Journal: Cell Death & Disease

Article Title: Genome-wide CRISPR screen identifies BUB1 kinase as a druggable vulnerability in malignant pleural mesothelioma

doi: 10.1038/s41419-025-07587-z

Figure Lengend Snippet: A Dot plots indicate the CRISPR viability scores of the BUB1 gene in all four cell lines. B Competitive cell proliferation assay results of MeT-5A, H2052, H2452, and H28 cells infected with gRNAs targeting the BUB1 gene. The percentage of Venus-positive cells at Day 0 was normalized to 100%, and the following measurements were calculated accordingly. Bar graphs are presented as the mean ± SD of three replicates. C Western blot assessment of BUB1 knockout in MeT-5A, H2052, H2452, and H28 cells. Cells were infected with lentiCRISPR v2 gRen (control) or lentiCRISPR v2 BUB1 (g1, g2, g3, and g4) lentiviral vectors and selected with puromycin for 3 days. BUB1 levels were detected at 6 days post-infection. β-actin was used as the equal loading control. D The MTT assay measures the proliferation rates of BUB1-depleted MeT-5A, H2052, H2452, and H28 cells. Data are presented as the mean ± SD of six replicates. Two-tailed Student’s t -test was used for statistical analysis. * p < 0.05, *** p < 0.001, ns not significant. E Correlation analysis of expression levels between BUB1 and proliferation markers Ki67 and PCNA in the TCGA MPM dataset. Log2 transformed mRNA levels were compared through cBioPortal. Pearson correlation coefficient ( r ) for Ki67 is 0.91 ( p value = 1.08e-33), and for PCNA, it is 0.68 ( p value = 3.09e-13). F Representative images showing decreased 2D colony formation capacity of MeT-5A, H2052, H2452, and H28 cell lines upon BUB1 depletion. Colony formation assay was performed in triplicates in 12-well cell culture plates for 10–14 days. High-resolution images of the plates were acquired by LI-COR Odyssey CLx Imaging System. G Crystal violet intensity data showing the relative difference in 2D colony forming capacity of gRen and BUB1 gRNA expressing cells. Image Studio software was used to measure signal intensities. Bar graphs are presented as the mean ± SD of three replicates. Two-tailed Student’s t -test was used for statistical analysis. ** p < 0.01, *** p < 0.001, ns not significant.

Article Snippet: The selective BUB1 kinase inhibitor, BAY-1816032 (HY-103020, MedChemExpress), was reconstituted in DMSO (472301, Sigma Aldrich) under sterile conditions, aliquoted, and stored at −80 °C until used.

Techniques: CRISPR, Proliferation Assay, Infection, Western Blot, Knock-Out, Control, MTT Assay, Two Tailed Test, Expressing, Transformation Assay, Colony Assay, Cell Culture, Imaging, Software

Journal: Cell Death & Disease

Article Title: Genome-wide CRISPR screen identifies BUB1 kinase as a druggable vulnerability in malignant pleural mesothelioma

doi: 10.1038/s41419-025-07587-z

Figure Lengend Snippet: A Representative image of BrdU incorporation assay showing reduced cell proliferation index (BrdU positivity, green, 12 h labeling) of BUB1-depleted cells. DAPI was used as a nuclear counterstain (Blue). Scale bar: 50 μm. B BrdU-positive cell percentages are presented as the mean ± SD, n = 6. Two-tailed Student’s t -test was used for statistical analysis. ** p < 0.01, *** p < 0.001, ns not significant. C Distribution of cell cycle phases of BUB1-depleted cells. Data are presented as mean ± SD, n = 3. Statistical analysis for multiple comparisons was performed by two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant. GSEA plots from H2052 and H2452 cell lines. The plots indicate significantly depleted E2F ( D ), MYC ( E ), and G2/M checkpoint ( F ) target gene expression, all reflecting repression of cell proliferation in MPM cell lines with BUB1 knockout ( BUB1 KO). The y -axis represents the enrichment score (ES). The significance of correlation is depicted on the x -axis, by the red color for positive and the blue color for negative correlation. Normalized enrichment score (NES), false discovery rate (FDR), and p values are shown. G Representative images of SA-β-Gal staining (blue) assay identifying increased senescence in BUB1-depleted cells compared to gRen control cells. Scale bar: 100 µm. H Percentage of SA-β-Gal staining. All cell populations as well as SA-β-Gal stained cells were counted and the percentage of SA-β-Gal staining was calculated by proportioning the number of stained cells to the total number of cells. Data are presented as mean ± SD, n = 4. Two-tailed Student’s t -test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. I Bar graphs of the Annexin V/PI apoptosis assay revealing increased apoptosis in BUB1-depleted cells compared to gRen control cells. The percentage of apoptotic cells was calculated as the sum of early and late apoptosis. Data are presented as mean ± SD, n = 3. Two-tailed Student’s t -test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant.

Article Snippet: The selective BUB1 kinase inhibitor, BAY-1816032 (HY-103020, MedChemExpress), was reconstituted in DMSO (472301, Sigma Aldrich) under sterile conditions, aliquoted, and stored at −80 °C until used.

Techniques: BrdU Incorporation Assay, Labeling, Two Tailed Test, Targeted Gene Expression, Knock-Out, Staining, Control, Apoptosis Assay

Journal: Cell Death & Disease

Article Title: Genome-wide CRISPR screen identifies BUB1 kinase as a druggable vulnerability in malignant pleural mesothelioma

doi: 10.1038/s41419-025-07587-z

Figure Lengend Snippet: A Representative image of soft agar colony formation assay. Scale bar: 100 µm. B Bar graphs showing the number of colonies with a diameter greater than 35 µm. Data are presented as mean ± SD, n = 4. Two-tailed Student’s t -test was used for statistical analysis. *** p < 0.001, ns not significant. Representative images of transwell migration ( C ) and invasion ( E ) assays in BUB1-depleted cells, with their relative controls. Scale bar: 100 μm. Migrated ( D ) and invaded ( F ) number of cells per field. ImageJ software was used for manual cell counting. Data are presented as the mean ± SD, n = 6. Two-tailed Student’s t -test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant. G Representative GSEA plots from H2052, H2452, and H28 cell lines. The plots show significantly depleted EMT target genes, reflecting decreased cell motility in MPM cells with BUB1 knockout (BUB1 KO). The y -axis represents the enrichment score (ES). The degree of correlation is represented on the x -axis, by the red color for positive and the blue color for negative correlation. Normalized enrichment score (NES), false discovery rate (FDR), and p values are shown.

Article Snippet: The selective BUB1 kinase inhibitor, BAY-1816032 (HY-103020, MedChemExpress), was reconstituted in DMSO (472301, Sigma Aldrich) under sterile conditions, aliquoted, and stored at −80 °C until used.

Techniques: Soft Agar Assay, Two Tailed Test, Migration, Software, Cell Counting, Knock-Out

Journal: Cell Death & Disease

Article Title: Genome-wide CRISPR screen identifies BUB1 kinase as a druggable vulnerability in malignant pleural mesothelioma

doi: 10.1038/s41419-025-07587-z

Figure Lengend Snippet: A Representative image of hanging drop assay. BUB1 depletion impaired the spheroid formation capacity of MPM cells. Spheroids ( n = 13, for each condition) were imaged using a stereo microscope. Scale bar: 200 μm. B The Box–Whisker plot of BUB1 mRNA expression levels in GSE2549, GSE42977, GSE51024, and GSE117668 datasets. C Representative IHC images depicting different immunoreactivity scores (IRS, ranging from 0 to 6) of BUB1 staining in MPM tissues. IRS was calculated by the multiplication of proportion of positive cells and the intensity of staining. Scale bar: 20 μm. D IHC-based analysis of BUB1 protein expression in human MPM tumor tissues ( n = 10) compared to noncancerous normal samples ( n = 10). MM: mesothelioma of other tissues. Two-tailed Student’s t -test was used for statistical analysis. * p < 0.05, ** p < 0.01. E The Kaplan–Meier survival plots of BUB1 in the TCGA MPM and GSE2549 datasets compare high gene expression (orange) relative to the low/medium or low patients (blue). p values are shown on the plots.

Article Snippet: The selective BUB1 kinase inhibitor, BAY-1816032 (HY-103020, MedChemExpress), was reconstituted in DMSO (472301, Sigma Aldrich) under sterile conditions, aliquoted, and stored at −80 °C until used.

Techniques: Microscopy, Whisker Assay, Expressing, Staining, Two Tailed Test, Gene Expression

Journal: Cell Death & Disease

Article Title: Genome-wide CRISPR screen identifies BUB1 kinase as a druggable vulnerability in malignant pleural mesothelioma

doi: 10.1038/s41419-025-07587-z

Figure Lengend Snippet: A BUB1 protein levels in empty vector (Vector) and BUB1 overexpression vector (BUB1) transduced H2052, H2452, and H28 cells. β-actin was used as a loading control. B Representative images showing increased 2D colony formation capacity of H2052, H2452, and H28 cell lines upon BUB1 overexpression. Colony formation assay was performed in triplicates in 12-well cell culture plates for 10–14 days. High-resolution images of the plates were acquired by LI-COR Odyssey CLx Imaging System. C Crystal violet intensity data showing the relative difference in 2D colony forming capacity of Vector and BUB1-overexpressing cells. Image Studio software was used to measure signal intensities. Bar graphs are presented as the mean ± SD of three replicates. Two-tailed Student’s t -test was used for statistical analysis. * p < 0.05 and ** p < 0.01. D Representative images of BrdU incorporation assay identifying increased cell proliferation index (BrdU positivity, red, 12 h incubation) in BUB1-overexpressing H2052, H2452, and H28 cells compared to Vector control cells. DAPI was used as the nuclear counterstain (Blue). Scale bar: 100 μm. E BrdU-positive cell percentages are presented as the mean ± SD, n = 6, n = 5 for H2452. Two-tailed Student’s t -test was used for statistical analysis. ** p < 0.01 and *** p < 0.001, ns not significant. F Representative images of soft agar colony formation assay. Scale bar: 100 µm. G Bar graphs showing the number of colonies with a diameter greater than 35 µm. Data are presented as mean ± SD, n = 5 for H2052, n = 4 for H2452. Two-tailed Student’s t -test was used for statistical analysis. * p < 0.05 and ** p < 0.01. Representative images of transwell migration ( H ) and invasion ( J ) assays upon BUB1 overexpression with their relative controls. Scale bar: 100 μm. Migrated ( I ) and invaded ( K ) number of cells per field. ImageJ software was used for manual cell counting. Data are presented as the mean ± SD, n = 6. Two-tailed Student’s t -test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The selective BUB1 kinase inhibitor, BAY-1816032 (HY-103020, MedChemExpress), was reconstituted in DMSO (472301, Sigma Aldrich) under sterile conditions, aliquoted, and stored at −80 °C until used.

Techniques: Plasmid Preparation, Over Expression, Control, Colony Assay, Cell Culture, Imaging, Software, Two Tailed Test, BrdU Incorporation Assay, Incubation, Soft Agar Assay, Migration, Cell Counting

Journal: Cell Death & Disease

Article Title: Genome-wide CRISPR screen identifies BUB1 kinase as a druggable vulnerability in malignant pleural mesothelioma

doi: 10.1038/s41419-025-07587-z

Figure Lengend Snippet: A Heatmap depicting the expression levels of BUB1 local network genes (highlighted in Fig. ) in BUB1 knockout (BUB1 KO) H2052 and H2452 cells. B BUB1, Cyclin B, Cyclin A, CDC20 and p21 protein levels in BUB1 WT vs BUB1 KO cells. β-actin was used as a loading control. C Localization of MAD1, MAD2, and SGO1 in mitotic MPM cells demonstrated through immunofluorescence staining. Cells were synchronized with a 12 h thymidine block followed by a 12 h release in a complete culture medium or 150 mM nocodazole for H28 cells. The images were captured as z-stacks using Apotome 3 (Zeiss) and subsequently processed into stacked projections. D Time-lapse live cell microscopy illustrating mitotic progression of BUB1 knockout H2452 cells compared to control. Cells were stained with Hoechst and monitored using confocal microscopy by acquiring z-stacks every 10 min for 20 h. Representative image sequences were aligned on the time axis and presented in the figures.

Article Snippet: The selective BUB1 kinase inhibitor, BAY-1816032 (HY-103020, MedChemExpress), was reconstituted in DMSO (472301, Sigma Aldrich) under sterile conditions, aliquoted, and stored at −80 °C until used.

Techniques: Expressing, Knock-Out, Control, Immunofluorescence, Staining, Blocking Assay, Microscopy, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: Genome-wide CRISPR screen identifies BUB1 kinase as a druggable vulnerability in malignant pleural mesothelioma

doi: 10.1038/s41419-025-07587-z

Figure Lengend Snippet: A MTT assay measuring the IC50 values of H2052, H2452, and H28 cells. Cells were treated with increasing doses of BAY-1816032 ranging from 0.25 to 10 μM. Control cells were treated with DMSO. The experiment was performed in 2 biological and 6 technical replicates. B Representative images showing 2D colony formation capacity of H2052, H2452, and H28 cell lines upon BAY-1816032 treatment. Experiments were performed in triplicates in 12-well plates. Cells were treated for 3 days with IC50 and two doses under IC50 of BAY-1816032 and cultured for another 7–11 days. High-resolution images were captured with the LI-COR Odyssey CLx Imaging System. C Crystal violet intensity data showing the relative difference in 2D colony forming capacity of Vehicle (DMSO) and BAY-1816032 treated cells. Image Studio software was used to measure signal intensities. Bar graphs are presented as the mean ± SD of three replicates. Two-tailed Student’s t -test was used for statistical analysis. *** p < 0.001. D Bar graphs showing the decrease in size of spheroids upon pharmacological BUB1 inhibition. Data are presented as mean ± SD, n = 6. Two-tailed Student’s t-test was used for statistical analysis. *** p < 0.001. E Distribution of cell cycle phases of BAY-1816032 treated H2052, H2452, and H28 cells. Data are presented as mean ± SD, n = 3. Statistical analysis was performed by two-way ANOVA multiple comparison. * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant. Representative images of transwell migration ( F ) and invasion ( H ) assays of BAY-1816032 treated cells, with their relative controls. Scale bar: 100 μm. Migrated ( G ) and invaded ( I ) number of cells per field. ImageJ software was used for manual cell counting. Data are presented as the mean ± SD, n = 6. Two-tailed Student’s t -test was used for statistical analysis. *** p < 0.001.

Article Snippet: The selective BUB1 kinase inhibitor, BAY-1816032 (HY-103020, MedChemExpress), was reconstituted in DMSO (472301, Sigma Aldrich) under sterile conditions, aliquoted, and stored at −80 °C until used.

Techniques: MTT Assay, Control, Cell Culture, Imaging, Software, Two Tailed Test, Inhibition, Comparison, Migration, Cell Counting