Journal: The Journal of Cell Biology

Article Title: High-content phenotyping reveals Golgi dynamics and their role in cell cycle regulation

doi: 10.1083/jcb.202503083

Figure Lengend Snippet: Morphological features of cellular and subcellular structures for cell phenotype profiling. (A) Example of the cell segmentation process and extraction of single cells. HeLa cells were segmented using contrast-enhanced images of microtubules and DAPI-stained nuclei. Each cell was assigned an identifier, and two representative cells (#38 and #81) are shown. Scale bar, 50 µm. (B and C) Examples of abstraction-based analysis of Golgi structure. Golgi morphology was characterized from GM130 images by analyzing fluorescence peaks (B) and by extracting skeletonized line objects (C). The boxed regions in the nucleus–Golgi distance measurements correspond to the cropped areas shown in . Scale bar, 50 µm. (D) Timeline for the analysis of mitotic spindle defects in metaphase-arrested HeLa cells. HeLa cells were synchronized with a double thymidine block, followed by ProTAME treatment to accumulate cells in metaphase. To induce specific spindle assembly defects, cells were treated with DMSO (control), taxol, or monastrol for 6 h, when most cells were expected to be in G2 phase. (E) Representative cell images. The left panel displays a full image of DMSO-treated cells, while the right panel shows magnified views of typical mitotic cells. Scale bars: 50 µm (full image) and 20 µm (magnified image). (F) Correlation matrix of all features used for cell phenotype profiling, as shown in . See for descriptions of individual features.

Article Snippet: The following reagents were used in cell culture or treatment: DAPI (400 ng/ml; D8417; Sigma-Aldrich) and Alexa Fluor 488–conjugated phalloidin (1:800; A12379; Thermo Fisher Scientific) for fluorescence staining; thymidine (2 mM; T1895; Sigma-Aldrich) and ProTAME (12 μM; 1362911; R&D Systems) for cell cycle synchronization; taxol (50 nM for mitosis or 1 μM for G2; HY-B0015; MedChemExpress), monastrol (100 nM; HY-101071A; MedChemExpress), SP600125 (50 μM; S5567; Sigma-Aldrich), cytochalasin D (100 ng/ml; C8273; Sigma-Aldrich), and brefeldin A (200 ng/ml; B7651; Sigma-Aldrich) for pharmacological inhibition.

Techniques: Extraction, Staining, Fluorescence, Blocking Assay, Control

Journal: bioRxiv

Article Title: Mitotic slippage causes nuclear instability in polyploid cells

doi: 10.1101/2025.07.21.665898

Figure Lengend Snippet: (A) Stills of time lapse movies showing RPE-1 cells stably expressing mCherry-H2B (in magenta) performing MS and treated with DMSO (upper panel) or 2µM Methylstat (lower panel). Microtubules were labelled using SPY650-Tubulin (in cyan). The white arrows indicate the microtubules transiently accumulating at the nuclear envelope. (B) Schematic workflow presenting the method used to treat cells with Methylstat before or after MS. (C) Super resolution representative images showing diploid and MS-generated tetraploid RPE-1 cells treated with DMSO or with 4µM Methylstat before and after MS. Lamin A/C in yellow hot, DNA in magenta. (D-E) Graph showing nuclear circularity and solidity in diploid and in MS-generated tetraploid cells (in grey and blue, respectively) treated with DMSO or with 4µM Methylstat. (F-G) Graph showing nuclear circularity and solidity in diploid and in MS-generated tetraploid cells (in grey and blue, respectively) treated with DMSO or with (F) GSK-J4 or (G) JIB. Mean□±□SEM, >70 G1 cells analysed per condition, three independent experiments. (H) Western blots of total protein extracts obtained from RPE-1 cells transfected with siCtrl or siPHF8 (upper panel) or overexpressing HA-G9a (lower panel). (I-J) Graphs showing nuclear circularity and solidity in diploid and in MS-generated tetraploid cells (in grey and blue, respectively) (I) transfected with siCtrl or siPHF8 or (J) overexpressing HA-G9a. Mean□±□SEM, >140 G1 cells analysed per condition, three independent experiments. (K) Schematic workflow presenting the method used to extend mitotic duration using Monastrol treatment. (L) Left panel – Super resolution representative images showing RPE-1 cells in prometaphase treated with DMSO or with 50µM Monastrol for 16hrs. H3S10 in green, DNA in magenta. Right panel – graph showing H3S10 mean intensity in early mitosis in RPE-1 diploid cells treated with DMSO or with 50µM Monastrol for 16hrs. Mean□±□SEM, >100 mitotic cells analysed, three independent experiments. (M) Graph showing nuclear circularity and solidity in diploid and in MS-generated tetraploid cells (in grey and blue, respectively) treated with DMSO or with 50µM Monastrol. Mean□±□SEM, >100 G1 cells analysed per condition, three independent experiments. (N) Representative images showing RPE-1 diploid cells treated with DMSO or with 5nM Calyculin. H3S10 and H3K9me2 in green and cyan hot, respectively, DNA in magenta. (O) Graph presenting nuclear circularity and solidity in RPE-1 diploid cells treated with DMSO or with 5nM Calyculin. Mean□±□SEM, >230 G1 cells analysed per condition, three independent experiments. Scale bars: 10µm. D=diploid; T=tetraploid; MS=mitotic slippage; ON=overnight. ( D,E,F,G,I,J,M ) Anova-test (one-sided). ( L,O,P ) t-test (two-sided). ns=not significant. *=P ≤ 0.05. **= P ≤ 0.01. ***= P ≤ 0.001. ****= P ≤ 0.0001.

Article Snippet: 48hrs later, the coverslips were transferred to new wells and incubated with DMSO (D8418 from Sigma Aldrich) or with 50μM Monastrol (S8439 from Selleckchem) + 1μM MPI-0479605 (S7488 from Selleckchem) to generate tetraploid cells.

Techniques: Stable Transfection, Expressing, Generated, Western Blot, Transfection