks, 2NG1, 4NG1, and >4NG1 populations. Quantification indicates a dose-dependent increase in 4N and >4N G1 cells upon infection. Mean ± SD from 3 independent experiments is shown. N = 23,000 nuclei per condition. (H) Confocal imaging of AdV-C5-GFP-E4orf4-infected primary HBA basal cells stained with Hoechst 33342 (DNA) and wheat germ agglutinin (WGA) to delineate cell boundaries. Z-sections illustrate enlarged, irregular nuclei within single-cell boundaries, consistent with polyploidization following cytokinesis failure. Scale bar, 10µm. (I) Time-lapse imaging of AdV-C5-GFP-E4orf4-infected primary HBA basal cells showing progression through mitosis, followed by cleavage furrow regression, binucleated cells. GFP-E4orf4 signal marks infected cells. Dashed outlines indicate cell boundaries and time is shown as hours post infection. Scale bar, 10µm.

Article Snippet: To find if regressing cells accumulate more viral proteins than non-regressing cells during AdV infection, we performed live time-lapse imaging of AdV-infected cells lines (HeLa FUCCI, HeLa H2B-mCherry, or HeLa WT) were grown on 96-well imaging plates (Greiner Biosciences and CellVis glass bottom plates), infected with indicated AdV strain, followed by timelapse imaging with high-content microscope.

Techniques: Infection, Expressing, Generated, Membrane, Single Cell, Binding Assay, Mutagenesis, Activity Assay, Plasmid Preparation, Microscopy, Imaging, Staining

Journal: bioRxiv

Article Title: Viral rewiring of APC/C-CDC20 drives Aurora B hyperubiquitination, mitotic regression and polyploidy

doi: 10.64898/2026.02.13.705602

Figure Lengend Snippet: (A) Polyploid cells generated by cleavage furrow regression during AdV infection remain viable and progress through the cell cycle. Time-lapse imaging of AdV-infected HeLa FUCCI cells shows that regressed cells do not undergo cell death but instead complete mitosis, re-enter interphase, and reach the G1/S boundary, similar to non-regressing infected cells. Time shown as hours post infection. Scale bar, 100 µm. (B) Quantification of cell cycle distribution in non-infected (NI) and AdV-infected HeLa FUCCI cells 50 h post infection. Stacked bar plots show the proportion of cells in G1, G1/S, and G2/M phases, with total cell numbers (N) indicated above each condition. Nor: not regressed, Reg: regressed. (C) Correlative time-lapse and fixed-cell immunofluorescence analysis of viral late protein expression in regressed versus normally dividing cells. HeLa FUCCI cells infected with AdV-C5-wt (MOI = 5) were tracked by live imaging to identify regressing and non-regressing divisions, followed by fixation and immunostaining for the viral late protein VI. Fixed-cell images were computationally aligned to the final time point of the time-lapse sequence to assign protein VI intensity to individual tracked cells. Scale bar, 20 µm. (D) Quantification of integrated viral protein VI intensity per cell for non-infected cells, infected non-regressing cells, infected regressing cells, and randomly selected infected cells. Regressed cells show significantly elevated levels of viral late protein expression compared to non-regressing infected cells. Significance was calculated using ordinary one-way ANOVA. ** = p<0.01, *** = p<0.001, **** = p<0.0001. Rd: random; Nor: not regressed, Reg: regressed.

Article Snippet: To find if regressing cells accumulate more viral proteins than non-regressing cells during AdV infection, we performed live time-lapse imaging of AdV-infected cells lines (HeLa FUCCI, HeLa H2B-mCherry, or HeLa WT) were grown on 96-well imaging plates (Greiner Biosciences and CellVis glass bottom plates), infected with indicated AdV strain, followed by timelapse imaging with high-content microscope.

Techniques: Generated, Infection, Imaging, Immunofluorescence, Expressing, Immunostaining, Sequencing

Journal: bioRxiv

Article Title: Viral rewiring of APC/C-CDC20 drives Aurora B hyperubiquitination, mitotic regression and polyploidy

doi: 10.64898/2026.02.13.705602

Figure Lengend Snippet: (A) Schematic of the workflow used to define the nucleo-cytoplasmic E4orf4 interactome by affinity purification-mass spectrometry. Mock- or AdV-C5-GFP-E4orf4-infected cells were fractionated into nuclear and cytoplasmic compartments, followed by GFP-Trap–based affinity enrichment and mass spectrometric identification of interacting proteins. (B) Immunoblot analysis validating nuclear and cytoplasmic fractionation during AdV-C5-GFP-E4orf4 infection of HeLa cells. GAPDH and Lamin A serve as cytoplasmic and nuclear markers, respectively, confirming enrichment of GFP-E4orf4 in the cytoplasmic fraction. (C) Volcano plot showing proteins enriched in cytoplasmic versus nuclear GFP-E4orf4 interactomes identified by mass spectrometry. Previously reported E4orf4 interactors are highlighted, with CDC20 emerging as a significantly enriched cytoplasmic binding partner. (D) Co-immunoprecipitation of CDC20 with E4orf4 in infected cells. A549 cells expressing CDC20-mScarlet were infected with AdV-C5-GFP-E4orf4, and GFP-Trap immunoprecipitates were analyzed by immunoblotting, demonstrating enrichment of CDC20 in GFP-E4orf4 complexes. (E) Quantification of CDC20 enrichment in GFP-E4orf4 immunoprecipitates relative to input, showing significant association upon AdV infection. (F) Quantification of cleavage furrow regression following CDC20 depletion with RNAi. Time-lapse imaging of AdV-C5-wt–infected A549-Sec61B-GFP cells transfected with non-targeting (NT) or CDC20-specific siRNA shows a significant reduction in the percentage of regressed divisions upon CDC20 knockdown. Each point represents approximately 50 cell divisions, in total 600 cell divisions each condition. Right panel shows reduction in CDC20 mRNA levels following siRNA transfection using quantitative RT-PCR. (G) In vitro GST pull-down assay demonstrating direct binding between CDC20 and E4orf4. GST-CDC20 immobilized on glutathione beads was incubated with purified FLAG–E4orf4 and full-length E1A, followed by SDS-PAGE analysis of input, eluate, and flow-through fractions. (H) Immunoblot validation of GST pull-down assays shown in (G), probing for CDC20 (anti-HA), E4orf4 (anti-FLAG), and E1A (anti-M58), confirming a direct and specific interaction between CDC20 and E4orf4.

Article Snippet: To find if regressing cells accumulate more viral proteins than non-regressing cells during AdV infection, we performed live time-lapse imaging of AdV-infected cells lines (HeLa FUCCI, HeLa H2B-mCherry, or HeLa WT) were grown on 96-well imaging plates (Greiner Biosciences and CellVis glass bottom plates), infected with indicated AdV strain, followed by timelapse imaging with high-content microscope.

Techniques: Affinity Purification, Mass Spectrometry, Infection, Western Blot, Fractionation, Binding Assay, Immunoprecipitation, Expressing, Imaging, Transfection, Knockdown, Quantitative RT-PCR, In Vitro, Pull Down Assay, Incubation, Purification, SDS Page, Biomarker Discovery

Journal: bioRxiv

Article Title: Viral rewiring of APC/C-CDC20 drives Aurora B hyperubiquitination, mitotic regression and polyploidy

doi: 10.64898/2026.02.13.705602

Figure Lengend Snippet: (A) Cell cycle-stage-specific distribution of endogenous CDC20 in HeLa FUCCI cells. Asynchronously cycling HeLa FUCCI cells were fixed and immunostained with an anti-CDC20 antibody. Cells were classified into sub-G1, G1, S, and G2/M phases using a semi-supervised machine-learning classifier (CellProfiler) based on DNA content and FUCCI signals, and CDC20 intensities were mapped onto DNA content-based cell cycle plots. Equal numbers of cells (n = 2,000 per bin) were grouped by increasing CDC20 intensity. (B) Quantification of mean CDC20 intensity per cell across cell cycle stages. Scatter plots show single-cell CDC20 intensities in G1, S, and G2/M phases, revealing highest CDC20 abundance in G2/M cells. Red lines indicate median values; Significance was calculated using ordinary one-way ANOVA. **** = p<0.0001. (C) CDC20 abundance is increased in adenovirus-infected G2/M cells. HeLa FUCCI cells were mock-treated or infected with AdV-C5-wt, synchronized in G2/M by double-thymidine block, fixed, immunostained for CDC20, and quantified as in (A). Scatter plots show increased CDC20 intensity per cell upon AdV infection. Significance was calculated using ordinary one-way ANOVA. **** = p<0.0001. (D) CDC20 stabilization in cytokinetic cells during adenovirus infection. Mitotic metaphase, anaphase, and cytokinetic cells were identified in mock- and AdV-infected A549 cells using the classification strategy described in (A). Quantification of CDC20 intensity shows selective stabilization of CDC20 in cytokinetic cells following AdV infection. ns, not significant; Significance was calculated using ordinary one-way ANOVA. **** = p<0.0001. (E) Live-cell imaging of CDC20-E4orf4 co-dynamics during cleavage furrow regression. A549 cells expressing CDC20-mScarlet were infected with AdV-C5-GFP-E4orf4 and imaged by spinning-disk confocal microscopy. Representative time-lapse images show co-accumulation of CDC20-mScarlet and GFP-E4orf4 in a cytokinetic cell, coinciding with midbody collapse and cleavage furrow regression. Scale bar, 10µm.

Article Snippet: To find if regressing cells accumulate more viral proteins than non-regressing cells during AdV infection, we performed live time-lapse imaging of AdV-infected cells lines (HeLa FUCCI, HeLa H2B-mCherry, or HeLa WT) were grown on 96-well imaging plates (Greiner Biosciences and CellVis glass bottom plates), infected with indicated AdV strain, followed by timelapse imaging with high-content microscope.

Techniques: Single Cell, Infection, Blocking Assay, Live Cell Imaging, Expressing, Confocal Microscopy

Journal: bioRxiv

Article Title: Viral rewiring of APC/C-CDC20 drives Aurora B hyperubiquitination, mitotic regression and polyploidy

doi: 10.64898/2026.02.13.705602

Figure Lengend Snippet: (A) Live-cell imaging of GFP-E4orf4 dynamics during AdV infection. Representative time-lapse images of HeLa-H2B-mCherry cells infected with AdV-C5-GFP-E4orf4 show fluctuating levels of GFP-E4orf4 over time (shown as hours post infection). Pseudocolor indicates relative fluorescence intensity (low to high). Scale bar, 10 µm. (B) Time-aligned single-cell analysis of GFP–E4orf4 expression dynamics. GFP-E4orf4 intensity traces from individual cells were aligned to the time point of maximal E4orf4 expression, revealing a common oscillatory pattern and a peak coinciding with cleavage furrow regression (green shaded region). (C) Co-localization of GFP-E4orf4 and ubiquitin in infected cells. HeLa cells stably expressing mCherry-ubiquitin were infected with AdV-C5-GFP-E4orf4 for 24 h, fixed, and imaged by confocal microscopy, revealing accumulation of ubiquitin-rich structures in E4orf4-positive cells. Scale bar, 10 µm. (D) E4orf4 itself is not detectably ubiquitylated during adenovirus infection. HEK293T cells transfected with His-tagged ubiquitin were infected with AdV-C5-GFP-E4orf4 for 24 h, lysed under denaturing conditions, and ubiquitylated proteins were enriched by Ni-NTA pulldown. Immunoblotting with anti-GFP antibody shows no high-molecular-weight ubiquitylated E4orf4 species. (E) Aurora B undergoes hyperubiquitination during adenovirus infection. HEK293T cells transfected with His–ubiquitin and AurB-HA were infected and processed as in (D). Ni-NTA pulldown followed by immunoblotting with AurB-specific antibodies reveals extensive high-molecular-weight ubiquitylated Aurora B species. (F) Schematic representation of APC/C-CDC20 inhibition by Apcin and proTAME. Based on the APC/C-CDC20 structure (PDB: 5g04), Apcin blocks the D-box substrate–binding interface of CDC20, whereas proTAME interferes with CDC20 binding to APC/C TPR subunits (APC3, APC6, APC8). Inhibitor positions are schematic and indicate functional inhibition interfaces rather than exact atomic contacts. (G) Formation of an E4orf4-CDC20-APC/C-AurB complex requires an APC/C-bound CDC20 state. HEK293T cells were transfected with plasmids expressing wild-type or ΔE4orf4 adenoviral constructs together with CDC20-HA and AurB-mScarlet and treated with Apcin or proTAME as indicated. CDC20 immunoprecipitates (anti-HA) were analyzed by immunoblotting, demonstrating E4orf4- and APC/C-dependent association of AurB with CDC20. (H) E4orf4 directs APC/C-CDC20 activity toward AurB hyperubiquitination. HEK293T cells were transfected with His–ubiquitin, AurB-HA, and wild-type or ΔE4orf4 constructs, treated with Apcin or proTAME, and briefly exposed to the proteasome inhibitor MLN9708. Ubiquitylated proteins were enriched by Ni-NTA pulldown and immunoblotted, showing E4orf4- and APC/C-CDC20-dependent hyperubiquitination of AurB.

Article Snippet: To find if regressing cells accumulate more viral proteins than non-regressing cells during AdV infection, we performed live time-lapse imaging of AdV-infected cells lines (HeLa FUCCI, HeLa H2B-mCherry, or HeLa WT) were grown on 96-well imaging plates (Greiner Biosciences and CellVis glass bottom plates), infected with indicated AdV strain, followed by timelapse imaging with high-content microscope.

Techniques: Live Cell Imaging, Infection, Fluorescence, Single-cell Analysis, Expressing, Ubiquitin Proteomics, Stable Transfection, Confocal Microscopy, Transfection, Western Blot, High Molecular Weight, Inhibition, Binding Assay, Functional Assay, Construct, Activity Assay

Journal: bioRxiv

Article Title: Viral rewiring of APC/C-CDC20 drives Aurora B hyperubiquitination, mitotic regression and polyploidy

doi: 10.64898/2026.02.13.705602

Figure Lengend Snippet: (A) AdV infection reduces endogenous AurB and active AurB (pT232) levels at the midbody of cytokinetic cells. HeLa cells were infected with AdV-C5-GFP-E4orf4 for 24 h, fixed, and immunostained for AurB and AurB pT232. Cytokinetic cells with an ingressed cleavage furrow were identified by confocal microscopy and imaged. Scale bar, 10 µm. (B) Quantification of AurB and AurB pT232 signal intensity at the midbody in mock- and AdV-infected cytokinetic cells shown in (A). Significance was calculated using ordinary one-way ANOVA. **** = p<0.0001. (C–D) AdV infection alters midbody ultrastructure. AurB-mScarlet-expressing A549 cells were seeded on 3.5-mm gridded dishes and mock-treated (C) or infected with AdV-C5-GFP-E4orf4 for 24 h (D). Cells with ingressed cleavage furrows were identified by live imaging, fixed, and processed for correlative light and electron microscopy (CLEM). Fluorescence and electron micrographs were aligned using lipid droplets as fiducial markers (cyan). Yellow boxes indicate regions shown at higher magnification. Serial 70-nm sections are shown for infected cells. Scale bar, 10µm. (E) Endogenously tagged AurB-mScarlet is removed from the midbody prior to cleavage furrow regression in AdV-infected cells. HEK293T cells carrying a C-terminal mScarlet knock-in at the AURKB locus (generated by microhomology-mediated end joining) were infected with AdV-C5-GFP-E4orf4 and imaged by spinning-disk confocal microscopy. Representative time-lapse images show AurB and GFP-E4orf4 localization during cytokinesis in non-infected and infected cells. Kymograms (right) illustrate cleavage furrow ingression in both conditions, followed by abscission in non-infected cells and regression in infected cells. Scale bar, 10µm. (F) Live-cell imaging of AurB-mScarlet–expressing A549 cells showing loss of Aurora B from the midbody preceding cleavage furrow regression in AdV-infected cells. Scale bar, 10 µm.

Article Snippet: To find if regressing cells accumulate more viral proteins than non-regressing cells during AdV infection, we performed live time-lapse imaging of AdV-infected cells lines (HeLa FUCCI, HeLa H2B-mCherry, or HeLa WT) were grown on 96-well imaging plates (Greiner Biosciences and CellVis glass bottom plates), infected with indicated AdV strain, followed by timelapse imaging with high-content microscope.

Techniques: Infection, Confocal Microscopy, Expressing, Imaging, Electron Microscopy, Fluorescence, Knock-In, Generated, Live Cell Imaging

Journal: bioRxiv

Article Title: Viral rewiring of APC/C-CDC20 drives Aurora B hyperubiquitination, mitotic regression and polyploidy

doi: 10.64898/2026.02.13.705602

Figure Lengend Snippet: (A-B) Prolonged ubiquitin residence at midbody-localized Aurora B during AdV infection. A549 cells expressing Ub-GFP and AurB-mScarlet were mock-treated (A) or infected with AdV-C5-wt (B) and imaged by spinning-disk confocal microscopy. Representative time-lapse sequences show progression from cleavage furrow ingression to abscission in non-infected cells or to furrow regression in infected cells. Corresponding kymograms (bottom) indicate the temporal alignment of individual frames and reveal sustained ubiquitin association with AurB at the midbody in infected cells prior to its removal and subsequent regression. Scale bar, 10µm. (C) Quantification of ubiquitin residence time at the midbody. Box-and-whisker plots show the duration of Ub-GFP persistence at AurB-positive midbodies in normal divisions of non-infected cells and regressed divisions of AdV-infected cells. Each point represents one cell. Significance was calculated using ordinary one-way ANOVA. **** = p<0.0001. (D) APC/C inhibition suppresses adenovirus-induced cleavage furrow regression via distinct mechanisms. A549-Sec61β-GFP cells were mock-treated or infected with AdV-C5-wt and treated with DMSO, Apcin, or proTAME as indicated. Single-cell time-lapse analysis shows the timing and outcome of cell divisions, with normal and regressed divisions plotted separately. Apcin significantly reduces the frequency of cleavage furrow regression, whereas proTAME prevents infected cells from progressing beyond mitosis. Percentages of regressed divisions and numbers of analyzed and divided cells are indicated.

Article Snippet: To find if regressing cells accumulate more viral proteins than non-regressing cells during AdV infection, we performed live time-lapse imaging of AdV-infected cells lines (HeLa FUCCI, HeLa H2B-mCherry, or HeLa WT) were grown on 96-well imaging plates (Greiner Biosciences and CellVis glass bottom plates), infected with indicated AdV strain, followed by timelapse imaging with high-content microscope.

Techniques: Ubiquitin Proteomics, Infection, Expressing, Confocal Microscopy, Whisker Assay, Inhibition, Single Cell